U.S. patent application number 17/379793 was filed with the patent office on 2022-04-21 for antisense oligomers and uses thereof.
The applicant listed for this patent is Cold Spring Harbor Laboratory, Stoke Therapeutics, Inc.. Invention is credited to Isabel Aznarez, Adrian Krainer, Huw M. Nash.
Application Number | 20220118000 17/379793 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-21 |
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United States Patent
Application |
20220118000 |
Kind Code |
A1 |
Aznarez; Isabel ; et
al. |
April 21, 2022 |
ANTISENSE OLIGOMERS AND USES THEREOF
Abstract
Provided herein are methods and compositions for increasing the
expression of a protein, and for treating a subject in need
thereof, e.g., a subject with deficient protein expression or a
subject having a disease described herein.
Inventors: |
Aznarez; Isabel; (Jamaica
Plain, MA) ; Nash; Huw M.; (Lexington, MA) ;
Krainer; Adrian; (East Northport, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Stoke Therapeutics, Inc.
Cold Spring Harbor Laboratory |
Bedford
Cold Spring Harbor |
MA
NY |
US
US |
|
|
Appl. No.: |
17/379793 |
Filed: |
July 19, 2021 |
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Application
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16007435 |
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11096956 |
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17379793 |
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PCT/US2016/066721 |
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PCT/US2016/066691 |
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62267210 |
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62267252 |
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62267261 |
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62267256 |
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62319011 |
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62267259 |
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International
Class: |
A61K 31/7125 20060101
A61K031/7125; A61K 47/68 20060101 A61K047/68; C12N 15/113 20060101
C12N015/113; A61P 43/00 20060101 A61P043/00; A61K 38/00 20060101
A61K038/00; A61K 38/17 20060101 A61K038/17; C12N 15/62 20060101
C12N015/62; C12N 15/11 20060101 C12N015/11 |
Claims
1-164. (canceled)
165. A method of increasing expression of a target protein by human
cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA)
that encodes the target protein, wherein the RIC pre-mRNA comprises
a retained intron, an exon flanking 5' splice site of the retained
intron, and an exon flanking 3' splice site of the retained intron,
the method comprising contacting the human cells with an antisense
oligomer (ASO) that binds to a targeted portion of the RIC pre-mRNA
encoding the target protein, whereby the retained intron is
constitutively spliced from the RIC pre-mRNA encoding the target
protein, thereby increasing level of mRNA encoding the target
protein, and increasing the expression of the target protein in the
human cells, wherein the target protein is encoded by a gene
selected from the group consisting of: ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA and STX1B.
166. The method of claim 165, wherein the targeted portion of the
RIC pre-mRNA is: (a) in the retained intron within a region +6
relative to the 5' splice site to -16 relative to the 3' splice
site of the retained intron; (b) a region +2e to -4e in the exon
flanking the 5' splice site of the retained intron; or (c) a region
+2e to -4e in the exon flanking the 3' splice site of the retained
intron.
167. The method of claim 165, wherein the target protein is encoded
by a SHANK3 gene, and wherein the retained intron comprises a
sequence with at least 90% sequence identity to SEQ ID NO:
30630.
168. The method of claim 165, wherein the targeted portion of the
RIC pre-mRNA is in the retained intron within a region +6 to +500
relative to the 5' splice site of the retained intron, or in the
retained intron within a region -16 to -500 relative to the 3'
splice site of the retained intron.
169. The method of claim 165, wherein the ASO comprises a sequence
that is complementary to at least 8 contiguous nucleotides of the
targeted portion of the RIC pre-mRNA.
170. The method of claim 165, wherein the target protein is encoded
by a SHANK3 gene, and wherein the ASO comprises a sequence selected
from the group consisting of SEQ ID NOs: 29621-29808.
171. The method of claim 165, wherein the ASO increases amount of
the target protein in the cells by at least about 1.1 fold.
172. The method of claim 165, wherein the ASO does not increase
amount of the target protein in the cells by modulating alternative
splicing of pre-mRNA transcribed from a gene encoding the target
protein.
173. The method of claim 165, wherein the ASO does not increase
amount of the target protein in the cells by modulating aberrant
splicing resulting from mutation of a gene encoding the target
protein.
174. The method of claim 165, wherein the cells are contacted with
the ASO ex vivo.
175. The method of claim 165, wherein the ASO comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage.
176. The method of claim 165, wherein the ASO comprises a
phosphorodiamidate morpholino, a locked nucleic acid, a peptide
nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl
moiety.
177. The method of claim 165, wherein the ASO consists of from 8 to
50 nucleobases.
178. A pharmaceutical composition comprising: an antisense oligomer
(ASO) or a viral vector encoding the ASO, wherein the antisense
oligomer hybridizes to a targeted portion of a
retained-intron-containing pre-mRNA (RIC pre-mRNA) that encodes a
target protein, wherein the RIC pre-mRNA comprises a retained
intron, an exon flanking 5' splice site of the retained intron, and
an exon flanking 3' splice site of the retained intron, and wherein
the antisense oligomer is configured to induce splicing out of the
retained intron from the RIC pre-mRNA, wherein the target protein
is encoded by a gene selected from the group consisting of: ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA
and STX1B.
179. The pharmaceutical composition of claim 178, wherein the
targeted portion of the RIC pre-mRNA is: (a) in the retained intron
within a region +6 relative to the 5' splice site to -16 relative
to the 3' splice site of the retained intron; (b) a region +2e to
-4e in the exon flanking the 5' splice site of the retained intron;
or (c) a region +2e to -4e in the exon flanking the 3' splice site
of the retained intron.
180. The pharmaceutical composition of claim 178, wherein the ASO
comprises a backbone modification comprising a phosphorothioate
linkage or a phosphorodiamidate linkage.
181. The pharmaceutical composition of claim 178, wherein the ASO
comprises a phosphorodiamidate morpholino, a locked nucleic acid, a
peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a
2'-O-methoxyethyl moiety.
182. The pharmaceutical composition of claim 178, wherein the ASO
comprises a sequence that is complementary to at least 8 contiguous
nucleotides of the targeted portion of the RIC pre-mRNA.
183. The pharmaceutical composition of claim 178, wherein the
target protein is encoded by a SHANK3 gene, and wherein the ASO
comprises a sequence selected from the group consisting of SEQ ID
NOs: 29621-29808.
184. The pharmaceutical composition of claim 178, wherein the
target protein is encoded by a SHANK3 gene, and wherein the
retained intron comprises a sequence with at least 90% sequence
identity to a sequence selected from the group consisting of SEQ ID
NOs: 30630.
185. The pharmaceutical composition of claim 178, wherein the
targeted portion of the RIC pre-mRNA is in the retained intron
within a region +6 to +500 relative to the 5' splice site of the
retained intron, or in the retained intron within a region -16 to
-500 relative to the 3' splice site of the retained intron.
186. The pharmaceutical composition of claim 178, wherein the ASO
consists of from 8 to 50 nucleobases.
Description
CROSS-REFERENCE
[0001] This application is a Continuation-in-Part of: PCT
Application No. PCT/US2016/066414 filed Dec. 13, 2016, which claims
the benefit of U.S. Provisional Patent Application No. 62/267,210
filed Dec. 14, 2015; PCT Application No. PCT/US2016/066705 filed
Dec. 14, 2016, which claims the benefit of U.S. Provisional Patent
Application No. 62/267,212 filed Dec. 14, 2015; PCT Application No.
PCT/US2016/066417 filed Dec. 13, 2016, which claims the benefit of
U.S. Provisional Patent Application No. 62/267,252 filed Dec. 14,
2015; PCT Application No. PCT/US2016/066684 filed Dec. 14, 2016,
which claims the benefit of U.S. Provisional Patent Application No.
62/267,261 filed Dec. 14, 2015; PCT Application No.
PCT/US2016/066721 filed Dec. 14, 2016, which claims the benefit of
U.S. Provisional Patent Application No. 62/267,256 filed Dec. 14,
2015 and U.S. Provisional Patent Application No. 62/319,011 filed
Apr. 6, 2016; PCT Application No. PCT/US2016/066691 filed Dec. 14,
2016, which claims the benefit of U.S. Provisional Patent
Application No. 62/267,259 filed Dec. 14, 2015 and U.S. Provisional
Patent Application No. 62/318,958 filed Apr. 6, 2016; PCT
Application No. PCT/US2016/066564 filed Dec. 14, 2016, which claims
the benefit of U.S. Provisional Patent Application No. 62/267,238
filed Dec. 14, 2015 and U.S. Provisional Patent Application No.
62/319,015 filed Apr. 6, 2016; and PCT Application No.
PCT/US2016/066576 filed Dec. 14, 2016, which claims the benefit of
U.S. Provisional Patent Application No. 62/267,242 filed Dec. 14,
2015, which applications are incorporated herein by reference in
their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 13, 2018 is named 47991_705_501_SL.txt and is 65,978,019
bytes in size.
BACKGROUND OF THE INVENTION
[0003] Alagille syndrome (ALGS), also known as arteriohepatic
dysplasia, is a rare, debilitating, autosomal dominant, multisystem
disorder (Tumpenny and Ellard, Eur. J. Hum. Gen. 2012, 20,
251-257). Patients suffer from liver damage caused by abnormalities
in the bile ducts. Other effects include heart disease, vascular
anomalies, skeletal anomalies, ophthalmic features, facial
features, renal anomalies, growth retardation, and pancreatic
insufficiency. The reported ALGS prevalence of 1:70,000 is thought
to be an underestimate because of the variability and reduced
penetrance of the condition.
[0004] Mutations of genes involved in Notch signaling have been
reported to cause ALGS. Mutations in JAG1 cause ALGS type 1, while
mutations in NOTCH2 cause ALGS type 2, which is less prevalent than
ALGS type 1. JAG1 encodes JAG1 protein, a cell surface ligand for
the Notch transmembrane receptors. Binding of JAG1 protein to the
Notch receptors triggers a signaling cascade that results in
transcription of genes involved in cell fate determination and
differentiation.
[0005] Tuberous sclerosis complex (TSC) is a disorder characterized
by growth of benign tumors in multiple organ systems (Au, K., et
al., J. Child Neurol., 2004, 19: 699-709). Tumors of the central
nervous system (CNS) are the leading cause of morbidity and
mortality, followed by renal disease. Patients can suffer from
abnormalities of the brain that may include seizures, intellectual
disability, and developmental delay, as well as abnormalities of
the skin, lung, kidneys, and heart. The disorder affects as many as
25,000 to 40,000 individuals in the United States and about 1 to 2
million individuals worldwide, with an estimated prevalence of one
in 6,000 newborns.
[0006] TSC is a genetic disorder with an autosomal dominant
inheritance pattern, caused by inherited defects or de novo
mutations that occur on two genes, TSC1 and TSC2. Only one of the
genes needs to be affected for TSC to be present. The TSC1 gene, on
chromosome 9, produces a protein called hamartin. The TSC2 gene,
discovered in 1993, is on chromosome 16 and produces the protein
tuberin. Scientists believe these proteins act in a complex as
growth suppressors by inhibiting the activation of a master,
evolutionarily conserved kinase called mTOR. Loss of regulation of
mTOR occurs in cells lacking either hamartin or tuberin, and this
leads to abnormal differentiation and development, and to the
generation of enlarged cells, as are seen in TSC brain lesions.
[0007] Autosomal dominant polycystic kidney disease (ADPKD), is one
of the most common inherited renal cystic diseases, conditions
characterized by the development of renal cysts and a variety of
extrarenal manifestations (Torres and Harris, 2009, Kidney
International (2009) 76, 149-168). Patients suffering from ADPKD
generally develop end-stage renal disease (ESRD) by age 70, which
ultimately requires interventions such as renal dialysis. The
prevalence of ADPKD at birth is estimated to be between 1:400 and
1:1,000, affecting about 600,000 people in the US.
[0008] Mutations in either the PKD1 or PKD2 gene have been shown to
manifest as ADPKD, with mutations in PKD2 being responsible for the
late onset form of ADPKD. The PKD1 and PKD2 genes encode the PC-1
and PC-2 proteins, respectively. These proteins are believed to be
essential to maintain the differentiated phenotype of the tubular
epithelium (Tones and Harris, 2009).
[0009] Retinitis pigmentosa (RP) describes a group of diseases that
have similar clinical phenotypes and are associated with
genetically heterogeneous causes. RP frequently manifests as a loss
of night vision, and can progress to peripheral blindness,
resulting in tunnel vision. As the disorder progresses, subjects
may lose a significant portion of their photoreceptors before
experiencing loss of visual acuity, and can eventually experience
complete blindness. RP-associated genes PRPF3, PRPF8, PRPF31, and
PAP1 are involved in the assembly of spliceosomes. Although the
functional properties of several RP-associated genes have been
extensively studied, genotype-phenotype correlations are
incompletely understood. However, it is known that disease-causing
mutations in the PRPF3, PRPF8, PRPF31 and PAP1 genes have a
dominant pattern of inheritance (Berger et al., 2010, Progress in
Retinal Eye Research 29, 335-375).
[0010] Certain diseases affecting central nervous system function
are associated with a deficiency in the expression of a gene, and
in turn, a deficiency in the gene product. Examples of gene
products for which increased expression can provide benefit in
central nervous system diseases or conditions include, ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA
and STX1B.
[0011] Certain diseases affecting eye function are associated with
a deficiency in the expression of a gene, and in turn, a deficiency
in the gene product. Examples of gene products for which increased
expression can provide benefit in eye diseases or conditions
include ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 and IDUA.
[0012] Liver disease is a debilitating and often fatal group of
conditions with an estimated mortality rate of 80%. The liver is
vital for many functions in the body including, but not limited to
clearing the blood of harmful toxins, storing and releasing
glucose, production of bile, storage of iron and aiding resistance
to infections. Dysfunction of the liver ultimately leads to failure
of other major organs and ultimately death. While liver
transplantation can often prevent mortality, the odds of receiving
a donor liver is typically low.
[0013] While there are a large number of diseases and conditions
associated with the liver, a subset of liver diseases have been
shown to proceed via a deficiency in the expression of a gene, and
in turn, a deficiency in the gene product. Examples of gene
products for which increased expression can provide benefit in
liver diseases or conditions include AMT, ADA, PPOX, UROD, HMBS,
ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1,
HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE,
ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5.
[0014] Kidney disease is a debilitating and potentially fatal group
of conditions associated with the damaging of the kidneys. The
kidney is vital for many functions in the body including, but not
limited to cleansing of the blood by removing waste and excess
fluid, maintaining the balance of salt and minerals in the blood,
and regulation of blood pressure. Incidence of kidney disease often
results in the buildup of fluid and waste products, vomiting,
weakness, poor sleep and shortness of breath, and ultimately could
lead to death. While kidney transplantation can often prevent
mortality, the odds of receiving a donor kidney are typically
low.
[0015] While there are a large number of diseases and conditions
associated with the kidney, a subset of kidney diseases have been
shown to proceed via a deficiency in the expression of a gene, and
in turn, a deficiency in the gene prod, for example, CTNS, PAX2,
CYP24A1 and PPARD.
SUMMARY OF THE INVENTION
[0016] The invention provides compositions and methods for treating
Alagille syndrome, including antisense oligomers (ASOs) that
promote constitutive splicing at intron splice sites of a JAG1
retained-intron-containing pre-mRNA (RIC pre-mRNA). The invention
further provides compositions and methods for increasing production
of mature JAG1 mRNA and, in turn, JAG1 protein, in cells of a
subject in need thereof, for example, a subject that can benefit
from increased production of JAG1 protein. The described methods
may be used to treat subjects having Alagille Syndrome caused by a
mutation(s) in the JAG1 gene, including missense, splicing,
frameshift and nonsense mutations, as well as whole gene deletions
that result in deficient JAG1 protein production. In other
embodiments, the compositions and methods of the present invention
are used to treat subjects having a muscular dystrophy, who can
benefit from increased production of JAG1 protein.
[0017] Disclosed herein, in certain embodiments, is a method of
treating Alagille syndrome in a subject in need thereof by
increasing the expression of a target protein or functional RNA by
cells of the subject, wherein the cells have a
retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre-mRN
6489362A comprising a retained intron, an exon flanking the 5'
splice site, an exon flanking the 3' splice site, and wherein the
RIC pre-mRNA encodes the target protein or functional RNA, the
method comprising contacting the cells of the subject with an
antisense oligomer (ASO) complementary to a targeted portion of the
RIC pre-mRNA encoding the target protein or functional RNA, whereby
the retained intron is constitutively spliced from the RIC pre-mRNA
encoding the target protein or functional RNA, thereby increasing
the level of mRNA encoding the target protein or functional RNA,
and increasing the expression of the target protein or functional
RNA in the cells of the subject. In some embodiments, also
disclosed herein is a method of increasing expression of a target
protein, wherein the target protein is JAG1, by cells having a
retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC
pre-mRNA comprising a retained intron, an exon flanking the 5'
splice site of the retained intron, an exon flanking the 3' splice
site of the retained intron, and wherein the RIC pre-mRNA encodes
JAG1 protein, the method comprising contacting the cells with an
antisense oligomer (ASO) complementary to a targeted portion of the
RIC pre-mRNA encoding JAG1 protein, whereby the retained intron is
constitutively spliced from the RIC pre-mRNA encoding JAG1 protein,
thereby increasing the level of mRNA encoding JAG1 protein, and
increasing the expression of JAG1 protein in the cells. In some
embodiments, the target protein is JAG1. In some embodiments, the
target protein or the functional RNA is a compensating protein or a
compensating functional RNA that functionally augments or replaces
a target protein or functional RNA that is deficient in amount or
activity in the subject. In some embodiments, the cells are in or
from a subject having a condition caused by a deficient amount or
activity of JAG1 protein. In some embodiments, the deficient amount
of the target protein is caused by haploinsufficiency of the target
protein, wherein the subject has a first allele encoding a
functional target protein, and a second allele from which the
target protein is not produced, or a second allele encoding a
nonfunctional target protein, and wherein the antisense oligomer
binds to a targeted portion of a RIC pre-mRNA transcribed from the
first allele. In some embodiments, the subject has a condition
caused by a disorder resulting from a deficiency in the amount or
function of the target protein, wherein the subject has a) a first
mutant allele from which (i) the target protein is produced at a
reduced level compared to production from a wild-type allele, (ii)
the target protein is produced in a form having reduced function
compared to an equivalent wild-type protein, or (iii) the target
protein is not produced, and b) a second mutant allele from which
(i) the target protein is produced at a reduced level compared to
production from a wild-type allele, (ii) the target protein is
produced in a form having reduced function compared to an
equivalent wild-type protein, or (iii) the target protein is not
produced, and wherein when the subject has a first mutant allele
a.iii., the second mutant allele is b.i. or b.ii., and wherein when
the subject has a second mutant allele b.iii., the first mutant
allele is a.i. or a.ii., and wherein the RIC pre-mRNA is
transcribed from either the first mutant allele that is a.i. or
a.ii., and/or the second allele that is b.i. or b.ii. In some
embodiments, the target protein is produced in a form having
reduced function compared to the equivalent wild-type protein. In
some embodiments, the target protein is produced in a form that is
fully-functional compared to the equivalent wild-type protein. In
some embodiments, the targeted portion of the RIC pre-mRNA is in
the retained intron within the region +6 relative to the 5' splice
site of the retained intron to -16 relative to the 3' splice site
of the retained intron. In some embodiments, the targeted portion
of the RIC pre-mRNA is in the retained intron within: (a) the
region +6 to +500, +6 to +400, +6 to 300, +6 to 200, or +6 to +100
relative to the 5' splice site of the retained intron; or (b) the
region -16 to -500, -16 to -400, -16 to -300, -16 to -200, or -16
to -100 relative to the 3' splice site of the retained intron. In
some embodiments, the targeted portion of the RIC pre-mRNA is
within: (a) the region +2e to -4e in the exon flanking the 5'
splice site of the retained intron; or (b) the region +2e to -4e in
the exon flanking the 3' splice site of the retained intron. In
some embodiments, the RIC pre-mRNA is encoded by a genetic sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO: 1. In some embodiments, the RIC
pre-mRNA comprises a sequence with at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ
ID NO: 2. In some embodiments, the antisense oligomer does not
increase the amount of the target protein or the functional RNA by
modulating alternative splicing of pre-mRNA transcribed from a gene
encoding the functional RNA or target protein. In some embodiments,
the antisense oligomer does not increase the amount of the target
protein or the functional RNA by modulating aberrant splicing
resulting from mutation of the gene encoding the target protein or
the functional RNA. In some embodiments, the RIC pre-mRNA was
produced by partial splicing of a full-length pre-mRNA or partial
splicing of a wild-type pre-mRNA. In some embodiments, the mRNA
encoding the target protein or functional RNA is a full-length
mature mRNA, or a wild-type mature mRNA. In some embodiments, the
target protein produced is full-length protein, or wild-type
protein. In some embodiments, the total amount of the mRNA encoding
the target protein or functional RNA produced in the cell contacted
with the antisense oligomer is increased about 1.1 to about
10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold,
about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to
about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold,
about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to
about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold,
about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about
6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3
to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold,
about 4 to about 9-fold, at least about 1.1-fold, at least about
1.5-fold, at least about 2-fold, at least about 2.5-fold, at least
about 3-fold, at least about 3.5-fold, at least about 4-fold, at
least about 5-fold, or at least about 10-fold, compared to the
total amount of the mRNA encoding the target protein or functional
RNA produced in a control cell. In some embodiments, the total
amount of target protein produced by the cell contacted with the
antisense oligomer is increased about 1.1 to about 10-fold, about
1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about
10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about
1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about
8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2
to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold,
about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about
7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4
to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold,
at least about 1.1-fold, at least about 1.5-fold, at least about
2-fold, at least about 2.5-fold, at least about 3-fold, at least
about 3.5-fold, at least about 4-fold, at least about 5-fold, or at
least about 10-fold, compared to the total amount of target protein
produced by a control cell. In some embodiments, the antisense
oligomer comprises a backbone modification comprising a
phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, the antisense oligomer comprises a phosphorodiamidate
morpholino, a locked nucleic acid, a peptide nucleic acid, a
2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some
embodiments, the antisense oligomer comprises at least one modified
sugar moiety. In some embodiments, each sugar moiety is a modified
sugar moiety. In some embodiments, the antisense oligomer consists
of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35
nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20
nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40
nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25
nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30
nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15
nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35
nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20
nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40
nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25
nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some
embodiments, the antisense oligomer is at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
complementary to the targeted portion of the RIC pre-mRNA encoding
the protein. In some embodiments, the targeted portion of the RIC
pre-mRNA is within a sequence selected from SEQ ID NOs: 437-439. In
some embodiments, the antisense oligomer comprises a nucleotide
sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID
NOs: 3-436. In some embodiments, the antisense oligomer comprises a
nucleotide sequence selected from SEQ ID NOs: 3-436. In some
embodiments, the cell comprises a population of RIC pre-mRNAs
transcribed from the gene encoding the target protein or functional
RNA, wherein the population of RIC pre-mRNAs comprises two or more
retained introns, and wherein the antisense oligomer binds to the
most abundant retained intron in the population of RIC pre-mRNAs.
In some embodiments, the binding of the antisense oligomer to the
most abundant retained intron induces splicing out of the two or
more retained introns from the population of RIC pre-mRNAs to
produce mRNA encoding the target protein or functional RNA. In some
embodiments, the cell comprises a population of RIC pre-mRNAs
transcribed from the gene encoding the target protein or functional
RNA, wherein the population of RIC pre-mRNAs comprises two or more
retained introns, and wherein the antisense oligomer binds to the
second most abundant retained intron in the population of RIC
pre-mRNAs. In some embodiments, the binding of the antisense
oligomer to the second most abundant retained intron induces
splicing out of the two or more retained introns from the
population of RIC pre-mRNAs to produce mRNA encoding the target
protein or functional RNA. In some embodiments, the condition is a
disease or disorder. In some embodiments, the disease or disorder
is Alagille syndrome or a muscular dystrophy. In some embodiments,
the target protein and the RIC pre-mRNA are encoded by the JAG1
gene. In some embodiments, the method further comprises assessing
JAG1 protein expression. In some embodiments, the antisense
oligomer binds to a targeted portion of a JAG1 RIC pre-mRNA,
wherein the targeted portion is within a sequence selected from SEQ
ID NOS: 49, 50, 51, 52, 53, 54, 55, 56, and 57. In some
embodiments, the subject is a human. In some embodiments, the
subject is a non-human animal. In some embodiments, the subject is
a fetus, an embryo, or a child. In some embodiments, the cells are
ex vivo. In some embodiments, the antisense oligomer is
administered by intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection of the subject. In
some embodiments, the 9 nucleotides at -3e to -1e of the exon
flanking the 5' splice site and +1 to +6 of the retained intron,
are identical to the corresponding wild-type sequence. In some
embodiments, the 16 nucleotides at -15 to -1 of the retained intron
and +1e of the exon flanking the 3' splice site are identical to
the corresponding wild-type sequence.
[0018] Disclosed herein, in certain embodiments, is an antisense
oligomer as used in a method described above.
[0019] Disclosed herein, in certain embodiments, is an antisense
oligomer comprising a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to any one of SEQ ID NOs:
3-436.
[0020] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising the antisense oligomer
described above and an excipient.
[0021] Disclosed herein, in certain embodiments, is a method of
treating a subject in need thereof by administering the
pharmaceutical composition described above by intraperitoneal
injection, intramuscular injection, subcutaneous injection, or
intravenous injection.
[0022] Disclosed herein, in certain embodiments, is a composition
comprising an antisense oligomer for use in a method of increasing
expression of a target protein or a functional RNA by cells to
treat Alagille syndrome in a subject in need thereof associated
with a deficient protein or deficient functional RNA, wherein the
deficient protein or deficient functional RNA is deficient in
amount or activity in the subject, wherein the antisense oligomer
enhances constitutive splicing of a retained intron-containing
pre-mRNA (RIC pre-mRNA) encoding the target protein or the
functional RNA, wherein the target protein is: (a) the deficient
protein; or (b) a compensating protein which functionally augments
or replaces the deficient protein or in the subject; and wherein
the functional RNA is: (a) the deficient RNA; or (b) a compensating
functional RNA which functionally augments or replaces the
deficient functional RNA in the subject; wherein the RIC pre-mRNA
comprises a retained intron, an exon flanking the 5' splice site
and an exon flanking the 3' splice site, and wherein the retained
intron is spliced from the RIC pre-mRNA encoding the target protein
or the functional RNA, thereby increasing production or activity of
the target protein or the functional RNA in the subject. In some
embodiments, also disclosed herein is a composition comprising an
antisense oligomer for use in a method of treating a condition
associated with JAG1 protein in a subject in need thereof, the
method comprising the step of increasing expression of JAG1 protein
by cells of the subject, wherein the cells have a
retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a
retained intron, an exon flanking the 5' splice site of the
retained intron, an exon flanking the 3' splice site of the
retained intron, and wherein the RIC pre-mRNA encodes the JAG1
protein, the method comprising contacting the cells with the
antisense oligomer, whereby the retained intron is constitutively
spliced from the RIC pre-mRNA transcripts encoding JAG1 protein,
thereby increasing the level of mRNA encoding JAG1, and increasing
the expression of JAG1 protein, in the cells of the subject. In
some embodiments, the condition is a disease or disorder. In some
embodiments, the disease or disorder is Alagille syndrome or a
muscular dystrophy. In some embodiments, the target protein and RIC
pre-mRNA are encoded by the JAG1 gene. In some embodiments, the
antisense oligomer targets a portion of the RIC pre-mRNA that is in
the retained intron within the region +6 relative to the 5' splice
site of the retained intron to -16 relative to the 3' splice site
of the retained intron. In some embodiments, the antisense oligomer
targets a portion of the RIC pre-mRNA that is in the retained
intron within: (a) the region +6 to +100 relative to the 5' splice
site of the retained intron; or (b) the region -16 to -100 relative
to the 3' splice site of the retained intron. In some embodiments,
the antisense oligomer targets a portion of the RIC pre-mRNA that
is within the region about 100 nucleotides downstream of the 5'
splice site of the at least one retained intron, to about 100
nucleotides upstream of the 3' splice site of the at least one
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is within: (a) the region +2e to -4e in the exon
flanking the 5' splice site of the retained intron; or (b) the
region +2e to -4e in the exon flanking the 3' splice site of the
retained intron. In some embodiments, the RIC pre-mRNA is encoded
by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In some
embodiments, the RIC pre-mRNA comprises a sequence with at least
about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NO: 2. In some embodiments, the
antisense oligomer does not increase the amount of target protein
or functional RNA by modulating alternative splicing of the
pre-mRNA transcribed from a gene encoding the target protein or
functional RNA. In some embodiments, the antisense oligomer does
not increase the amount of the functional RNA or functional protein
by modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or functional RNA. In some embodiments,
the RIC pre-mRNA was produced by partial splicing from a
full-length pre-mRNA or a wild-type pre-mRNA. In some embodiments,
the mRNA encoding the target protein or functional RNA is a
full-length mature mRNA, or a wild-type mature mRNA. In some
embodiments, the target protein produced is full-length protein, or
wild-type protein. In some embodiments, the retained intron is a
rate-limiting intron. In some embodiments, the retained intron is
the most abundant retained intron in the RIC pre-mRNA. In some
embodiments, the retained intron is the second most abundant
retained intron in the RIC pre-mRNA. In some embodiments, the
antisense oligomer comprises a backbone modification comprising a
phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, the antisense oligomer is an antisense
oligonucleotide. In some embodiments, the antisense oligomer
comprises a phosphorodiamidate morpholino, a locked nucleic acid, a
peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a
2'-O-methoxyethyl moiety. In some embodiments, the antisense
oligomer comprises at least one modified sugar moiety. In some
embodiments, each sugar moiety is a modified sugar moiety. In some
embodiments, the antisense oligomer consists of from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
the targeted portion of the RIC pre-mRNA encoding the protein. In
some embodiments, the antisense oligomer binds to a targeted
portion of a JAG1 RIC pre-mRNA, wherein the targeted portion is in
a sequence selected from SEQ ID NOs 437-439. In some embodiments,
the antisense oligomer comprises a nucleotide sequence that is at
least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or 99% sequence identity to any one of SEQ ID NOs: 3-436. In some
embodiments, the antisense oligomer comprises a nucleotide sequence
selected from SEQ ID NOs: 3-436.
[0023] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising the antisense oligomer of any
of the compositions described above, and an excipient. In some
embodiments, also described herein is a method of treating a
subject in need thereof by administering the pharmaceutical
composition by intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection.
[0024] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising: an antisense oligomer that
hybridizes to a target sequence of a deficient JAG1 mRNA
transcript, wherein the deficient JAG1 mRNA transcript comprises a
retained intron, wherein the antisense oligomer induces splicing
out of the retained intron from the deficient JAG1 mRNA transcript;
and a pharmaceutical acceptable excipient. In some embodiments, the
deficient JAG1 mRNA transcript is a JAG1 RIC pre-mRNA transcript.
In some embodiments, the targeted portion of the JAG1 RIC pre-mRNA
transcript is in the retained intron within the region +6 relative
to the 5' splice site of the retained intron to -16 relative to the
3' spliced site of the retained intron. In some embodiments, the
JAG1 RIC pre-mRNA transcript is encoded by a genetic sequence with
at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO: 1. In some embodiments, the JAG1
RIC pre-mRNA transcript comprises a sequence with at least about
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to
any one of SEQ ID NO: 2. In some embodiments, the antisense
oligomer comprises a backbone modification comprising a
phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, the antisense oligomer is an antisense
oligonucleotide. In some embodiments, the antisense oligomer
comprises a phosphorodiamidate morpholino, a locked nucleic acid, a
peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a
2'-O-methoxyethyl moiety. In some embodiments, the antisense
oligomer comprises at least one modified sugar moiety. In some
embodiments, the antisense oligomer comprises from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
a targeted portion of the JAG1 RIC pre-mRNA transcript. In some
embodiments, the targeted portion of the JAG1 RIC pre-mRNA
transcript is within a sequence selected from SEQ ID NOs: 437-439.
In some embodiments, the antisense oligomer comprises a nucleotide
sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID
NOs: 3-436. In some embodiments, the antisense oligomer comprises a
nucleotide sequence selected from SEQ ID NOs: 3-436. In some
embodiments, the pharmaceutical composition is formulated for
intrathecal injection, intracerebroventricular injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0025] Disclosed herein, in certain embodiments, is a method of
inducing processing of a deficient JAG1 mRNA transcript to
facilitate removal of a retained intron to produce a fully
processed JAG1 mRNA transcript that encodes a functional form of a
tuberin protein, the method comprising: (a) contacting an antisense
oligomer to a target cell of a subject; (b) hybridizing the
antisense oligomer to the deficient JAG1 mRNA transcript, wherein
the deficient JAG1 mRNA transcript is capable of encoding the
functional form of tuberin protein and comprises at least one
retained intron; (c) removing the at least one retained intron from
the deficient JAG1 mRNA transcript to produce the fully processed
JAG1 mRNA transcript that encodes the functional form of tuberin
protein; and (d) translating the functional form of tuberin protein
from the fully processed JAG1 mRNA transcript. In some embodiments,
the retained intron is an entire retained intron. In some
embodiments, the deficient JAG1 mRNA transcript is a JAG1 RIC
pre-mRNA transcript.
[0026] Disclosed herein, in certain embodiments, is a method of
treating a subject having a condition caused by a deficient amount
or activity of JAG1 protein comprising: administering to the
subject an antisense oligomer comprising a nucleotide sequence with
at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436.
[0027] The invention provides compositions and methods for treating
tuberous sclerosis complex, including antisense oligomers (ASOs)
that promote constitutive splicing at intron splice sites of a TSC2
retained-intron-containing pre-mRNA (RIC pre-mRNA). The invention
further provides compositions and methods for increasing production
of mature TSC2 mRNA and, in turn, TSC2 protein, in cells of a
subject in need thereof, for example, a subject that can benefit
from increased production of TSC2 protein. The described methods
may be used to treat subjects having tuberous sclerosis complex
caused by mutations in the TSC2 gene, including missense, splicing,
frameshift and nonsense mutations, as well as whole gene deletions
that result in deficient tuberin protein production.
[0028] Disclosed herein, in certain embodiments, is a method of
treating tuberous sclerosis complex in a subject in need thereof,
by increasing the expression of a target protein or functional RNA
by cells of the subject, wherein the cells have a
retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC
pre-mRNA comprising a retained intron, an exon flanking the 5'
splice site, an exon flanking the 3' splice site, and wherein the
RIC pre-mRNA encodes the target protein or functional RNA, the
method comprising contacting the cells of the subject with an
antisense oligomer (ASO) complementary to a targeted portion of the
RIC pre-mRNA encoding the target protein or functional RNA, whereby
the retained intron is constitutively spliced from the RIC pre-mRNA
encoding the target protein or functional RNA, thereby increasing
the level of mRNA encoding the target protein or functional RNA,
and increasing the expression of the target protein or functional
RNA in the cells of the subject. In some embodiments, also
described herein is a method of increasing expression of a target
protein, wherein the target protein is tuberin, by cells having a
retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC
pre-mRNA comprising a retained intron, an exon flanking the 5'
splice site of the retained intron, an exon flanking the 3' splice
site of the retained intron, and wherein the RIC pre-mRNA encodes
tuberin protein, the method comprising contacting the cells with an
antisense oligomer (ASO) complementary to a targeted portion of the
RIC pre-mRNA encoding tuberin protein, whereby the retained intron
is constitutively spliced from the RIC pre-mRNA encoding tuberin
protein, thereby increasing the level of mRNA encoding tuberin
protein, and increasing the expression of tuberin protein in the
cells. In some embodiments, the target protein is tuberin. In some
embodiments, the target protein or the functional RNA is a
compensating protein or a compensating functional RNA that
functionally augments or replaces a target protein or functional
RNA that is deficient in amount or activity in the subject. In some
embodiments, the cells are in or from a subject having a condition
caused by a deficient amount or activity of tuberin protein. In
some embodiments, the deficient amount of the target protein is
caused by haploinsufficiency of the target protein, wherein the
subject has a first allele encoding a functional target protein,
and a second allele from which the target protein is not produced,
or a second allele encoding a nonfunctional target protein, and
wherein the antisense oligomer binds to a targeted portion of a RIC
pre-mRNA transcribed from the first allele. In some embodiments,
the subject has a condition caused by a disorder resulting from a
deficiency in the amount or function of the target protein, wherein
the subject has (a) a first mutant allele from which (i) the target
protein is produced at a reduced level compared to production from
a wild-type allele, (ii) the target protein is produced in a form
having reduced function compared to an equivalent wild-type
protein, or (iii) the target protein is not produced, and (b) a
second mutant allele from which (i) the target protein is produced
at a reduced level compared to production from a wild-type allele,
(ii) the target protein is produced in a form having reduced
function compared to an equivalent wild-type protein, or (iii) the
target protein is not produced, and wherein when the subject has a
first mutant allele a.iii., the second mutant allele is b.i. or
b.ii., and wherein when the subject has a second mutant allele
b.iii., the first mutant allele is a.i. or a.ii., and wherein the
RIC pre-mRNA is transcribed from either the first mutant allele
that is a.i. or a.ii., and/or the second allele that is b.i. or
b.ii. In some embodiments, the target protein is produced in a form
having reduced function compared to the equivalent wild-type
protein. In some embodiments, the target protein is produced in a
form that is fully-functional compared to the equivalent wild-type
protein. In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +6 relative to
the 5' splice site of the retained intron to -16 relative to the 3'
splice site of the retained intron. In some embodiments, the
targeted portion of the RIC pre-mRNA is in the retained intron
within the region +500 relative to the 5' splice site of the
retained intron to -500 relative to the 3' splice site of the
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is in the retained intron within: (a) the region +6 to
+500, +6 to +495, or +6 to +100 relative to the 5' splice site of
the retained intron; or (b) the region -16 to -500, -16 to -400, or
-16 to -100 relative to the 3' splice site of the retained intron.
In some embodiments, the targeted portion of the RIC pre-mRNA is
within: (a) the region +2e to -4e in the exon flanking the 5'
splice site of the retained intron; or (b) the region +2e to -4e in
the exon flanking the 3' splice site of the retained intron. In
some embodiments, the RIC pre-mRNA is encoded by a genetic sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO: 440. In some embodiments, the RIC
pre-mRNA comprises a sequence with at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ
ID NOs: 441-447. In some embodiments, the antisense oligomer does
not increase the amount of the target protein or the functional RNA
by modulating alternative splicing of pre-mRNA transcribed from a
gene encoding the functional RNA or target protein. In some
embodiments, the antisense oligomer does not increase the amount of
the target protein or the functional RNA by modulating aberrant
splicing resulting from mutation of the gene encoding the target
protein or the functional RNA. In some embodiments, the RIC
pre-mRNA was produced by partial splicing of a full-length pre-mRNA
or partial splicing of a wild-type pre-mRNA. In some embodiments,
the mRNA encoding the target protein or functional RNA is a
full-length mature mRNA, or a wild-type mature mRNA. In some
embodiments, the target protein produced is full-length protein, or
wild-type protein. In some embodiments, the total amount of the
mRNA encoding the target protein or functional RNA produced in the
cell contacted with the antisense oligomer is increased about 1.1
to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
total amount of the mRNA encoding the target protein or functional
RNA produced in a control cell. In some embodiments, the total
amount of target protein produced by the cell contacted with the
antisense oligomer is increased about 1.1 to about 10-fold, about
1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about
10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about
1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about
8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2
to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold,
about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about
7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4
to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold,
at least about 1.1-fold, at least about 1.5-fold, at least about
2-fold, at least about 2.5-fold, at least about 3-fold, at least
about 3.5-fold, at least about 4-fold, at least about 5-fold, or at
least about 10-fold, compared to the total amount of target protein
produced by a control cell. In some embodiments, the antisense
oligomer comprises a backbone modification comprising a
phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, the antisense oligomer comprises a phosphorodiamidate
morpholino, a locked nucleic acid, a peptide nucleic acid, a
2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some
embodiments, the antisense oligomer comprises at least one modified
sugar moiety. In some embodiments, each sugar moiety is a modified
sugar moiety. In some embodiments, the antisense oligomer consists
of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35
nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20
nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40
nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25
nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30
nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15
nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35
nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20
nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40
nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25
nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some
embodiments, the antisense oligomer is at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
complementary to the targeted portion of the RIC pre-mRNA encoding
the protein. In some embodiments, the targeted portion of the RIC
pre-mRNA is within a sequence selected from SEQ ID NOs: 5536-5544.
In some embodiments, the antisense oligomer comprises a nucleotide
sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID
NOs: 448-5535. In some embodiments, the antisense oligomer
comprises a nucleotide sequence selected from SEQ ID NOs: 448-5535.
In some embodiments, the cell comprises a population of RIC
pre-mRNAs transcribed from the gene encoding the target protein or
functional RNA, wherein the population of RIC pre-mRNAs comprises
two or more retained introns, and wherein the antisense oligomer
binds to the most abundant retained intron in the population of RIC
pre-mRNAs. In some embodiments, the binding of the antisense
oligomer to the most abundant retained intron induces splicing out
of the two or more retained introns from the population of RIC
pre-mRNAs to produce mRNA encoding the target protein or functional
RNA. In some embodiments, the cell comprises a population of RIC
pre-mRNAs transcribed from the gene encoding the target protein or
functional RNA, wherein the population of RIC pre-mRNAs comprises
two or more retained introns, and wherein the antisense oligomer
binds to the second most abundant retained intron in the population
of RIC pre-mRNAs. In some embodiments, the binding of the antisense
oligomer to the second most abundant retained intron induces
splicing out of the two or more retained introns from the
population of RIC pre-mRNAs to produce mRNA encoding the target
protein or functional RNA. In some embodiments, the condition is a
disease or disorder. In some embodiments, the disease or disorder
is tuberous sclerosis complex. In some embodiments, the target
protein and the RIC pre-mRNA are encoded by the TSC2 gene. In some
embodiments, the method further comprises assessing TSC2 protein
expression. In some embodiments, the antisense oligomer binds to a
targeted portion of a tuberin RIC pre-mRNA, wherein the targeted
portion is within a sequence selected from SEQ ID NOS: 488, 489,
490, 491, 492, 493, 494, 495, and 496. In some embodiments, the
subject is a human. In some embodiments, the subject is a non-human
animal. In some embodiments, the subject is a fetus, an embryo, or
a child. In some embodiments, the cells are ex vivo. In some
embodiments, the antisense oligomer is administered by topical
application to the skin, pulmonary delivery to the lung,
intrathecal injection, intracerebroventricular injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection of the subject. In some
embodiments, the 9 nucleotides at -3e to -1e of the exon flanking
the 5' splice site and +1 to +6 of the retained intron, are
identical to the corresponding wild-type sequence. In some
embodiments, the 16 nucleotides at -15 to -1 of the retained intron
and +1e of the exon flanking the 3' splice site are identical to
the corresponding wild-type sequence.
[0029] Disclosed herein, in certain embodiments, is an antisense
oligomer as used in a method described above.
[0030] Disclosed herein, in certain embodiments, is an antisense
oligomer comprising a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to any one of SEQ ID NOs:
448-5535.
[0031] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising the antisense oligomer
described above, and an excipient. In some embodiments, also
described herein is a method of treating a subject in need thereof,
by administering the pharmaceutical composition by topical
application to the skin, pulmonary delivery to the lung,
intrathecal injection, intracerebroventricular injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0032] Disclosed herein, in certain embodiments, is a composition
comprising an antisense oligomer for use in a method of increasing
expression of a target protein or a functional RNA by cells to
treat tuberous sclerosis complex in a subject in need thereof,
associated with a deficient protein or deficient functional RNA,
wherein the deficient protein or deficient functional RNA is
deficient in amount or activity in the subject, wherein the
antisense oligomer enhances constitutive splicing of a retained
intron-containing pre-mRNA (RIC pre-mRNA) encoding the target
protein or the functional RNA, wherein the target protein is: (a)
the deficient protein; or (b) a compensating protein which
functionally augments or replaces the deficient protein or in the
subject; and wherein the functional RNA is: (a) the deficient RNA;
or (b) a compensating functional RNA which functionally augments or
replaces the deficient functional RNA in the subject; wherein the
RIC pre-mRNA comprises a retained intron, an exon flanking the 5'
splice site and an exon flanking the 3' splice site, and wherein
the retained intron is spliced from the RIC pre-mRNA encoding the
target protein or the functional RNA, thereby increasing production
or activity of the target protein or the functional RNA in the
subject. In some embodiments, also disclosed herein is a
composition comprising an antisense oligomer for use in a method of
treating a condition associated with tuberin protein in a subject
in need thereof, the method comprising the step of increasing
expression of tuberin protein by cells of the subject, wherein the
cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA)
comprising a retained intron, an exon flanking the 5' splice site
of the retained intron, an exon flanking the 3' splice site of the
retained intron, and wherein the RIC pre-mRNA encodes the tuberin
protein, the method comprising contacting the cells with the
antisense oligomer, whereby the retained intron is constitutively
spliced from the RIC pre-mRNA transcripts encoding tuberin protein,
thereby increasing the level of mRNA encoding the tuberin protein,
and increasing the expression of tuberin protein, in the cells of
the subject. In some embodiments, the condition is a disease or
disorder. In some embodiments, the disease or disorder is tuberous
sclerosis complex. In some embodiments, the target protein and RIC
pre-mRNA are encoded by the TSC2 gene. In some embodiments, the
antisense oligomer targets a portion of the RIC pre-mRNA that is in
the retained intron within the region +6 relative to the 5' splice
site of the retained intron to -16 relative to the 3' splice site
of the retained intron. In some embodiments, the antisense oligomer
targets a portion of the RIC pre-mRNA that is in the retained
intron within: (a) the region +6 to +100 relative to the 5' splice
site of the retained intron; or (b) the region -16 to -100 relative
to the 3' splice site of the retained intron. In some embodiments,
the antisense oligomer targets a portion of the RIC pre-mRNA that
is within the region about 500, 400, 300, 200, or 100 nucleotides
downstream of the 5' splice site of the at least one retained
intron, to about 100, 200, 300, 400, or 500 nucleotides upstream of
the 3' splice site of the at least one retained intron. In some
embodiments, the targeted portion of the RIC pre-mRNA is within:
(a) the region +2e to -4e in the exon flanking the 5' splice site
of the retained intron; or (b) the region +2e to -4e in the exon
flanking the 3' splice site of the retained intron. In some
embodiments, the RIC pre-mRNA is encoded by a genetic sequence with
at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO: 440. In some embodiments, the RIC
pre-mRNA comprises a sequence with at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ
ID NOs: 441-447. In some embodiments, the antisense oligomer does
not increase the amount of target protein or functional RNA by
modulating alternative splicing of the pre-mRNA transcribed from a
gene encoding the target protein or functional RNA. In some
embodiments, the antisense oligomer does not increase the amount of
the functional RNA or functional protein by modulating aberrant
splicing resulting from mutation of the gene encoding the target
protein or functional RNA. In some embodiments, the RIC pre-mRNA
was produced by partial splicing from a full-length pre-mRNA or a
wild-type pre-mRNA. In some embodiments, the mRNA encoding the
target protein or functional RNA is a full-length mature mRNA, or a
wild-type mature mRNA. In some embodiments, the target protein
produced is full-length protein, or wild-type protein. In some
embodiments, the retained intron is a rate-limiting intron. In some
embodiments, said retained intron is the most abundant retained
intron in said RIC pre-mRNA. In some embodiments, the retained
intron is the second most abundant retained intron in said RIC
pre-mRNA. In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, said antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, each sugar moiety is a modified sugar moiety. In
some embodiments, the antisense oligomer consists of from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
the targeted portion of the RIC pre-mRNA encoding the protein. In
some embodiments, the antisense oligomer binds to a targeted
portion of a tuberin RIC pre-mRNA, wherein the targeted portion is
within a sequence selected from SEQ ID NOs: 5536-5544. In some
embodiments, the antisense oligomer comprises a nucleotide sequence
that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:
448-5535. In some embodiments, the antisense oligomer comprises a
nucleotide sequence selected from SEQ ID NOs: 448-5535.
[0033] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising the antisense oligomer
described above, and an excipient. In some embodiments, also
described herein is a method of treating a subject in need thereof,
by administering the pharmaceutical composition by topical
application to the skin, pulmonary delivery to the lung,
intrathecal injection, intracerebroventricular injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0034] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising: an antisense oligomer that
hybridizes to a target sequence of a deficient TSC2 mRNA
transcript, wherein the deficient TSC2 mRNA transcript comprises a
retained intron, wherein the antisense oligomer induces splicing
out of the retained intron from the deficient TSC2 mRNA transcript;
and a pharmaceutical acceptable excipient. In some embodiments, the
deficient TSC2 mRNA transcript is a TSC2 RIC pre-mRNA transcript.
In some embodiments, the targeted portion of the TSC2 RIC pre-mRNA
transcript is in the retained intron within the region +500
relative to the 5' splice site of the retained intron to -500
relative to the 3' spliced site of the retained intron. In some
embodiments, the TSC2 RIC pre-mRNA transcript is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO: 440. In some
embodiments, the TSC2 RIC pre-mRNA transcript comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs: 2-8. In some
embodiments, the antisense oligomer comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, the antisense oligomer comprises from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
a targeted portion of the TSC2 RIC pre-mRNA transcript. In some
embodiments, the targeted portion of the TSC2 RIC pre-mRNA
transcript is within a sequence selected from SEQ ID NOs:
5536-5544. In some embodiments, the antisense oligomer comprises a
nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one
of SEQ ID NOs: 448-5535. In some embodiments, the antisense
oligomer comprises a nucleotide sequence selected from SEQ ID NOs:
448-5535. In some embodiments, the pharmaceutical composition is
formulated for intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection.
[0035] Disclosed herein, in certain embodiments, is a method of
inducing processing of a deficient TSC2 mRNA transcript to
facilitate removal of a retained intron to produce a fully
processed TSC2 mRNA transcript that encodes a functional form of a
tuberin protein, the method comprising: (a) contacting an antisense
oligomer to a target cell of a subject; (b) hybridizing the
antisense oligomer to the deficient TSC2 mRNA transcript, wherein
the deficient TSC2 mRNA transcript is capable of encoding the
functional form of tuberin protein and comprises at least one
retained intron; (c) removing the at least one retained intron from
the deficient TSC2 mRNA transcript to produce the fully processed
TSC2 mRNA transcript that encodes the functional form of tuberin
protein; and (d) translating the functional form of tuberin protein
from the fully processed TSC2 mRNA transcript. In some embodiments,
the retained intron is an entire retained intron. In some
embodiments, the deficient TSC2 mRNA transcript is a TSC2 RIC
pre-mRNA transcript.
[0036] Disclosed herein, in certain embodiments, is a method of
treating a subject having a condition caused by a deficient amount
or activity of tuberin protein comprising: administering to the
subject an antisense oligomer comprising a nucleotide sequence with
at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to any one of SEQ ID NOs:
448-5535.
[0037] Disclosed herein, in some embodiments, are methods of
treating Polycystic Kidney Disease in a subject in need thereof, by
increasing the expression of a target protein or functional RNA by
cells of the subject, wherein the cells have a
retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC
pre-mRNA comprising a retained intron, an exon flanking the 5'
splice site, an exon flanking the 3' splice site, and wherein the
RIC pre-mRNA encodes the target protein or functional RNA, the
method comprising contacting the cells of the subject with an
antisense oligomer (ASO) complementary to a targeted portion of the
RIC pre-mRNA encoding the target protein or functional RNA, whereby
the retained intron is constitutively spliced from the RIC pre-mRNA
encoding the target protein or functional RNA, thereby increasing
the level of mRNA encoding the target protein or functional RNA,
and increasing the expression of the target protein or functional
RNA in the cells of the subject.
[0038] Also disclosed herein, in some embodiments, are methods of
increasing expression of a target protein, wherein the target
protein is PC-2, by cells having a retained-intron-containing
pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained
intron, an exon flanking the 5' splice site of the retained intron,
an exon flanking the 3' splice site of the retained intron, and
wherein the RIC pre-mRNA encodes PC-2 protein, the method
comprising contacting the cells with an antisense oligomer (ASO)
complementary to a targeted portion of the RIC pre-mRNA encoding
PC-2 protein, whereby the retained intron is constitutively spliced
from the RIC pre-mRNA encoding PC-2 protein, thereby increasing the
level of mRNA encoding PC-2 protein, and increasing the expression
of PC-2 protein in the cells.
[0039] In some embodiments of any of the aforementioned methods,
the target protein is PC-2. In some embodiments, the target protein
or the functional RNA is a compensating protein or a compensating
functional RNA that functionally augments or replaces a target
protein or functional RNA that is deficient in amount or activity
in the subject. In some embodiments, the cells are in or from a
subject having a condition caused by a deficient amount or activity
of PC-2 protein.
[0040] In some embodiments of any of the aforementioned methods,
the deficient amount of the target protein is caused by
haploinsufficiency of the target protein, wherein the subject has a
first allele encoding a functional target protein, and a second
allele from which the target protein is not produced, or a second
allele encoding a nonfunctional target protein, and wherein the
antisense oligomer binds to a targeted portion of a RIC pre-mRNA
transcribed from the first allele. In some embodiments, the subject
has a condition caused by a disorder resulting from a deficiency in
the amount or function of the target protein, wherein the subject
has (a) a first mutant allele from which (i) the target protein is
produced at a reduced level compared to production from a wild-type
allele, (ii) the target protein is produced in a form having
reduced function compared to an equivalent wild-type protein, or
(iii) the target protein is not produced, and (b) a second mutant
allele from which (i) the target protein is produced at a reduced
level compared to production from a wild-type allele, (ii) the
target protein is produced in a form having reduced function
compared to an equivalent wild-type protein, or (iii) the target
protein is not produced, and wherein when the subject has a first
mutant allele (a)(iii), the second mutant allele is (b)(i) or
(b)(ii), and wherein when the subject has a second mutant allele
(b)(iii), the first mutant allele is (a)(i) or (a)(ii), and wherein
the RIC pre-mRNA is transcribed from either the first mutant allele
that is (a)(i) or (a)(ii), and/or the second allele that is (b)(i)
or (b)(ii). In some embodiments, the target protein is produced in
a form having reduced function compared to the equivalent wild-type
protein. In some embodiments, the target protein is produced in a
form that is fully-functional compared to the equivalent wild-type
protein.
[0041] In some embodiments of any of the aforementioned methods,
the targeted portion of the RIC pre-mRNA is in the retained intron
within the region +6 relative to the 5' splice site of the retained
intron to -16 relative to the 3' splice site of the retained
intron. In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within: (a) the region +6 to
+497 relative to the 5' splice site of the retained intron; or (b)
the region -16 to -496 relative to the 3' splice site of the
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is within: (a) the region +2e to -4e in the exon
flanking the 5' splice site of the retained intron; or (b) the
region +2e to -4e in the exon flanking the 3' splice site of the
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is in the retained intron within: (a) the region -4e
to -1,054e relative to the 5' splice site of the retained intron;
(b) the region +6 to +499 relative to the 5' splice site of the
retained intron; (c) the region -16 to -496 relative to the 3'
splice site of the retained intron; or (d) the region +2e to
+1,912e relative to the 3' splice site of the retained intron. In
some embodiments, the target protein is PC-2.
[0042] In some embodiments of any of the aforementioned methods,
the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 5546. In
some embodiments, the RIC pre-mRNA is encoded by a genetic sequence
with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence
identity to SEQ ID NO: 5545. In some embodiments, the targeted
portion of the RIC pre-mRNA comprises a sequence with at least 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to a region
comprising at least 8 contiguous nucleic acids of SEQ ID NO: 5825.
In some embodiments, the ASO comprises a sequence that is at least
about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of
SEQ ID NOs: 5547-5824. In some embodiments, the targeted portion of
the RIC pre-mRNA is within the region -204e to +497 relative to the
5' splice site of the retained intron 5 or within the region -496
to +212e relative to the 3' splice site of the retained intron 5.
In some embodiments, the ASO comprises a sequence that is at least
about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of
SEQ ID NOs: 5547-5824. In some embodiments, the targeted portion of
the RIC pre-mRNA is in exon 5 within the region -204e to -4e
relative to the 5' splice site of the retained intron 5. In some
embodiments, the ASO comprises a sequence that is at least about
80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID
NOs: 5547-5587. In some embodiments, the targeted portion of the
RIC pre-mRNA is in retained intron 5 within the region +6 to +497
relative to the 5' splice site of the retained intron. In some
embodiments, the ASO comprises a sequence that is at least about
80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID
NOs:5588-5684. In some embodiments, the targeted portion of the RIC
pre-mRNA is in retained intron 5 within the region -16 to -496
relative to the 3' splice site of the retained intron. In some
embodiments, the ASO comprises a sequence that is at least about
80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID
NOs: 5685-5781. In some embodiments, the targeted portion of the
RIC pre-mRNA is in exon 6 within the region +2e to +212e relative
to the 3' splice site of the retained intron 5. In some
embodiments, the ASO comprises a sequence that is at least about
80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID
NOs: 238-280.
[0043] In some embodiments of any of the aforementioned methods,
the antisense oligomer does not increase the amount of the target
protein or the functional RNA by modulating alternative splicing of
pre-mRNA transcribed from a gene encoding the functional RNA or
target protein. In some embodiments, the antisense oligomer does
not increase the amount of the target protein or the functional RNA
by modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or the functional RNA. In some
embodiments, the RIC pre-mRNA was produced by partial splicing of a
full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.
In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA. In some embodiments, the target protein produced is
full-length protein, or wild-type protein. In some embodiments, the
total amount of the mRNA encoding the target protein or functional
RNA produced in the cell contacted with the antisense oligomer is
increased about 1.1 to about 10-fold, about 1.5 to about 10-fold,
about 2 to about 10-fold, about 3 to about 10-fold, about 4 to
about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the total amount of the mRNA encoding
the target protein or functional RNA produced in a control cell. In
some embodiments, the total amount of target protein produced by
the cell contacted with the antisense oligomer is increased about
1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
total amount of target protein produced by a control cell.
[0044] In some embodiments of any of the aforementioned methods,
the antisense oligomer comprises a backbone modification comprising
a phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, the antisense oligomer comprises a phosphorodiamidate
morpholino, a locked nucleic acid, a peptide nucleic acid, a
2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some
embodiments, the antisense oligomer comprises at least one modified
sugar moiety. In some embodiments, each sugar moiety is a modified
sugar moiety. In some embodiments, the antisense oligomer consists
of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35
nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20
nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40
nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25
nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30
nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15
nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35
nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20
nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40
nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25
nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some
embodiments, the antisense oligomer is at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
complementary to the targeted portion of the RIC pre-mRNA encoding
the protein.
[0045] In some embodiments of any of the aforementioned methods,
the cell comprises a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein or functional RNA, wherein the
population of RIC pre-mRNAs comprises at least one retained intron,
and wherein the antisense oligomer binds to the most abundant
retained intron in the population of RIC pre-mRNAs. In some
embodiments, the binding of the antisense oligomer to the most
abundant retained intron induces splicing out of the at least one
retained intron from the population of RIC pre-mRNAs to produce
mRNA encoding the target protein or functional RNA. In some
embodiments, the cell comprises a population of RIC pre-mRNAs
transcribed from the gene encoding the target protein or functional
RNA, wherein the population of RIC pre-mRNAs comprises two or more
retained introns, and wherein the antisense oligomer binds to the
second most abundant retained intron in the population of RIC
pre-mRNAs. In some embodiments, the binding of the antisense
oligomer to the second most abundant retained intron induces
splicing out of the two or more retained introns from the
population of RIC pre-mRNAs to produce mRNA encoding the target
protein or functional RNA. In some embodiments, the method further
comprises assessing PC-2 protein expression.
[0046] In some embodiments of any of the aforementioned methods,
the antisense oligomer binds to a targeted portion of a PKD2 RIC
pre-mRNA, wherein the targeted portion is in a sequence selected
from SEQ ID NOs: 5547-5824. In some embodiments, the subject is a
human. In some embodiments, the subject is a non-human animal. In
some embodiments, the subject is a fetus, an embryo, or a child. In
some embodiments, the cells are ex vivo. In some embodiments, the
antisense oligomer is administered by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection of the subject. In some embodiments, the 9 nucleotides at
-3e to -1e of the exon flanking the 5' splice site and +1 to +6 of
the retained intron, are identical to the corresponding wild-type
sequence. In some embodiments, the 16 nucleotides at -15 to -1 of
the retained intron and +1e of the exon flanking the 3' splice site
are identical to the corresponding wild-type sequence. Disclosed
herein, in some embodiments, are antisense oligomers as described
in any of the aforementioned methods.
[0047] Disclosed herein, in some embodiments, are antisense
oligomers comprising a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to any one of SEQ ID NOs:
5547-5824.
[0048] Also disclosed herein, in some embodiments, are
pharmaceutical compositions comprising any of the aforementioned
antisense oligomers and an excipient.
[0049] Disclosed herein, in some embodiments, are methods of
treating a subject in need thereof, by administering any of the
aforementioned pharmaceutical compositions by intraperitoneal
injection, intramuscular injection, subcutaneous injection, or
intravenous injection.
[0050] Disclosed herein, in some embodiments, are compositions
comprising an antisense oligomer for use in a method of increasing
expression of a target protein or a functional RNA by cells to
treat Polycystic Kidney Disease in a subject in need thereof,
associated with a deficient protein or deficient functional RNA,
wherein the deficient protein or deficient functional RNA is
deficient in amount or activity in the subject, wherein the
antisense oligomer enhances constitutive splicing of a retained
intron-containing pre-mRNA (RIC pre-mRNA) encoding the target
protein or the functional RNA, wherein the target protein is: (a)
the deficient protein; or (b) a compensating protein which
functionally augments or replaces the deficient protein or in the
subject; and wherein the functional RNA is: (c) the deficient RNA;
or (d) a compensating functional RNA which functionally augments or
replaces the deficient functional RNA in the subject; wherein the
RIC pre-mRNA comprises a retained intron, an exon flanking the 5'
splice site and an exon flanking the 3' splice site, and wherein
the retained intron is spliced from the RIC pre-mRNA encoding the
target protein or the functional RNA, thereby increasing production
or activity of the target protein or the functional RNA in the
subject.
[0051] Disclosed herein, in some embodiments, are compositions
comprising an antisense oligomer for use in a method of treating a
condition associated with PC-2 protein in a subject in need
thereof, the method comprising the step of increasing expression of
PC-2 protein by cells of the subject, wherein the cells have a
retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a
retained intron, an exon flanking the 5' splice site of the
retained intron, an exon flanking the 3' splice site of the
retained intron, and wherein the RIC pre-mRNA encodes the PC-2
protein, the method comprising contacting the cells with the
antisense oligomer, whereby the retained intron is constitutively
spliced from the RIC pre-mRNA transcripts encoding PC-2 protein,
thereby increasing the level of mRNA encoding the PC-2 protein, and
increasing the expression of PC-2 protein, in the cells of the
subject. In some embodiments, the condition is a disease or
disorder. In some embodiments, the disease or disorder is
Polycystic Kidney Disease. In some embodiments, the target protein
and RIC pre-mRNA are encoded by the PKD2 gene.
[0052] In some embodiments of any of the aforementioned
compositions, the antisense oligomer targets a portion of the RIC
pre-mRNA that is in the retained intron within the region +6
relative to the 5' splice site of the retained intron to -16
relative to the 3' splice site of the retained intron. In some
embodiments, the antisense oligomer targets a portion of the RIC
pre-mRNA that is in the retained intron within: (a) the region +6
to +497 relative to the 5' splice site of the retained intron; or
(b) the region -16 to -496 relative to the 3' splice site of the
retained intron. In some embodiments, the antisense oligomer
targets a portion of the RIC pre-mRNA that is within the region
about 100 nucleotides downstream of the 5' splice site of the at
least one retained intron, to about 100 nucleotides upstream of the
3' splice site of the at least one retained intron. In some
embodiments, the targeted portion of the RIC pre-mRNA is within:
(a) the region +2e to -4e in the exon flanking the 5' splice site
of the retained intron; or (b) the region +2e to -4e in the exon
flanking the 3' splice site of the retained intron. In some
embodiments, the targeted portion of the RIC pre-mRNA is within:
(a) the region -4e to -1,054e relative to the 5' splice site of the
retained intron; (b) the region +6 to +499 relative to the 5'
splice site of the retained intron; (c) the region -16 to -496
relative to the 3' splice site of the retained intron; or (d) the
region +2e to +1,912e relative to the 3' splice site of the
retained intron. In some embodiments, the target protein is PC-2.
In some embodiments, the RIC pre-mRNA comprises a sequence with at
least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to
SEQ ID NO: 5546. In some embodiments, the RIC pre-mRNA is encoded
by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97%,
or 100% sequence identity to SEQ ID NO: 5545. In some embodiments,
the targeted portion of the RIC pre-mRNA comprises a sequence with
at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a
region comprising at least 8 contiguous nucleic acids of SEQ ID NO:
5825. In some embodiments, the ASO comprises a sequence that is at
least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any
one of SEQ ID NOs: 5547-5824. In some embodiments, the targeted
portion of the RIC pre-mRNA is within the region -204e to +497
relative to the 5' splice site of the retained intron 5 or within
the region -496 to +212e relative to the 3' splice site of the
retained intron 5. In some embodiments, the ASO comprises a
sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100%
complimentary to any one of SEQ ID NOs: 5547-5824. In some
embodiments, the targeted portion of the RIC pre-mRNA is in exon 5
within the region -204e to -4e relative to the 5' splice site of
the retained intron 5. In some embodiments, the ASO comprises a
sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100%
complimentary to any one of SEQ ID NOs: 5547-5587. In some
embodiments, the targeted portion of the RIC pre-mRNA is in
retained intron 5 within the region +6 to +497 relative to the 5'
splice site of the retained intron. In some embodiments, the ASO
comprises a sequence that is at least about 80%, 85%, 90%, 95%,
97%, or 100% complimentary to any one of SEQ ID NOs:5588-5684. In
some embodiments, the targeted portion of the RIC pre-mRNA is in
retained intron 5 within the region -16 to -496 relative to the 3'
splice site of the retained intron. In some embodiments, the ASO
comprises a sequence that is at least about 80%, 85%, 90%, 95%,
97%, or 100% complimentary to any one of SEQ ID NOs: 5685-5781. In
some embodiments, the targeted portion of the RIC pre-mRNA is in
exon 6 within the region +2e to +212e relative to the 3' splice
site of the retained intron 5. In some embodiments, the ASO
comprises a sequence that is at least about 80%, 85%, 90%, 95%,
97%, or 100% complimentary to any one of SEQ ID NOs: 5782-5824.
[0053] In some embodiments of any of the aforementioned
compositions, the antisense oligomer does not increase the amount
of target protein or functional RNA by modulating alternative
splicing of the pre-mRNA transcribed from a gene encoding the
target protein or functional RNA. In some embodiments, the
antisense oligomer does not increase the amount of the functional
RNA or functional protein by modulating aberrant splicing resulting
from mutation of the gene encoding the target protein or functional
RNA. In some embodiments, the RIC pre-mRNA was produced by partial
splicing from a full-length pre-mRNA or a wild-type pre-mRNA. In
some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA. In some embodiments, the target protein produced is
full-length protein, or wild-type protein. In some embodiments, the
retained intron is a rate-limiting intron. In some embodiments, the
retained intron is the most abundant retained intron in said RIC
pre-mRNA. In some embodiments, the retained intron is the second
most abundant retained intron in said RIC pre-mRNA.
[0054] In some embodiments of any of the aforementioned
compositions, the antisense oligomer comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, each sugar moiety is a modified sugar moiety. In
some embodiments, the antisense oligomer consists of from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases.
[0055] Disclosed herein, in some embodiments, are pharmaceutical
compositions comprising any of the aforementioned antisense
oligomers and an excipient. Disclosed herein, in some embodiments,
are methods of treating a subject in need thereof, by administering
the aforementioned pharmaceutical compositions by intraperitoneal
injection, intramuscular injection, subcutaneous injection, or
intravenous injection.
[0056] Disclosed herein, in some embodiments, are pharmaceutical
compositions comprising: an antisense oligomer that hybridizes to a
target sequence of a deficient PKD2 mRNA transcript, wherein the
deficient PKD2 mRNA transcript comprises a retained intron, wherein
the antisense oligomer induces splicing out of the retained intron
from the deficient PKD2 mRNA transcript; and a pharmaceutical
acceptable excipient. In some embodiments, the deficient PKD2 mRNA
transcript is a PKD2 RIC pre-mRNA transcript. In some embodiments,
the targeted portion of the PKD2 RIC pre-mRNA transcript is in the
retained intron within the region +500 relative to the 5' splice
site of the retained intron to -500 relative to the 3' spliced site
of the retained intron. In some embodiments, the PKD2 RIC pre-mRNA
transcript is encoded by a genetic sequence with at least about
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to
SEQ ID NO: 5545. In some embodiments, the PKD2 RIC pre-mRNA
transcript comprises a sequence with at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:
5546. In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, the antisense oligomer comprises from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
a targeted portion of the PKD2 RIC pre-mRNA transcript. In some
embodiments, the targeted portion of the PKD2 RIC pre-mRNA
transcript is within SEQ ID NO: 5825. In some embodiments, the
antisense oligomer comprises a nucleotide sequence that is at least
about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to any one of SEQ ID NOs: 5547-5824. In some
embodiments, the antisense oligomer comprises a nucleotide sequence
selected from SEQ ID NOs: 5547-5824. In some embodiments, the
pharmaceutical composition is formulated for intrathecal injection,
intracerebroventricular injection, intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection.
[0057] Disclosed herein, in some embodiments, are methods of
inducing processing of a deficient PKD2 mRNA transcript to
facilitate removal of a retained intron to produce a fully
processed PKD2 mRNA transcript that encodes a functional form of a
PC-2 protein, the method comprising: (a) contacting an antisense
oligomer to a target cell of a subject; (b) hybridizing the
antisense oligomer to the deficient PKD2 mRNA transcript, wherein
the deficient PKD2 mRNA transcript is capable of encoding the
functional form of a PC-2 protein and comprises at least one
retained intron; (c) removing the at least one retained intron from
the deficient PKD2 mRNA transcript to produce the fully processed
PKD2 mRNA transcript that encodes the functional form of PC-2
protein; and (d) translating the functional form of PC-2 protein
from the fully processed PKD2 mRNA transcript. In some embodiments,
the retained intron is an entire retained intron. In some
embodiments, the deficient PKD2 mRNA transcript is a PKD2 pre-mRNA
transcript.
[0058] Disclosed herein, in some embodiments, are methods of
treating a subject having a condition caused by a deficient amount
or activity of PC-2 protein comprising administering to the subject
an antisense oligomer comprising a nucleotide sequence with at
least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
or 99% sequence identity to any one of SEQ ID NOs: 5547-5824.
[0059] Disclosed herein, in some embodiments, are methods of
treating Retinitis Pigmentosa 18 (RP18) or Retinitis Pigmentosa 13
(RP13) in a subject in need thereof, by increasing the expression
of a target protein or functional RNA by cells of the subject,
wherein the cells have a retained-intron-containing pre-mRNA (RIC
pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon
flanking the 5' splice site, an exon flanking the 3' splice site,
and wherein the RIC pre-mRNA encodes the target protein or
functional RNA, the method comprising contacting the cells of the
subject with an antisense oligomer (ASO) complementary to a
targeted portion of the RIC pre-mRNA encoding the target protein or
functional RNA, whereby the retained intron is constitutively
spliced from the RIC pre-mRNA encoding the target protein or
functional RNA, thereby increasing the level of mRNA encoding the
target protein or functional RNA, and increasing the expression of
the target protein or functional RNA in the cells of the
subject.
[0060] Also disclosed herein are methods of increasing expression
of a target protein, wherein the target protein is PRPF3 or PRPF8,
by cells having a retained-intron-containing pre-mRNA (RIC
pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon
flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and wherein the
RIC pre-mRNA encodes PRPF3 or PRPF8 protein, the method comprising
contacting the cells with an antisense oligomer (ASO) complementary
to a targeted portion of the RIC pre-mRNA encoding PRPF3 or PRPF8
protein, whereby the retained intron is constitutively spliced from
the RIC pre-mRNA encoding PRPF3 or PRPF8 protein, thereby
increasing the level of mRNA encoding PRPF3 or PRPF8 protein, and
increasing the expression of PRPF3 or PRPF8 protein in the
cells.
[0061] In some embodiments of any of the aforementioned methods,
when the method is a method of treating RP18 the target protein is
PRPF3. In some embodiments of any of the aforementioned methods,
when the method is a method of treating RP13 the target protein is
PRPF8. In some embodiments, the target protein or the functional
RNA is a compensating protein or a compensating functional RNA that
functionally augments or replaces a target protein or functional
RNA that is deficient in amount or activity in the subject. In some
embodiments, the cells are in or from a subject having a condition
caused by a deficient amount or activity of PRPF3 or PRPF8
protein.
[0062] In some embodiments of any of the aforementioned methods,
the deficient amount of the target protein is caused by
haploinsufficiency of the target protein, wherein the subject has a
first allele encoding a functional target protein, and a second
allele from which the target protein is not produced, or a second
allele encoding a nonfunctional target protein, and wherein the
antisense oligomer binds to a targeted portion of a RIC pre-mRNA
transcribed from the first allele. In some embodiments, the subject
has a condition caused by a disorder resulting from a deficiency in
the amount or function of the target protein, wherein the subject
has (a) a first mutant allele from which (i) the target protein is
produced at a reduced level compared to production from a wild-type
allele, (ii) the target protein is produced in a form having
reduced function compared to an equivalent wild-type protein, or
(iii) the target protein is not produced, and (b) a second mutant
allele from which (i) the target protein is produced at a reduced
level compared to production from a wild-type allele, (ii) the
target protein is produced in a form having reduced function
compared to an equivalent wild-type protein, or (iii) the target
protein is not produced, and wherein when the subject has a first
mutant allele a(iii), the second mutant allele is b(i) or b(ii),
and wherein when the subject has a second mutant allele b(iii), the
first mutant allele is a(i) or a(ii), and wherein the RIC pre-mRNA
is transcribed from either the first mutant allele that is a(i) or
a(ii), and/or the second allele that is b(i) or b(ii). In some
embodiments, the target protein is produced in a form having
reduced function compared to the equivalent wild-type protein. In
some embodiments, the target protein is produced in a form that is
fully-functional compared to the equivalent wild-type protein.
[0063] In some embodiments of any of the aforementioned methods,
the targeted portion of the RIC pre-mRNA is in the retained intron
within the region +6 relative to the 5' splice site of the retained
intron to -16 relative to the 3' splice site of the retained
intron. In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +498 relative
to the 5' splice site of the retained intron to -496 relative to
the 3' splice site of the retained intron. In some embodiments, the
targeted portion of the RIC pre-mRNA is in the retained intron
within the region +498 relative to the 5' splice site of the
retained intron to -496 relative to the 3' splice site of the
retained intron.
[0064] In some embodiments of any of the aforementioned methods,
the target protein is PRPF3. In some embodiments, the targeted
portion of the RIC pre-mRNA comprises a sequence that is at least
about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of
SEQ ID NOs 5830-6148. In some embodiments, the in the targeted
portion of the RIC pre-mRNA comprises a sequence with at least 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to a region
comprising at least 8 contiguous nucleic acids of SEQ ID NO 6272 or
SEQ ID NO 6271. In some embodiments, the ASO comprises a sequence
with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence
identity to any one of SEQ ID NOs 5830-6148. In some embodiments,
the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
90%, 95%, 97%, or 100% sequence identity to SEQ ID NO 5828. In some
embodiments, the RIC pre-mRNA is encoded by a genetic sequence with
at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity
to SEQ ID NO 5826.
[0065] In some embodiments, the target protein is PRPF8. In some
embodiments, the targeted portion of the RIC pre-mRNA is in the
retained intron within the region +156 relative to the 5' splice
site of the retained intron to -156 relative to the 3' splice site
of the retained intron. In some embodiments, the targeted portion
of the RIC pre-mRNA comprises a sequence that is at least about
80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID
NOs 6149-6270. In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
97%, or 100% sequence identity to a region comprising at least 8
contiguous nucleic acids of SEQ ID NO 6273. In some embodiments,
the ASO comprises a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to any one of SEQ ID NOs
6149-6270. In some embodiments, the RIC pre-mRNA comprises a
sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100%
sequence identity to SEQ ID NO 5829. In some embodiments, the RIC
pre-mRNA is encoded by a genetic sequence with at least about 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO
5827.
[0066] In some embodiments of any of the aforementioned methods,
the targeted portion of the RIC pre-mRNA is in the retained intron
within: (a) the region +6 to +100 relative to the 5' splice site of
the retained intron; or (b) the region -16 Table 6to -100 relative
to the 3' splice site of the retained intron. In some embodiments,
the antisense oligomer targets a portion of the RIC pre-mRNA that
is within the region about 100 nucleotides downstream of the 5'
splice site of the at least one retained intron, to about 100
nucleotides upstream of the 3' splice site of the at least one
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is the retained intron within the: (a) the region +2e
to -4e in the exon flanking the 5' splice site of the retained
intron; or (b) the region +2e to -4e in the exon flanking the 3'
splice site of the retained intron.
[0067] In some embodiments of any of the aforementioned methods,
the antisense oligomer does not increase the amount of the target
protein or the functional RNA by modulating alternative splicing of
pre-mRNA transcribed from a gene encoding the functional RNA or
target protein. In some embodiments, the antisense oligomer does
not increase the amount of the target protein or the functional RNA
by modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or the functional RNA. In some
embodiments, the RIC pre-mRNA was produced by partial splicing of a
full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.
In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA. In some embodiments, the target protein produced is
full-length protein, or wild-type protein. In some embodiments, the
total amount of the mRNA encoding the target protein or functional
RNA produced in the cell contacted with the antisense oligomer is
increased about 1.1 to about 10-fold, about 1.5 to about 10-fold,
about 2 to about 10-fold, about 3 to about 10-fold, about 4 to
about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the total amount of the mRNA encoding
the target protein or functional RNA produced in a control cell. In
some embodiments, the total amount of target protein produced by
the cell contacted with the antisense oligomer is increased about
1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
total amount of target protein produced by a control cell.
[0068] In some embodiments of any of the aforementioned methods,
the antisense oligomer comprises a backbone modification comprising
a phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, the antisense oligomer comprises a phosphorodiamidate
morpholino, a locked nucleic acid, a peptide nucleic acid, a
2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some
embodiments, the antisense oligomer comprises at least one modified
sugar moiety. In some embodiments, each sugar moiety is a modified
sugar moiety. In some embodiments, the antisense oligomer consists
of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35
nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20
nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40
nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25
nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30
nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15
nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35
nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20
nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40
nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25
nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some
embodiments, the antisense oligomer is at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
complementary to the targeted portion of the RIC pre-mRNA encoding
the protein.
[0069] In some embodiments of any of the aforementioned methods,
the cell comprises a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein or functional RNA, wherein the
population of RIC pre-mRNAs comprises two or more retained introns,
and wherein the antisense oligomer binds to the most abundant
retained intron in the population of RIC pre-mRNAs. In some
embodiments, the binding of the antisense oligomer to the most
abundant retained intron induces splicing out of the two or more
retained introns from the population of RIC pre-mRNAs to produce
mRNA encoding the target protein or functional RNA. In some
embodiments, the cell comprises a population of RIC pre-mRNAs
transcribed from the gene encoding the target protein or functional
RNA, wherein the population of RIC pre-mRNAs comprises two or more
retained introns, and wherein the antisense oligomer binds to the
second most abundant retained intron in the population of RIC
pre-mRNAs. In some embodiments, the binding of the antisense
oligomer to the second most abundant retained intron induces
splicing out of the two or more retained introns from the
population of RIC pre-mRNAs to produce mRNA encoding the target
protein or functional RNA. In some embodiments, the condition is a
disease or disorder. In some embodiments, the disease or disorder
is RP18 or RP13. In some embodiments, the target protein and the
RIC pre-mRNA are encoded by the PRPF3 gene or PRPF8 gene. In some
embodiments, the method further comprises assessing protein
expression. In some embodiments, the subject is a human. In some
embodiments, the subject is a non-human animal. In some
embodiments, the subject is a fetus, an embryo, or a child. In some
embodiments, the cells are ex vivo. In some embodiments, the
antisense oligomer is administered by intravitreal injection,
subretinal injection, topical application, implantation,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection of the subject. In some
embodiments, the 9 nucleotides at -3e to -1e of the exon flanking
the 5' splice site and +1 to +6 of the retained intron, are
identical to the corresponding wild-type sequence. In some
embodiments, the 16 nucleotides at -15 to -1 of the retained intron
and +1e of the exon flanking the 3' splice site are identical to
the corresponding wild-type sequence.
[0070] Disclosed herein, in some embodiments, are antisense
oligomers as used in the methods described herein. In some
embodiments, the antisense oligomer comprises a sequence with at
least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to
any one of SEQ ID NOs 5830-6270.
[0071] Also disclosed herein, in some embodiments, are
pharmaceutical compositions comprising any of the aforementioned
antisense oligomers and an excipient.
[0072] Disclosed herein, in some embodiments, are methods of
treating a subject in need thereof, by administering any of the
aforementioned pharmaceutical compositions by intravitreal
injection, subretinal injection, topical application, implantation,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0073] Disclosed herein, in some embodiments, are compositions
comprising an antisense oligomer for use in a method of increasing
expression of a target protein or a functional RNA by cells to
treat RP18 or RP13 in a subject in need thereof, associated with a
deficient protein or deficient functional RNA, wherein the
deficient protein or deficient functional RNA is deficient in
amount or activity in the subject, wherein the antisense oligomer
enhances constitutive splicing of a retained intron-containing
pre-mRNA (RIC pre-mRNA) encoding the target protein or the
functional RNA, wherein the target protein is: the deficient
protein; or a compensating protein which functionally augments or
replaces the deficient protein or in the subject; and wherein the
function RNA is: the deficient RNA; or a compensating functional
RNA which functionally augments or replaces the deficient
functional RNA in the subject; wherein the RIC pre-mRNA comprises a
retained intron, an exon flanking the 5' splice site and an exon
flanking the 3' splice site, and wherein the retained intron is
spliced from the RIC pre-mRNA encoding the target protein or the
functional RNA, thereby increasing production or activity of the
target protein or the functional RNA in the subject.
[0074] Disclosed herein, in some embodiments, are compositions
comprising an antisense oligomer for use in a method of treating a
condition associated with PRPF3 or PRPF8 protein in a subject in
need thereof, the method comprising the step of increasing
expression of PRPF3 or PRPF8 protein by cells of the subject,
wherein the cells have a retained-intron-containing pre-mRNA (RIC
pre-mRNA) comprising a retained intron, an exon flanking the 5'
splice site of the retained intron, an exon flanking the 3' splice
site of the retained intron, and wherein the RIC pre-mRNA encodes
the PRPF3 or PRPF8 protein, the method comprising contacting the
cells with the antisense oligomer, whereby the retained intron is
constitutively spliced from the RIC pre-mRNA transcripts encoding
PRPF3 or PRPF8 protein, thereby increasing the level of mRNA
encoding PRPF3 or PRPF8 protein, and increasing the expression of
PRPF3 or PRPF8 protein, in the cells of the subject. In some
embodiments, the target protein PRPF3 is encoded by the sequence at
NM_004698 or PRPF8 or encoded by the sequence at NM_006445. In some
embodiments, the condition is a disease or disorder. In some
embodiments, the disease or disorder is RP18 or RP13. In some
embodiments, the target protein and RIC pre-mRNA are encoded by the
PRPF3 or PRPF8 gene. In some embodiments, the antisense oligomer
targets a portion of the RIC pre-mRNA that is in the retained
intron within the region +6 relative to the 5' splice site of the
retained intron to -16 relative to the 3' splice site of the
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is in the retained intron within the region +498
relative to the 5' splice site of the retained intron to -496
relative to the 3' splice site of the retained intron. In some
embodiments, the target protein in PRPF3. In some embodiments, the
targeted portion of the RIC pre-mRNA is in the retained intron
within the region +498 relative to the 5' splice site of the
retained intron to -496 relative to the 3' splice site of the
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA comprises a sequence that is at least about 80%, 85%,
90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs
5830-6148. In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
97%, or 100% sequence identity to a region comprising at least 8
contiguous nucleic acids of SEQ ID NO 6272 or SEQ ID NO 6271. In
some embodiments, the ASO comprises a sequence with at least about
80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of
SEQ ID NOs 5830-6148. In some embodiments, the RIC pre-mRNA
comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%,
or 100% sequence identity to SEQ ID NO 5828. In some embodiments,
the RIC pre-mRNA is encoded by a genetic sequence with at least
about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID
NO 5826. In some embodiments, the target protein is PRPF8. In some
embodiments, the targeted portion of the RIC pre-mRNA is in the
retained intron within the region +156 relative to the 5' splice
site of the retained intron to -156 relative to the 3' splice site
of the retained intron. In some embodiments, the targeted portion
of the RIC pre-mRNA comprises a sequence that is at least about
80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID
NOs 6149-6270. In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
97%, or 100% sequence identity to a region comprising at least 8
contiguous nucleic acids of SEQ ID NO 6273. In some embodiments,
the ASO comprises a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to any one of SEQ ID NOs
6149-6270. In some embodiments, the RIC pre-mRNA comprises a
sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100%
sequence identity to SEQ ID NO 5829. In some embodiments, the RIC
pre-mRNA is encoded by a genetic sequence with at least about 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO 5827. In
some embodiments, the antisense oligomer targets a portion of the
RIC pre-mRNA that is in the retained intron within: (a) the region
+6 to +100 relative to the 5' splice site of the retained intron;
or (b) the region -16 to -100 relative to the 3' splice site of the
retained intron. In some embodiments, the antisense oligomer
targets a portion of the RIC pre-mRNA that is within the region
about 100 nucleotides downstream of the 5' splice site of the at
least one retained intron, to about 100 nucleotides upstream of the
3' splice site of the at least one retained intron. In some
embodiments, the targeted portion of the RIC pre-mRNA is within:
(a) the region +2e to -4e in the exon flanking the 5' splice site
of the retained intron; or (b) the region +2e to -4e in the exon
flanking the 3' splice site of the retained intron.
[0075] In some embodiments of any of the aforementioned
compositions, the antisense oligomer does not increase the amount
of target protein or functional RNA by modulating alternative
splicing of the pre-mRNA transcribed from a gene encoding the
target protein or functional RNA. In some embodiments, the
antisense oligomer does not increase the amount of the functional
RNA or functional protein by modulating aberrant splicing resulting
from mutation of the gene encoding the target protein or functional
RNA. In some embodiments, the RIC pre-mRNA was produced by partial
splicing from a full-length pre-mRNA or a wild-type pre-mRNA. In
some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA. In some embodiments, the target protein produced is
full-length protein, or wild-type protein. In some embodiments, the
retained intron is a rate-limiting intron. In some embodiments, the
retained intron is the most abundant retained intron in the RIC
pre-mRNA. In some embodiments, the retained intron is the second
most abundant retained intron in the RIC pre-mRNA.
[0076] In some embodiments of any of the aforementioned
compositions, the antisense oligomer comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, each sugar moiety is a modified sugar moiety. In
some embodiments, the antisense oligomer consists of from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, n the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
the targeted portion of the RIC pre-mRNA encoding the protein.
[0077] Disclosed herein, in some embodiments, are pharmaceutical
compositions comprising the antisense oligomer of any of the
aforementioned compositions and an excipient.
[0078] Disclosed herein, in some embodiments, are methods of
treating a subject in need thereof, by administering any of the
aforementioned pharmaceutical compositions by intravitreal
injection, subretinal injection, topical application, implantation,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection of the subject.
[0079] Disclosed herein, in some embodiments, are pharmaceutical
compositions comprising: an antisense oligomer that hybridizes to a
target sequence of a deficient PRPF3 mRNA transcript, wherein the
deficient PRPF3 mRNA transcript comprises a retained intron,
wherein the antisense oligomer induces splicing out of the retained
intron from the deficient PRPF3 mRNA transcript; and a
pharmaceutical acceptable excipient. In some embodiments, the
deficient PRPF3 mRNA transcript is a PRPF3 RIC pre-mRNA transcript.
In some embodiments, the targeted portion of the PRPF3 RIC pre-mRNA
transcript is in the retained intron within the region +500
relative to the 5' splice site of the retained intron to -500
relative to the 3' spliced site of the retained intron. In some
embodiments, the PRPF3 RIC pre-mRNA transcript is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO: 5826. In some
embodiments, the PRPF3 RIC pre-mRNA transcript comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs: 5828-5829. In some
embodiments, the antisense oligomer comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, the antisense oligomer comprises from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
a targeted portion of the PRPF3 RIC pre-mRNA transcript. In some
embodiments, the targeted portion of the PRPF3 RIC pre-mRNA
transcript is within a sequence selected from SEQ ID NOs:
6271-6272. In some embodiments, the antisense oligomer comprises a
nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one
of SEQ ID NOs: 5830-6148. In some embodiments, the antisense
oligomer comprises a nucleotide sequence selected from SEQ ID NOs:
5830-6148. In some embodiments, the pharmaceutical composition is
formulated for intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection.
[0080] Disclosed herein, in some embodiments, are pharmaceutical
compositions comprising: an antisense oligomer that hybridizes to a
target sequence of a deficient PRPF8 mRNA transcript, wherein the
deficient PRPF8 mRNA transcript comprises a retained intron,
wherein the antisense oligomer induces splicing out of the retained
intron from the deficient PRPF8 mRNA transcript; and a
pharmaceutical acceptable excipient. In some embodiments, the
deficient PRPF8 mRNA transcript is a PRPF8 RIC pre-mRNA transcript.
In some embodiments, the targeted portion of the PRPF8 RIC pre-mRNA
transcript is in the retained intron within the region +500
relative to the 5' splice site of the retained intron to -500
relative to the 3' spliced site of the retained intron. In some
embodiments, the PRPF8 RIC pre-mRNA transcript is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO: 5827. In some
embodiments, the PRPF8 RIC pre-mRNA transcript comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs: 5828-5829. In some
embodiments, the antisense oligomer comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, the antisense oligomer comprises from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
a targeted portion of the PRPF8 RIC pre-mRNA transcript. In some
embodiments, the targeted portion of the PRPF8 RIC pre-mRNA
transcript is within a sequence selected from SEQ ID NOs: 6273. In
some embodiments, the antisense oligomer comprises a nucleotide
sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID
NOs: 6149-6270. In some embodiments, the antisense oligomer
comprises a nucleotide sequence selected from SEQ ID NOs:
6149-6270. In some embodiments, the pharmaceutical composition is
formulated for intravitreal injection, subretinal injection,
topical application, implantation, intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection.
[0081] Disclosed herein, in some embodiments, are methods of
inducing processing of a deficient PRPF3 mRNA transcript to
facilitate removal of a retained intron to produce a fully
processed PRPF3 mRNA transcript that encodes a functional form of a
PRPF3 protein, the method comprising: (a) contacting an antisense
oligomer to a target cell of a subject; (b) hybridizing the
antisense oligomer to the deficient PRPF3 mRNA transcript, wherein
the deficient PRPF3 mRNA transcript is capable of encoding the
functional form of a PRPF3 protein and comprises at least one
retained intron; (c) removing the at least one retained intron from
the deficient PRPF3 mRNA transcript to produce the fully processed
PRPF3 mRNA transcript that encodes the functional form of PRPF3
protein; and (d) translating the functional form of PRPF3 protein
from the fully processed PRPF3 mRNA transcript. In some
embodiments, the retained intron is an entire retained intron. In
some embodiments, the deficient PRPF3 mRNA transcript is a PRPF3
RIC pre-mRNA transcript.
[0082] Disclosed herein, in some embodiments, are methods of
treating a subject having a condition caused by a deficient amount
or activity of PRPF3 protein comprising administering to the
subject an antisense oligomer comprising a nucleotide sequence with
at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to any one of SEQ ID NOs:
5830-6148.
[0083] Disclosed herein, in some embodiments, are methods of
inducing processing of a deficient PRPF8 mRNA transcript to
facilitate removal of a retained intron to produce a fully
processed PRPF8 mRNA transcript that encodes a functional form of a
PRPF8 protein, the method comprising: (a) contacting an antisense
oligomer to a target cell of a subject; (b) hybridizing the
antisense oligomer to the deficient PRPF8 mRNA transcript, wherein
the deficient PRPF8 mRNA transcript is capable of encoding the
functional form of a PRPF8 protein and comprises at least one
retained intron; (c) removing the at least one retained intron from
the deficient PRPF8 mRNA transcript to produce the fully processed
PRPF8 mRNA transcript that encodes the functional form of PRPF8
protein; and (d) translating the functional form of PRPF8 protein
from the fully processed PRPF8 mRNA transcript. In some
embodiments, the retained intron is an entire retained intron. In
some embodiments, the deficient PRPF8 mRNA transcript is a PRPF8
RIC pre-mRNA transcript.
[0084] Disclosed herein, in some embodiments, are methods of
treating a subject having a condition caused by a deficient amount
or activity of PRPF8 protein comprising administering to the
subject an antisense oligomer comprising a nucleotide sequence with
at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to any one of SEQ ID NOs:
6149-6270.
[0085] The invention provides compositions and methods for treating
a central nervous system (CNS) disease or conditions, including
antisense oligomers (ASOs) that promote constitutive splicing at
intron splice sites of a retained-intron-containing pre-mRNA (RIC
pre-mRNA) encoding a gene product that is deficient in the central
nervous system disease or condition. The invention further provides
compositions and methods for increasing production of mature mRNA
encoding a gene product, wherein either the gene product is
deficient in a CNS disease or condition, or wherein increased
expression of the gene product could provide benefit in a subject
having a CNS disease or condition. Increasing the production of the
mature mRNA results in increased production of the encoded protein
in cells of a subject in need thereof, for example, any subject
that can benefit from increased production of the gene product. The
described methods may be used to treat subjects having a CNS
disease or condition caused by a gene mutation(s), including
missense, splicing, frameshift and nonsense mutations, as well as
whole gene deletions that result in deficient protein
production.
[0086] Disclosed herein, in certain embodiments, is a method of
treating a CNS disease in a subject in need thereof by increasing
the expression of a target protein or functional RNA by cells of
the subject, wherein the cells have a retained-intron-containing
pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained
intron, an exon flanking the 5' splice site, an exon flanking the
3' splice site, and wherein the RIC pre-mRNA encodes the target
protein or functional RNA, the method comprising contacting the
cells of the subject with an antisense oligomer (ASO) complementary
to a targeted portion of the RIC pre-mRNA encoding the target
protein or functional RNA, whereby the retained intron is
constitutively spliced from the RIC pre-mRNA encoding the target
protein or functional RNA, thereby increasing the level of mRNA
encoding the target protein or functional RNA, and increasing the
expression of the target protein or functional RNA in the cells of
the subject. In some embodiments, the CNS disease is Familial
hemiplegic migraine-2, Familial Basilar migraine, Alternating
hemiplegia of childhood, Episodic ataxia type 2, Familial
hemiplegic migraine, Spinocerebellar ataxia type 6, Mental
retardation-23, 3p25 microdeletion syndrome, Phelan-McDermid
syndrome, Schizophrenia-15, Neurofibromatosis, type 2, Meningioma,
NF2-related, Schwannomatosis 1, Hereditary sensory neuropathy type
IE, Autosomal dominant cerebellar ataxia, deafness, and narcolepsy,
Pitt-hopkins syndrome, Smith-magenis syndrome, peroxisome
biogenesis disorder 1a, Heimler syndrome-1, Metachromatic
Leukodystrophy, Leukoencephalopathy with vanishing white matter,
Niemann-Pick disease type C1 and Niemann-Pick disease type D,
Aicardi-Goutieres syndrome-6, early infantile epileptic
encephalopathy-4, progressive myoclonic epilepsy 5, familial
infantile convulsion with paroxysmal choreoathetosis, episodic
kinesigenic dyskinesia 1, benign familial infantile seizuers-2, or
generalized Epilepsy with febrile seizures plus type 9. In some
embodiments, also described herein is a method of increasing
expression of a target protein, wherein the target protein is
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, MFSD8,
IDUA, or STX1B expression by improving splicing efficiency of
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, MFSD8,
IDUA, or STX1B, by cells having a retained-intron-containing
pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained
intron, an exon flanking the 5' splice site of the retained intron,
an exon flanking the 3' splice site of the retained intron, and
wherein the RIC pre-mRNA encodes ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B expression by
improving splicing efficiency of ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B protein, the
method comprising contacting the cells with an antisense oligomer
(ASO) complementary to a targeted portion of the RIC pre-mRNA
encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, MFSD8, IDUA, or STX1B expression by improving splicing
efficiency of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B protein, whereby the
retained intron is constitutively spliced from the RIC pre-mRNA
encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, MFSD8, IDUA, or STX1B expression by improving splicing
efficiency of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B protein, thereby increasing
the level of mRNA encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B protein, and
increasing the expression of ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B expression by
improving splicing efficiency of ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B protein in the
cells. In some embodiments, the target protein is ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B. In some
embodiments, the target protein is ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B. In some embodiments, the target
protein or the functional RNA is a compensating protein or a
compensating functional RNA that functionally augments or replaces
a target protein or functional RNA that is deficient in amount or
activity in the subject. In some embodiments, the cells are in or
from a subject having a condition caused by a deficient amount or
activity of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or
STX1B protein. In some embodiments, the cells are in or from a
subject having a condition caused by a deficient amount or activity
of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
protein. In some embodiments, the deficient amount of the target
protein is caused by haploinsufficiency of the target protein,
wherein the subject has a first allele encoding a functional target
protein, and a second allele from which the target protein is not
produced, or a second allele encoding a nonfunctional target
protein, and wherein the antisense oligomer binds to a targeted
portion of a RIC pre-mRNA transcribed from the first allele. In
some embodiments, the subject has a condition caused by a disorder
resulting from a deficiency in the amount or function of the target
protein, wherein the subject has (a) a first mutant allele from
which (i) the target protein is produced at a reduced level
compared to production from a wild-type allele, (ii) the target
protein is produced in a form having reduced function compared to
an equivalent wild-type protein, or (iii) the target protein is not
produced, and (b) a second mutant allele from which (i) the target
protein is produced at a reduced level compared to production from
a wild-type allele, (ii) the target protein is produced in a form
having reduced function compared to an equivalent wild-type
protein, or (iii) the target protein is not produced, and wherein
when the subject has a first mutant allele a(iii), the second
mutant allele is b(i) or b(ii), and wherein when the subject has a
second mutant allele b(iii), the first mutant allele is a(i) or
a(ii), and wherein the RIC pre-mRNA is transcribed from either the
first mutant allele that is a(i) or a(ii), and/or the second allele
that is b(i) or b(ii). In some embodiments, the target protein is
produced in a form having reduced function compared to the
equivalent wild-type protein. In some embodiments, the target
protein is produced in a form that is fully-functional compared to
the equivalent wild-type protein. In some embodiments, the targeted
portion of the RIC pre-mRNA is in the retained intron within the
region +6 relative to the 5' splice site of the retained intron to
-16 relative to the 3' splice site of the retained intron. In some
embodiments, the targeted portion of the RIC pre-mRNA is in the
retained intron within: (a) the region +6 to +4000, +6 to +3000, +6
to +2000 or +6 to +100 relative to the 5' splice site of the
retained intron; or (b) the region -16 to -4000, -16 to -2000, -16
to -1000, -16 to -500 or -16 to -100 relative to the 3' splice site
of the retained intron. In some embodiments, the targeted portion
of the RIC pre-mRNA is within: (a) the region +2e to -4e in the
exon flanking the 5' splice site of the retained intron; or (b) the
region +2e to -4e in the exon flanking the 3' splice site of the
retained intron. In some embodiments, the RIC pre-mRNA is encoded
by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 6274-6292.
In some embodiments, the RIC pre-mRNA comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs: 6293-6353. In some embodiments,
the antisense oligomer does not increase the amount of the target
protein or the functional RNA by modulating alternative splicing of
pre-mRNA transcribed from a gene encoding the functional RNA or
target protein. In some embodiments, the antisense oligomer does
not increase the amount of the target protein or the functional RNA
by modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or the functional RNA. In some
embodiments, the RIC pre-mRNA was produced by partial splicing of a
full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.
In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA. In some embodiments, the target protein produced is
full-length protein, or wild-type protein. In some embodiments, the
total amount of the mRNA encoding the target protein or functional
RNA produced in the cell contacted with the antisense oligomer is
increased about 1.1 to about 10-fold, about 1.5 to about 10-fold,
about 2 to about 10-fold, about 3 to about 10-fold, about 4 to
about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the total amount of the mRNA encoding
the target protein or functional RNA produced in a control cell. In
some embodiments, the total amount of target protein produced by
the cell contacted with the antisense oligomer is increased about
1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
total amount of target protein produced by a control cell. In some
embodiments, the antisense oligomer comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer comprises a phosphorodiamidate morpholino, a locked
nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro,
or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense
oligomer comprises at least one modified sugar moiety. In some
embodiments, each sugar moiety is a modified sugar moiety. In some
embodiments, the antisense oligomer consists of from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, complementary to
the targeted portion of the RIC pre-mRNA encoding the protein. In
some embodiments, the targeted portion of the RIC pre-mRNA is
within a sequence selected from SEQ ID NOs: 30624-30627,
30629-30651, 30653-30659, or 34788-34789. In some embodiments, the
antisense oligomer comprises a nucleotide sequence that is at least
about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to any one of SEQ ID NOs: 6354-8948, 8949-9575,
9576-9825, 9826-10067, 10068-10305, 10306-12684, 12685-14026,
14027-14237, 14238-14326, 14327-15605, 15606-15873, 15874-16076,
30660-34787, 19965-20033, 20034-22187, 22188-23203, 23204-29620,
29621-29808, or 29809-30623. In some embodiments, the antisense
oligomer comprises a nucleotide sequence selected from SEQ ID NOs:
6354-8948, 8949-9575, 9576-9825, 9826-10067, 10068-10305,
10306-12684, 12685-14026, 14027-14237, 14238-14326, 14327-15605,
15606-15873, 15874-16076, 30660-34787, 19965-20033, 20034-22187,
22188-23203, 23204-29620, 29621-29808, or 29809-30623. In some
embodiments, the cell comprises a population of RIC pre-mRNAs
transcribed from the gene encoding the target protein or functional
RNA, wherein the population of RIC pre-mRNAs comprises two or more
retained introns, and wherein the antisense oligomer binds to the
most abundant retained intron in the population of RIC pre-mRNAs.
In some embodiments, the binding of the antisense oligomer to the
most abundant retained intron induces splicing out of the two or
more retained introns from the population of RIC pre-mRNAs to
produce mRNA encoding the target protein or functional RNA. In some
embodiments, the cell comprises a population of RIC pre-mRNAs
transcribed from the gene encoding the target protein or functional
RNA, wherein the population of RIC pre-mRNAs comprises two or more
retained introns, and wherein the antisense oligomer binds to the
second most abundant retained intron in the population of RIC
pre-mRNAs. In some embodiments, the binding of the antisense
oligomer to the second most abundant retained intron induces
splicing out of the two or more retained introns from the
population of RIC pre-mRNAs to produce mRNA encoding the target
protein or functional RNA. In some embodiments, the condition is a
disease or disorder. In some embodiments, the disease or disorder
is Familial hemiplegic migraine-2, Familial Basilar migraine,
Alternating hemiplegia of childhood, Episodic ataxia type 2,
Familial hemiplegic migraine, Spinocerebellar ataxia type 6, Mental
retardation-23, 3p25 microdeletion syndrome, Phelan-McDermid
syndrome, Schizophrenia-15, Neurofibromatosis, type 2, Meningioma,
NF2-related, Schwannomatosis 1, Hereditary sensory neuropathy type
IE, Autosomal dominant cerebellar ataxia, deafness, and narcolepsy,
Pitt-hopkins syndrome, Smith-magenis syndrome, peroxisome
biogenesis disorder 1a, Heimler syndrome-1, Metachromatic
Leukodystrophy, Leukoencephalopathy with vanishing white matter,
Niemann-Pick disease type C1 and Niemann-Pick disease type D,
Aicardi-Goutieres syndrome-6, early infantile epileptic
encephalopathy-4, progressive myoclonic epilepsy 5, familial
infantile convulsion with paroxysmal choreoathetosis, episodic
kinesigenic dyskinesia 1, benign familial infantile seizuers-2, or
generalized Epilepsy with febrile seizures plus type 9. In some
embodiments, the target protein and the RIC pre-mRNA are encoded by
the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
gene. In some embodiments, the method further comprises assessing
protein expression. In some embodiments, the antisense oligomer
binds to a targeted portion of a ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B RIC pre-mRNA. In some
embodiments, the subject is a human. In some embodiments, the
subject is a non-human animal. In some embodiments, the subject is
a fetus, an embryo, or a child. In some embodiments, the cells are
ex vivo. In some embodiments, the antisense oligomer is
administered by intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection of the subject. In
some embodiments, the 9 nucleotides at -3e to -1e of the exon
flanking the 5' splice site
and +1 to +6 of the retained intron, are identical to the
corresponding wild-type sequence. In some embodiments, the 16
nucleotides at -15 to -1 of the retained intron and +1e of the exon
flanking the 3' splice site are identical to the corresponding
wild-type sequence.
[0087] Described herein, in certain embodiments, is an antisense
oligomer as used in a method described above.
[0088] Described herein, in certain embodiments, is an antisense
oligomer comprising a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to any one of SEQ ID NOs:
6354-8948, 8949-9575, 9576-9825, 9826-10067, 10068-10305,
10306-12684, 12685-14026, 14027-14237, 14238-14326, 14327-15605,
15606-15873, 15874-16076, 30660-34787, 19965-20033, 20034-22187,
22188-23203, 23204-29620, 29621-29808, and 29809-30623.
[0089] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising the antisense oligomer
described above, and an excipient. In some embodiments, also
described herein is a method of treating a subject in need thereof
by administering the pharmaceutical composition by intrathecal
injection, intracerebroventricular injection, intraperitoneal
injection, intramuscular injection, subcutaneous injection, or
intravenous injection.
[0090] Disclosed herein, in certain embodiments, is a composition
comprising an antisense oligomer for use in a method of increasing
expression of a target protein or a functional RNA by cells to
treat Familial hemiplegic migraine-2, Familial Basilar migraine,
Alternating hemiplegia of childhood, Episodic ataxia type 2,
Familial hemiplegic migraine, Spinocerebellar ataxia type 6, Mental
retardation-23, 3p25 microdeletion syndrome, Phelan-McDermid
syndrome, Schizophrenia-15, Neurofibromatosis, type 2, Meningioma,
NF2-related, Schwannomatosis 1, Hereditary sensory neuropathy type
IE, Autosomal dominant cerebellar ataxia, deafness, and narcolepsy,
Pitt-hopkins syndrome, Smith-magenis syndrome, peroxisome
biogenesis disorder 1a, Heimler syndrome-1, Metachromatic
Leukodystrophy, Leukoencephalopathy with vanishing white matter,
Niemann-Pick disease type C1 and Niemann-Pick disease type D,
Aicardi-Goutieres syndrome-6, early infantile epileptic
encephalopathy-4, progressive myoclonic epilepsy 5, familial
infantile convulsion with paroxysmal choreoathetosis, episodic
kinesigenic dyskinesia 1, benign familial infantile seizuers-2, or
generalized Epilepsy with febrile seizures plus type 9 in a subject
in need thereof associated with a deficient protein or deficient
functional RNA, wherein the deficient protein or deficient
functional RNA is deficient in amount or activity in the subject,
wherein the antisense oligomer enhances constitutive splicing of a
retained intron-containing pre-mRNA (RIC pre-mRNA) encoding the
target protein or the functional RNA, wherein the target protein
is: (a) the deficient protein; or (b) a compensating protein which
functionally augments or replaces the deficient protein or in the
subject; and wherein the functional RNA is: (a) the deficient RNA;
or (b) a compensating functional RNA which functionally augments or
replaces the deficient functional RNA in the subject; wherein the
RIC pre-mRNA comprises a retained intron, an exon flanking the 5'
splice site and an exon flanking the 3' splice site, and wherein
the retained intron is spliced from the RIC pre-mRNA encoding the
target protein or the functional RNA, thereby increasing production
or activity of the target protein or the functional RNA in the
subject. In some embodiments, also described herein is a
composition comprising an antisense oligomer for use in a method of
treating a condition associated with ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein
in a subject in need thereof, the method comprising the step of
increasing expression of ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein by cells of
the subject, wherein the cells have a retained-intron-containing
pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon
flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and wherein the
RIC pre-mRNA encodes the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, the method
comprising contacting the cells with the antisense oligomer,
whereby the retained intron is constitutively spliced from the RIC
pre-mRNA transcripts encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, thereby
increasing the level of mRNA encoding the target protein or
functional RNA, and increasing the expression of ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein, in the cells of the subject. In some embodiments, the
target protein is ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B. In some embodiments, the target protein
is ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or
STX1B. In some embodiments, the condition is a disease or disorder.
In some embodiments, the disease or disorder is Familial hemiplegic
migraine-2, Familial Basilar migraine, Alternating hemiplegia of
childhood, Episodic ataxia type 2, Familial hemiplegic migraine,
Spinocerebellar ataxia type 6, Mental retardation-23, 3p25
microdeletion syndrome, Phelan-McDermid syndrome, Schizophrenia-15,
Neurofibromatosis, type 2, Meningioma, NF2-related, Schwannomatosis
1, Hereditary sensory neuropathy type IE, Autosomal dominant
cerebellar ataxia, deafness, and narcolepsy, Pitt-hopkins syndrome,
Smith-magenis syndrome, peroxisome biogenesis disorder 1a, Heimler
syndrome-1, Metachromatic Leukodystrophy, Leukoencephalopathy with
vanishing white matter, Niemann-Pick disease type C1 and
Niemann-Pick disease type D, Aicardi-Goutieres syndrome-6, early
infantile epileptic encephalopathy-4, progressive myoclonic
epilepsy 5, familial infantile convulsion with paroxysmal
choreoathetosis, episodic kinesigenic dyskinesia 1, benign familial
infantile seizuers-2, or generalized Epilepsy with febrile seizures
plus type 9. In some embodiments, the target protein and RIC
pre-mRNA are encoded by the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B gene. In some embodiments, the
target protein and RIC pre-mRNA are encoded by the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B gene. In some
embodiments, the antisense oligomer targets a portion of the RIC
pre-mRNA that is in the retained intron within the region +6
relative to the 5' splice site of the retained intron to -16
relative to the 3' splice site of the retained intron. In some
embodiments, the antisense oligomer targets a portion of the RIC
pre-mRNA that is in the retained intron within: (a) the region +6
to +100 relative to the 5' splice site of the retained intron; or
(b) the region -16 to -100 relative to the 3' splice site of the
retained intron. In some embodiments, the antisense oligomer
targets a portion of the RIC pre-mRNA that is within the region
about 100 nucleotides downstream of the 5' splice site of the at
least one retained intron, to about 100 nucleotides upstream of the
3' splice site of the at least one retained intron. In some
embodiments, the targeted portion of the RIC pre-mRNA is within:
(a) the region +2e to -4e in the exon flanking the 5' splice site
of the retained intron; or (b) the region +2e to -4e in the exon
flanking the 3' splice site of the retained intron. In some
embodiments, the RIC pre-mRNA is encoded by a genetic sequence with
at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NOs: 6274-6292. In some embodiments,
the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one
of SEQ ID NOs: 6293-6353. In some embodiments, the antisense
oligomer does not increase the amount of target protein or
functional RNA by modulating alternative splicing of the pre-mRNA
transcribed from a gene encoding the target protein or functional
RNA. In some embodiments, the antisense oligomer does not increase
the amount of the functional RNA or functional protein by
modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or functional RNA. In some embodiments,
the RIC pre-mRNA was produced by partial splicing from a
full-length pre-mRNA or a wild-type pre-mRNA. In some embodiments,
the mRNA encoding the target protein or functional RNA is a
full-length mature mRNA, or a wild-type mature mRNA. In some
embodiments, the target protein produced is full-length protein, or
wild-type protein. In some embodiments, the retained intron is a
rate-limiting intron. In some embodiments, said retained intron is
the most abundant retained intron in said RIC pre-mRNA. In some
embodiments, the retained intron is the second most abundant
retained intron in said RIC pre-mRNA. In some embodiments, the
antisense oligomer comprises a backbone modification comprising a
phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, said antisense oligomer is an antisense
oligonucleotide. In some embodiments, the antisense oligomer
comprises a phosphorodiamidate morpholino, a locked nucleic acid, a
peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a
2'-O-methoxyethyl moiety. In some embodiments, the antisense
oligomer comprises at least one modified sugar moiety. In some
embodiments, each sugar moiety is a modified sugar moiety. In some
embodiments, the antisense oligomer consists of from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
the targeted portion of the RIC pre-mRNA encoding the protein. In
some embodiments, the antisense oligomer binds to a targeted
portion of a tuberin RIC pre-mRNA, wherein the targeted portion of
the RIC pre-mRNA is within a sequence selected from SEQ ID NOs:
30624-30627, 30629-30651, 30653-30659, or 34788-34789. In some
embodiments, the antisense oligomer comprises a nucleotide sequence
that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:
6354-8948, 8949-9575, 9576-9825, 9826-10067, 10068-10305,
10306-12684, 12685-14026, 14027-14237, 14238-14326, 14327-15605,
15606-15873, 15874-16076, 30660-34787, 19965-20033, 20034-22187,
22188-23203, 23204-29620, 29621-29808, or 29809-30623. In some
embodiments, the antisense oligomer comprises a nucleotide sequence
selected from SEQ ID NOs: 6354-8948, 8949-9575, 9576-9825,
9826-10067, 10068-10305, 10306-12684, 12685-14026, 14027-14237,
14238-14326, 14327-15605, 15606-15873, 15874-16076, 30660-34787,
19965-20033, 20034-22187, 22188-23203, 23204-29620, 29621-29808, or
29809-30623. In some embodiments, the antisense oligomer binds to a
targeted portion of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B RIC pre-mRNA. In some embodiments, the
antisense oligomer binds to a targeted portion of a ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B RIC
pre-mRNA.
[0091] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising the antisense oligomer of any
of the compositions described above, and an excipient. In some
embodiments, also described herein is a method of treating a
subject in need thereof by administering the pharmaceutical
composition by intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection.
[0092] Disclosed herein, in certain embodiments, is a
pharmaceutical composition comprising: an antisense oligomer that
hybridizes to a target sequence of a deficient ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B
mRNA transcript, wherein the deficient ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B mRNA
transcript comprises a retained intron, wherein the antisense
oligomer induces splicing out of the retained intron from the
deficient ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, MFSD8, IDUA, or STX1B mRNA transcript; and a pharmaceutical
acceptable excipient. In some embodiments, the antisense oligomer
hybridizes to a target sequence of a deficient ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B mRNA
transcript. In some embodiments, the deficient ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B mRNA
transcript is a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B RIC pre-mRNA transcript. In some
embodiments, the antisense oligomer hybridizes to a target sequence
of a deficient ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B mRNA transcript. In some embodiments, the deficient
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B mRNA
transcript is a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B RIC pre-mRNA transcript. In some embodiments, the
targeted portion of the RIC pre-mRNA transcript is in the retained
intron within the region +4000 relative to the 5' splice site of
the retained intron to -2000 relative to the 3' spliced site of the
retained intron. In some embodiments, the RIC pre-mRNA transcript
is encoded by a genetic sequence with at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs:
6274-6292. In some embodiments, the RIC pre-mRNA transcript
comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:
6293-6353. In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, the antisense oligomer comprises from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
a targeted portion of the RIC pre-mRNA transcript. In some
embodiments, the targeted portion of the RIC pre-mRNA transcript is
within a sequence selected from SEQ ID NOs: 30624-30627,
30629-30651, 30653-30659, or 34788-34789. In some embodiments, the
antisense oligomer comprises a nucleotide sequence that is at least
about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to any one of SEQ ID NOs: 6354-8948, 8949-9575,
9576-9825, 9826-10067, 10068-10305, 10306-12684, 12685-14026,
14027-14237, 14238-14326, 14327-15605, 15606-15873, 15874-16076,
30660-34787, 19965-20033, 20034-22187, 22188-23203, 23204-29620,
29621-29808, or 29809-30623. In some embodiments, the antisense
oligomer comprises a nucleotide sequence selected from SEQ ID NOs:
6354-8948, 8949-9575, 9576-9825, 9826-10067, 10068-10305,
10306-12684, 12685-14026, 14027-14237, 14238-14326, 14327-15605,
15606-15873, 15874-16076, 30660-34787, 19965-20033, 20034-22187,
22188-23203, 23204-29620, 29621-29808, or 29809-30623. In some
embodiments, the pharmaceutical composition is formulated for
intrathecal injection, intracerebroventricular injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0093] Disclosed herein, in certain embodiments, is a method of
inducing processing of a mRNA transcript to facilitate removal of a
retained intron to produce a fully processed mRNA transcript that
encodes a functional form of a target protein, the method
comprising: (a) contacting an antisense oligomer to a target cell
of a subject; (b) hybridizing the antisense oligomer to the mRNA
transcript, wherein the mRNA transcript is capable of encoding the
functional form of the target protein and comprises at least one
retained intron; (c) removing the at least one retained intron from
the mRNA transcript to produce the fully processed mRNA transcript
that encodes the functional form of the target protein; and (d)
translating the functional form of the target protein from the
fully processed mRNA transcript; wherein the mRNA transcript is
selected from ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, MFSD8, IDUA, or STX1B mRNA transcript. In some
embodiments, the retained intron is an entire retained intron. In
some embodiments, the mRNA transcript is a RIC pre-mRNA transcript.
In some embodiments, the mRNA transcript is selected from ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B mRNA
transcript. In some embodiments, the mRNA transcript is selected
from ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
mRNA transcript.
[0094] Disclosed herein, in certain embodiments, is a method of
treating a subject having a condition caused by a deficient amount
or activity of a target protein comprising: administering to the
subject an antisense oligomer comprising a nucleotide sequence with
at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to any one of SEQ ID NOs: 6354-8948,
8949-9575, 9576-9825, 9826-10067, 10068-10305, 10306-12684,
12685-14026, 14027-14237, 14238-14326, 14327-15605, 15606-15873,
15874-16076, 30660-34787, 19965-20033, 20034-22187, 22188-23203,
23204-29620, 29621-29808, or 29809-30623.
[0095] Disclosed herein, in some embodiments, are methods of
treating an eye disease in a subject in need thereof by increasing
the expression of a target protein or functional RNA by cells of
the subject, wherein the cells have a retained-intron-containing
pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained
intron, an exon flanking the 5' splice site, an exon flanking the
3' splice site, and wherein the RIC pre-mRNA encodes the target
protein or functional RNA, the method comprising contacting the
cells of the subject with an antisense oligomer (ASO) complementary
to a targeted portion of the RIC pre-mRNA encoding the target
protein or functional RNA, whereby the retained intron is
constitutively spliced from the RIC pre-mRNA encoding the target
protein or functional RNA, thereby increasing the level of mRNA
encoding the target protein or functional RNA, and increasing the
expression of the target protein or functional RNA in the cells of
the subject. In some embodiments, the eye disease is Retinitis
pigmentosa-7, Sveinsson chorioretinal atrophy, Fundus
Albipunctatus, Retinitis pigmentosa 37, Aniridia, Coloboma of the
optic nerve, Ocular coloboma, Foveal hypoplasia-1, Bilateral optic
nerve hypoplasia, Cone-rod dystrophy-2, Leber congenital
amaurosis-7, Retinitis Pigmentosa 30, Stargardt disease-1,
Retinitis pigmentosa-19, Age-related macular degeneration-2,
Cone-rod dystrophy-3, Primary open angle glaucoma, Fuchs
endothelial corneal dystrophy-3, Macular dystrophy with central
cone involvement, Ocular nonnephropathic cystinosis, Leber
congenital amaurosis, Primary open angle glaucoma, Amyotrophic
lateral sclerosis 12, Bothnia retinal dystrophy, Fundus
albipunctatus, Retinitis punctata albescens, Leber congenital
amaurosis 2, Retinitis pigmentosa 20, Leber congenital amaurosis
14, Retinitis pigmentosa, Eye diseases with slow clearance or
accumulation of all-trans retinal (e.g. STGD1), Leber congenital
amaurosis13, Retinitis pigmentosa 44, Achromatopsia-2, Jet lag,
Alstrom syndrome, Attenuated MPS-1 (Hurler-scheie syndrome and
Scheie syndrome) or Bardet-biedl syndrome.
[0096] Also disclosed herein are methods of increasing expression
of a target protein, wherein the target protein is ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA expression by improving splicing efficiency of ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA, by cells having a retained-intron-containing
pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained
intron, an exon flanking the 5' splice site of the retained intron,
an exon flanking the 3' splice site of the retained intron, and
wherein the RIC pre-mRNA encodes ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
expression by improving splicing efficiency of ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein, the method comprising contacting the cells with an
antisense oligomer (ASO) complementary to a targeted portion of the
RIC pre-mRNA encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA expression by
improving splicing efficiency of ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein,
whereby the retained intron is constitutively spliced from the RIC
pre-mRNA encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA expression by
improving splicing efficiency of ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein,
thereby increasing the level of mRNA encoding ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein, and increasing the expression of ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA expression by improving splicing efficiency of ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein in the cells, wherein the target protein is
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA.
[0097] In some embodiments of any of the aforementioned methods,
the target protein is ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA. In some embodiments,
the target protein or the functional RNA is a compensating protein
or a compensating functional RNA that functionally augments or
replaces a target protein or functional RNA that is deficient in
amount or activity in the subject. In some embodiments, the cells
are in or from a subject having a condition caused by a deficient
amount or activity of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein.
[0098] In some embodiments of any of the aforementioned methods,
the deficient amount of the target protein is caused by
haploinsufficiency of the target protein, wherein the subject has a
first allele encoding a functional target protein, and a second
allele from which the target protein is not produced, or a second
allele encoding a nonfunctional target protein, and wherein the
antisense oligomer binds to a targeted portion of a RIC pre-mRNA
transcribed from the first allele. In some embodiments, the subject
has a condition caused by a disorder resulting from a deficiency in
the amount or function of the target protein, wherein the subject
has (a) a first mutant allele from which (i) the target protein is
produced at a reduced level compared to production from a wild-type
allele, (ii) the target protein is produced in a form having
reduced function compared to an equivalent wild-type protein, or
(iii) the target protein is not produced, and (b) a second mutant
allele from which (i) the target protein is produced at a reduced
level compared to production from a wild-type allele, (ii) the
target protein is produced in a form having reduced function
compared to an equivalent wild type protein, or (iii) the target
protein is not produced, and wherein when the subject has a first
mutant allele a(iii), the second mutant allele is b(i) or b(ii),
and wherein when the subject has a second mutant allele b(iii), the
first mutant allele is a(i) or a(ii), and wherein the RIC pre-mRNA
is transcribed from either the first mutant allele that is a(i) or
a(ii), and/or the second allele that is b(i) or b(ii). In some
embodiments, the target protein is produced in a form having
reduced function compared to the equivalent wild-type protein. In
some embodiments, the target protein is produced in a form that is
fully-functional compared to the equivalent wild-type protein.
[0099] In some embodiments of any of the aforementioned methods,
the targeted portion of the RIC pre-mRNA is in the retained intron
within the region +6 relative to the 5' splice site of the retained
intron to -16 relative to the 3' splice site of the retained
intron. In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +69 relative
to the 5' splice site of the retained intron to -79 relative to the
3' splice site of the retained intron.
[0100] In some embodiments of any of the aforementioned methods,
the target protein is (a) ABCA4, (b) RPE65, (c) MYOC, (d) CNGA3,
(e) MFSD8, (f) IDUA, (g) LRAT, (h) OPTN, (i) RGR, (j) TEAD1, (k)
PAX6, (l) ROM1, (m) RDH5, (n) RDH12, (o) NR2E3, (p) RLBP1, (q)
CTNS, (r) PER1, (s) FSCN2, (t) TCF4, (u) RDH8, (v) NXNL1, or (w)
CRX. In some embodiments, the targeted portion of the RIC pre-mRNA
comprises a sequence that is at least about 80%, 85%, 90%, 95%,
97%, or 100% complimentary to (a) any one of SEQ ID NOs
34873-35915, (b) any one of SEQ ID NOs 35916-36317, (c) any one of
SEQ ID NOs 36318-37107, (d) any one of SEQ ID NOs 37108-37559, (e)
any one of SEQ ID NOs 37560-38420, (f) any one of SEQ ID NOs
38421-39232, (g) any one of SEQ ID NOs 39233-41436, (h) any one of
SEQ ID NOs 41437-42368, (i) any one of SEQ ID NOs 42369-43747, (j)
any one of SEQ ID NOs 43748-43952, (k) any one of SEQ ID NOs
43953-49968, (l) any one of SEQ ID NOs 49969-50275, (m) any one of
SEQ ID NOs 50276-50991, (n) any one of SEQ ID NOs 50992-51247, (o)
any one of SEQ 5ID NOs 51248-52998, (p) any one of SEQ ID NOs
52999-53428, (q) any one of SEQ ID NOs 53429-54323, (r) any one of
SEQ ID NOs 54324-54634, (s) any one of SEQ ID NOs 54634-55638, (t)
any one of SEQ ID NOs 55639-59526, (u) any one of SEQ ID NOs
59527-59662, (v) any one of SEQ ID NOs 59663-25231, or (w) any one
of SEQ ID NOs 60020-61443. In some embodiments, the targeted
portion of the RIC pre-mRNA comprises a sequence with at least 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to a region
comprising at least 8 contiguous nucleic acids of (a) SEQ ID NO
61463, SEQ ID NO, SEQ ID NO 61445, SEQ ID NO 61470, or SEQ ID NO
61453, (b) SEQ ID NO 61480 or SEQ ID NO 61460, (c) SEQ ID NO 61458
or SEQ ID NO 61485, (d) SEQ ID NO 61500, (e) SEQ ID NO 61492 or SEQ
ID NO 61497, (f) SEQ ID NO 61457, SEQ ID NO 61468, SEQ ID NO 61489,
SEQ ID NO 61444, or SEQ ID NO 61452, (g) SEQ ID NO 61474, (h) SEQ
ID NO 61503, (i) SEQ ID NO 61446, SEQ ID NO 61476, or SEQ ID NO
61472, (j) SEQ ID NO 61461, (k) SEQ ID NO 61486, SEQ ID NO 61466,
SEQ ID NO 61496, SEQ ID NO 61467, SEQ ID NO 61502, SEQ ID NO 61483,
or SEQ ID NO 61448, (l) SEQ ID NO 61454, (m) SEQ ID NO 61493, SEQ
ID NO 61455, SEQ ID NO 61498, or SEQ ID NO 61473, (n) SEQ ID NO
61482, (o) SEQ ID NO 61491, SEQ ID NO 61449, SEQ ID NO 61494, SEQ
ID NO 61487, SEQ ID NO 61447, SEQ ID NO 61465, SEQ ID NO 61501 SEQ
ID NO 61490, (p) SEQ ID NO 61462 or SEQ ID NO 61456, (q) SEQ ID NO
61479, or SEQ ID NO 61481, (r) SEQ ID NO 61471 or SEQ ID NO 61499,
(s) SEQ ID NO 61459, SEQ ID NO 61451, or SEQ ID NO 61450, (t) SEQ
ID NO 61477, or SEQ ID NO 61478, (u) SEQ ID NO 61469, (v) SEQ ID NO
61488, or (w) SEQ ID NO 61484, or SEQ ID NO 61475. In some
embodiments, the ASO comprises a sequence with at least about 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to (a) any one of SEQ
ID NOs 34873-35915, (b) any one of SEQ ID NOs 35916-36317, (c) any
one of SEQ ID NOs 36318-37107, (d) any one of SEQ ID NOs
37108-37559, (e) any one of SEQ ID NOs 37560-38420, (f) any one of
SEQ ID NOs 38421-39232, (g) any one of SEQ ID NOs 39233-41436, (h)
any one of SEQ ID NOs 41437-42368, (i) any one of SEQ ID NOs
42369-43747, (j) any one of SEQ ID NOs 43748-43952, (k) any one of
SEQ ID NOs 43953-49968, (l) any one of SEQ ID NOs 49969-50275, (m)
any one of SEQ ID NOs 50276-50991, (n) any one of SEQ ID NOs
50992-51247, (o) any one of SEQ ID NOs 51248-52998, (p) any one of
SEQ ID NOs 52999-53427, (q) any one of SEQ ID NOs 53428-54323, (r)
any one of SEQ ID NOs 54324-54634, (s) any one of SEQ ID NOs
54635-55638, (t) any one of SEQ ID NOs 55639-59526, (u) any one of
SEQ ID NOs 59527-59662, (v) any one of SEQ ID NOs 59663-60020, or
(w) any one of SEQ ID NOs 60021-61443. In some embodiments, the RIC
pre-mRNA comprises a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to (a) SEQ ID NO 34813, (b) SEQ
ID NO 34814, (c) SEQ ID NO 34815, (d) SEQ ID NO 34816 or SEQ ID NO
34817, (e) SEQ ID NO 34818, (f) SEQ ID NO 34819 or SEQ ID NO 34820,
(g) SEQ ID NO 34821 or SEQ ID NO 34822, (h) SEQ ID NO 34823, SEQ ID
NO 34824, SEQ ID NO 34825, or SEQ ID NO 34826, (i) SEQ ID NO 34827,
SEQ ID NO 34828, or SEQ ID NO 34829, (j) SEQ ID NO 34830, (k) SEQ
ID NO 34831, SEQ ID NO 34832, SEQ ID NO 34833, SEQ ID NO 34834, SEQ
ID NO 34835, SEQ ID NO 34836, SEQ ID NO 34837, SEQ ID NO 34838, SEQ
ID NO 34839, SEQ ID NO 34840, or SEQ ID NO 34841, (l) SEQ ID NO
34842, (m) SEQ ID NO 34843 or SEQ ID NO 34844, (n) SEQ ID NO 34845,
(o) SEQ ID NO 34846 or SEQ ID NO 34847, (p) SEQ ID NO 34848, (q)
SEQ ID NO 34849 or SEQ ID NO 34850, (r) SEQ ID NO 34851, (s) SEQ ID
NO 34852 or SEQ ID NO 34853, (t) SEQ ID NO 34854, SEQ ID NO 34855,
SEQ ID NO 34856, SEQ ID NO 34857, SEQ ID NO 34858, SEQ ID NO 34859,
SEQ ID NO 34860, SEQ ID NO 34861, SEQ ID NO 34862, SEQ ID NO 34863,
SEQ ID NO 34864, SEQ ID NO 34865, SEQ ID NO 34866, SEQ ID NO 34867,
SEQ ID NO 34868, or SEQ ID NO 34869, (u) SEQ ID NO 34870, (v) SEQ
ID NO 34871, or (w) SEQ ID NO 34872. In some embodiments, the RIC
pre-mRNA is encoded by a genetic sequence with at least about 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to (a) SEQ ID NO
34790, (b) SEQ ID NO 34791, (c) SEQ ID NO 34792, (d) SEQ ID NO
34793, (e) SEQ ID NO 34794, (f) SEQ ID NO 34795, (g) SEQ ID NO
34796, (h) SEQ ID NO 34797, (i) SEQ ID NO 34798, (j) SEQ ID NO
34799, (k) SEQ ID NO 34800, (l) SEQ ID NO 34801, (m) SEQ ID NO
34802, (n) SEQ ID NO 34803, (o) SEQ ID NO 34804, (p) SEQ ID NO
34805, (q) SEQ ID NO 34806, (r) SEQ ID NO 34807, (s) SEQ ID NO
34808, (t) SEQ ID NO 34809, (u) SEQ ID NO 34810, (v) SEQ ID NO
34811, (w) SEQ ID NO 34812.
[0101] In some embodiments of any of the aforementioned methods,
the targeted portion of the RIC pre-mRNA is in the retained intron
within: (a) the region +6 to +100 relative to the 5' splice site of
the retained intron; or (b) the region -16 to -100 relative to the
3' splice site of the retained intron. In some embodiments, the
antisense oligomer targets a portion of the RIC pre-mRNA that is
within the region about 100 nucleotides downstream of the 5' splice
site of the at least one retained intron, to about 100 nucleotides
upstream of the 3' splice site of the at least one retained intron.
In some embodiments, the targeted portion of the RIC pre-mRNA is
within: (a) the region +2e to -4e in the exon flanking the 5'
splice site of the retained intron; or (b) the region +2e to -4e in
the exon flanking the 3' splice site of the retained intron.
[0102] In some embodiments of any of the aforementioned methods,
the antisense oligomer does not increase the amount of the target
protein or the functional RNA by modulating alternative splicing of
pre-mRNA transcribed from a gene encoding the functional RNA or
target protein. In some embodiments, the antisense oligomer does
not increase the amount of the target protein or the functional RNA
by modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or the functional RNA. In some
embodiments, the RIC pre-mRNA was produced by partial splicing of a
full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.
In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA. In some embodiments, the target protein produced is
full-length protein, or wild-type protein. In some embodiments, the
total amount of the mRNA encoding the target protein or functional
RNA produced in the cell contacted with the antisense oligomer is
increased about 1.1 to about 10-fold, about 1.5 to about 10-fold,
about 2 to about 10-fold, about 3 to about 10-fold, about 4 to
about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the total amount of the mRNA encoding
the target protein or functional RNA produced in a control cell. In
some embodiments, the total amount of target protein produced by
the cell contacted with the antisense oligomer is increased about
1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
total amount of target protein produced by a control cell.
[0103] In some embodiments, of any of the aforementioned methods,
the antisense oligomer comprises a backbone modification comprising
a phosphorothioate linkage or a phosphorodiamidate linkage. In some
embodiments, the antisense oligomer comprises a phosphorodiamidate
morpholino, a locked nucleic acid, a peptide nucleic acid, a
2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some
embodiments, the antisense oligomer comprises at least one modified
sugar moiety. In some embodiments, each sugar moiety is a modified
sugar moiety. In some embodiments, the antisense oligomer consists
of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35
nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20
nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40
nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25
nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30
nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15
nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35
nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20
nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40
nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25
nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some
embodiments, the antisense oligomer is at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, at least 99%, or 100%,
complementary to the targeted portion of the RIC pre-mRNA encoding
the protein.
[0104] In some embodiments of any of the aforementioned methods,
the cell comprises a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein or functional RNA, wherein the
population of RIC pre-mRNAs comprises two or more retained introns,
and wherein the antisense oligomer binds to the most abundant
retained intron in the population of RIC pre-mRNAs. In some
embodiments, the binding of the antisense oligomer to the most
abundant retained intron induces splicing out of the two or more
retained introns from the population of RIC pre-mRNAs to produce
mRNA encoding the target protein or functional RNA. In some
embodiments, the cell comprises a population of RIC pre-mRNAs
transcribed from the gene encoding the target protein or functional
RNA, wherein the population of RIC pre-mRNAs comprises two or more
retained introns, and wherein the antisense oligomer binds to the
second most abundant retained intron in the population of RIC
pre-mRNAs. In some embodiments, the binding of the antisense
oligomer to the second most abundant retained intron induces
splicing out of the two or more retained introns from the
population of RIC pre-mRNAs to produce mRNA encoding the target
protein or functional RNA. In some embodiments, the condition is a
disease or disorder. In some embodiments, the disease or disorder
is Retinitis pigmentosa-7, Sveinsson chorioretinal atrophy, Fundus
Albipunctatus, Retinitis pigmentosa 37, Aniridia, Coloboma of the
optic nerve, Ocular coloboma, Foveal hypoplasia-1, Bilateral optic
nerve hypoplasia, Cone-rod dystrophy-2, Leber congenital
amaurosis-7, Retinitis Pigmentosa 30, Stargardt disease-1,
Retinitis pigmentosa-19, Age-related macular degeneration-2,
Cone-rod dystrophy-3, Primary open angle glaucoma, Fuchs
endothelial corneal dystrophy-3, Macular dystrophy with central
cone involvement, Ocular nonnephropathic cystinosis, Leber
congenital amaurosis, Primary open angle glaucoma, Amyotrophic
lateral sclerosis 12, Bothnia retinal dystrophy, Fundus
albipunctatus, Retinitis punctata albescens, Leber congenital
amaurosis 2, Retinitis pigmentosa 20, Leber congenital amaurosis
14, Retinitis pigmentosa, Eye diseases with slow clearance or
accumulation of all-trans retinal (e.g. STGD1), Leber congenital
amaurosis13, Retinitis pigmentosa 44, Achromatopsia-2, Jet lag,
Alstrom syndrome, Attenuated MPS-1 (Hurler-scheie syndrome and
Scheie syndrome) or Bardet-biedl syndrome. In some embodiments, the
target protein and the RIC pre-mRNA are encoded by the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA gene.
[0105] In some embodiments, the method further comprises assessing
protein expression. In some embodiments, the antisense oligomer
binds to a targeted portion of a ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC
pre-mRNA. In some embodiments, the subject is a human. In some
embodiments, the subject is a non-human animal. In some
embodiments, the subject is a fetus, an embryo, or a child. In some
embodiments, the cells are ex vivo. In some embodiments, the
antisense oligomer is administered by intravitreal injection,
subretinal injection, topical application, implantation,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection of the subject. In some
embodiments, the 9 nucleotides at -3e to -1e of the exon flanking
the 5' splice site and +1 to +6 of the retained intron, are
identical to the corresponding wild-type sequence. In some
embodiments, the 16 nucleotides at -15 to -1 of the retained intron
and +1e of the exon flanking the 3' splice site are identical to
the corresponding wild-type sequence.
[0106] Disclosed herein, in some embodiments, are antisense
oligomers as used in the methods described herein. In some
embodiments, the antisense oligomer comprises a sequence with at
least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to
any one of SEQ ID NOs 34873-61443.
[0107] Also disclosed herein, in some embodiments, are
pharmaceutical compositions comprising any of the aforementioned
antisense oligomers and an excipient.
[0108] Disclosed herein, in some embodiments, are methods of
treating a subject in need thereof by administering any of the
aforementioned pharmaceutical compositions by intravitreal
injection, subretinal injection, topical application, implantation,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0109] Disclosed herein, in some embodiments, are compositions
comprising an antisense oligomer for use in a method of increasing
expression of a target protein or a functional RNA by cells to
treat Retinitis pigmentosa-7, Sveinsson chorioretinal atrophy,
Fundus Albipunctatus, Retinitis pigmentosa 37, Aniridia, Coloboma
of the optic nerve, Ocular coloboma, Foveal hypoplasia-1, Bilateral
optic nerve hypoplasia, Cone-rod dystrophy-2, Leber congenital
amaurosis-7, Retinitis Pigmentosa 30, Stargardt disease-1,
Retinitis pigmentosa-19, Age-related macular degeneration-2,
Cone-rod dystrophy-3, Primary open angle glaucoma, Fuchs
endothelial corneal dystrophy-3, Macular dystrophy with central
cone involvement, Ocular nonnephropathic cystinosis, Leber
congenital amaurosis, Primary open angle glaucoma, Amyotrophic
lateral sclerosis 12, Bothnia retinal dystrophy, Fundus
albipunctatus, Retinitis punctata albescens, Leber congenital
amaurosis 2, Retinitis pigmentosa 20, Leber congenital amaurosis
14, Retinitis pigmentosa, Eye diseases with slow clearance or
accumulation of all-trans retinal (e.g. STGD1), Leber congenital
amaurosis13, Retinitis pigmentosa 44, Achromatopsia-2, Jet lag,
Alstrom syndrome, Attenuated MPS-1 (Hurler-scheie syndrome and
Scheie syndrome) or Bardet-biedl syndrome in a subject in need
thereof associated with a deficient protein or deficient functional
RNA, wherein the deficient protein or deficient functional RNA is
deficient in amount or activity in the subject, wherein the
antisense oligomer enhances constitutive splicing of a retained
intron-containing pre-mRNA (RIC pre-mRNA) encoding the target
protein or the functional RNA, wherein the target protein is: (a)
the deficient protein; or (b) a compensating protein which
functionally augments or replaces the deficient protein or in the
subject; and wherein the functional RNA is: (a) the deficient RNA;
or (b) a compensating functional RNA which functionally augments or
replaces the deficient functional RNA in the subject; wherein the
RIC pre-mRNA comprises a retained intron, an exon flanking the 5'
splice site and an exon flanking the 3' splice site, and wherein
the retained intron is spliced from the RIC pre-mRNA encoding the
target protein or the functional RNA, thereby increasing production
or activity of the target protein or the functional RNA in the
subject.
[0110] Disclosed herein, in some embodiments, are compositions
comprising an antisense oligomer for use in a method of treating a
condition associated with ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein in a
subject in need thereof, the method comprising the step of
increasing expression of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein by cells
of the subject, wherein the cells have a retained-intron-containing
pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon
flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and wherein the
RIC pre-mRNA encodes the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, the
method comprising contacting the cells with the antisense oligomer,
whereby the retained intron is constitutively spliced from the RIC
pre-mRNA transcripts encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, thereby
increasing the level of mRNA encoding the target protein or
functional RNA, and increasing the expression of ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein, in the cells of the subject. In some embodiments, the
target protein is ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA. In some embodiments,
the condition is a disease or disorder. In some embodiments, the
disease or disorder is Retinitis pigmentosa-7, Sveinsson
chorioretinal atrophy, Fundus Albipunctatus, Retinitis pigmentosa
37, Aniridia, Coloboma of the optic nerve, Ocular coloboma, Foveal
hypoplasia-1, Bilateral optic nerve hypoplasia, Cone-rod
dystrophy-2, Leber congenital amaurosis-7, Retinitis Pigmentosa 30,
Stargardt disease-1, Retinitis pigmentosa-19, Age-related macular
degeneration-2, Cone-rod dystrophy-3, Primary open angle glaucoma,
Fuchs endothelial corneal dystrophy-3, Macular dystrophy with
central cone involvement, Ocular nonnephropathic cystinosis, Leber
congenital amaurosis, Primary open angle glaucoma, Amyotrophic
lateral sclerosis 12, Bothnia retinal dystrophy, Fundus
albipunctatus, Retinitis punctata albescens, Leber congenital
amaurosis 2, Retinitis pigmentosa 20, Leber congenital amaurosis
14, Retinitis pigmentosa, Eye diseases with slow clearance or
accumulation of all-trans retinal (e.g. STGD1), Leber congenital
amaurosis13, Retinitis pigmentosa 44, Achromatopsia-2, Jet lag,
Alstrom syndrome, Attenuated MPS-1 (Hurler-scheie syndrome and
Scheie syndrome) or Bardet-biedl syndrome. In some embodiments, the
target protein and RIC pre-mRNA are encoded by the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA gene. In some embodiments, the antisense oligomer
targets a portion of the RIC pre-mRNA that is in the retained
intron within the region +6 relative to the 5' splice site of the
retained intron to -16 relative to the 3' splice site of the
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is in the retained intron within the region +69
relative to the 5' splice site of the retained intron to -79
relative to the 3' splice site of the retained intron. In some
embodiments, the target protein is (a) ABCA4, (b) RPE65, (c) MYOC,
(d) CNGA3, (e) MFSD8, (f) IDUA, (g) LRAT, (h) OPTN, (i) RGR, (j)
TEAD1, (k) PAX6, (l) ROM1, (m) RDH5, (n) RDH12, (o) NR2E3, (p)
RLBP1, (q) CTNS, (r) PER1, (s) FSCN2, (t) TCF4, (u) RDH8, (v)
NXNL1, or (w) CRX. In some embodiments, the targeted portion of the
RIC pre-mRNA comprises a sequence that is at least about 80%, 85%,
90%, 95%, 97%, or 100% complimentary to (a) any one of SEQ ID NOs
34873-35915, (b) any one of SEQ ID NOs 35916-36317, (c) any one of
SEQ ID NOs 36318-37107, (d) any one of SEQ ID NOs 37108-37559, (e)
any one of SEQ ID NOs 37560-38420, (f) any one of SEQ ID NOs
38421-39232, (g) any one of SEQ ID NOs 39233-41436, (h) any one of
SEQ ID NOs 41437-42368, (i) any one of SEQ ID NOs 42369-43747, (j)
any one of SEQ ID NOs 43748-43952, (k) any one of SEQ ID NOs
43953-49968, (l) any one of SEQ ID NOs 49969-50275, (m) any one of
SEQ ID NOs 50276-50991, (n) any one of SEQ ID NOs 50992-51247, (o)
any one of SEQ ID NOs 51248-52998, (p) any one of SEQ ID NOs
52999-53427, (q) any one of SEQ ID NOs 53428-54323, (r) any one of
SEQ ID NOs 54324-54634, (s) any one of SEQ ID NOs 54635-55638, (t)
any one of SEQ ID NOs 55639-59526, (u) any one of SEQ ID NOs
59527-59662, (v) any one of SEQ ID NOs 59663-60020, or (w) any one
of SEQ ID NOs 60021-61443. In some embodiments, the targeted
portion of the RIC pre-mRNA comprises a sequence with at least 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to a region
comprising at least 8 contiguous nucleic acids of (a) SEQ ID NO
61463, SEQ ID NO 61495, SEQ ID NO 26656, SEQ ID NO 61470, or SEQ ID
NO 61453, (b) SEQ ID NO 61480, or SEQ ID NO 61460, (c) SEQ ID NO
61458 or SEQ ID NO 61485, (d) SEQ ID NO 61500, (e) SEQ ID NO 61492,
or SEQ ID NO 61497, (f) SEQ ID NO 61457, SEQ ID NO 61468, SEQ ID NO
61489, SEQ ID NO 61444, or SEQ ID NO 61452, (g) SEQ ID NO 61474,
(h) SEQ ID NO 61503, (i) SEQ ID NO 61446, SEQ ID NO 61476, or SEQ
ID NO 61472, (j) SEQ ID NO 61461, (k) SEQ ID NO 61486, SEQ ID NO
61466, SEQ ID NO 61496, SEQ ID NO 61467, SEQ ID NO 61502, SEQ ID NO
61483, or SEQ ID NO 61448, (l) SEQ ID NO 61454, (m) SEQ ID NO
61493, SEQ ID NO 61455, SEQ ID NO 61498, or SEQ ID NO 61473, (n)
SEQ ID NO 61482, (o) SEQ ID NO 61491, SEQ ID NO 61449, SEQ ID NO
61494, SEQ ID NO 61487, SEQ ID NO 61447, SEQ ID NO 61465, SEQ ID NO
61501, or SEQ ID NO 61490, (p) SEQ ID NO 61462, or SEQ ID NO 61456,
(q) SEQ ID NO 61479 or SEQ ID NO 61481, (r) SEQ ID NO 61471 or SEQ
ID NO 61499, (s) SEQ ID NO 61459, SEQ ID NO 61451, or SEQ ID NO
61450, (t) SEQ ID NO 61477 or SEQ ID NO 61478, (u) SEQ ID NO 61469,
(v) SEQ ID NO 61488, or (w) SEQ ID NO 61484 or SEQ ID NO 61475. In
some embodiments, the ASO comprises a sequence with at least about
80%, 85%, 90%, 95%, 97%, or 100% sequence identity to (a) any one
of SEQ ID NOs 34873-35915, (b) any one of SEQ ID NOs 35916-36317,
(c) any one of SEQ ID NOs 36318-37107, (d) any one of SEQ ID NOs
37108-37559, (e) any one of SEQ ID NOs 37560-38420, (f) any one of
SEQ ID NOs 38421-39232, (g) any one of SEQ ID NOs 39233-41436, (h)
any one of SEQ ID NOs 41437-42368, (i) any one of SEQ ID NOs
42369-43747, (j) any one of SEQ ID NOs 43748-43952, (k) any one of
SEQ ID NOs 43953-49968, (l) any one of SEQ ID NOs 49969-50275, (m)
any one of SEQ ID NOs 50276-50991, (n) any one of SEQ ID NOs
50992-51247, (o) any one of SEQ ID NOs 51248-52998, (p) any one of
SEQ ID NOs 52999-53427, (q) any one of SEQ ID NOs 53428-54323, (r)
any one of SEQ ID NOs 54324-54634, (s) any one of SEQ ID NOs
54635-55638, (t) any one of SEQ ID NOs 55639-59526, (u) any one of
SEQ ID NOs 59527-59662, (v) any one of SEQ ID NOs 59663-60020, or
(w) any one of SEQ ID NOs 60021-61443. In some embodiments, the RIC
pre-mRNA comprises a sequence with at least about 80%, 85%, 90%,
95%, 97%, or 100% sequence identity to (a) SEQ ID NO 34813, (b) SEQ
ID NO 34814, (c) SEQ ID NO 34815, (d) SEQ ID NO 34816 or SEQ ID NO
34817, (e) SEQ ID NO 34818, (f) SEQ ID NO 34819 or SEQ ID NO 34820,
(g) SEQ ID NO 34821 or SEQ ID NO 34822, (h) SEQ ID NO 34823, SEQ ID
NO 34824, SEQ ID NO 34825, or SEQ ID NO 34826, (i) SEQ ID NO 34827,
SEQ ID NO 34828, or SEQ ID NO 34829, (j) SEQ ID NO 34830, (k) SEQ
ID NO 34831, SEQ ID NO 34832, SEQ ID NO 34833, SEQ ID NO 34834, SEQ
ID NO 34835, SEQ ID NO 34836, SEQ ID NO 34837, SEQ ID NO 34838, SEQ
ID NO 34839, SEQ ID NO 34840, or SEQ ID NO 34841, (l) SEQ ID NO
34842, (m) SEQ ID NO 34843 or SEQ ID NO 34844, (n) SEQ ID NO 34845,
(o) SEQ ID NO 34846 or SEQ ID NO 34847, (p) SEQ ID NO 34848, (q)
SEQ ID NO 34849 or SEQ ID NO 34850, (r) SEQ ID NO 34851, (s) SEQ ID
NO 34852 or SEQ ID NO 34853, (t) SEQ ID NO 34854, SEQ ID NO 34855,
SEQ ID NO 34856, SEQ ID NO 34857, SEQ ID NO 34858, SEQ ID NO 34859,
SEQ ID NO 34860, SEQ ID NO 34861, SEQ ID NO 34862, SEQ ID NO 34863,
SEQ ID NO 34864, SEQ ID NO 34865, SEQ ID NO 34866, SEQ ID NO 34867,
SEQ ID NO 34868, or SEQ ID NO 34869, (u) SEQ ID NO 34870, (v) SEQ
ID NO 34871, or (w) SEQ ID NO 34872. In some embodiments, the RIC
pre-mRNA is encoded by a genetic sequence with at least about 80%,
85%, 90%, 95%, 97%, or 100% sequence identity to (a) SEQ ID NO
34790, (b) SEQ ID NO 34791, (c) SEQ ID NO 34792, (d) SEQ ID NO
34793, (e) SEQ ID NO 34794, (f) SEQ ID NO 34795, (g) SEQ ID NO
34796, (h) SEQ ID NO 34797, (i) SEQ ID NO 34798, (j) SEQ ID NO
34799, (k) SEQ ID NO 34800, (l) SEQ ID NO 34801, (m) SEQ ID NO
34802, (n) SEQ ID NO 34803, (o) SEQ ID NO 34804, (p) SEQ ID NO
34805, (q) SEQ ID NO 34806, (r) SEQ ID NO 34807, (s) SEQ ID NO
34808, (t) SEQ ID NO 34809, (u) SEQ ID NO 34810, (v) SEQ ID NO
34811, (w) SEQ ID NO 34812. In some embodiments, the antisense
oligomer targets a portion of the RIC pre-mRNA that is in the
retained intron within: (a) the region +6 to +100 relative to the
5' splice site of the retained intron; or (b) the region -16 to
-100 relative to the 3' splice site of the retained intron. In some
embodiments, the antisense oligomer targets a portion of the RIC
pre-mRNA that is within the region about 100 nucleotides downstream
of the 5' splice site of the at least one retained intron, to about
100 nucleotides upstream of the 3' splice site of the at least one
retained intron. In some embodiments, the targeted portion of the
RIC pre-mRNA is within: (a) the region +2e to -4e in the exon
flanking the 5' splice site of the retained intron; or (b) the
region +2e to -4e in the exon flanking the 3' splice site of the
retained intron.
[0111] Disclosed herein, in some embodiments, the antisense
oligomer does not increase the amount of target protein or
functional RNA by modulating alternative splicing of the pre-mRNA
transcribed from a gene encoding the target protein or functional
RNA. In some embodiments, the antisense oligomer does not increase
the amount of the functional RNA or functional protein by
modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or functional RNA. In some embodiments,
the RIC pre-mRNA was produced by partial splicing from a
full-length pre-mRNA or a wild-type pre-mRNA. In some embodiments,
the mRNA encoding the target protein or functional RNA is a
full-length mature mRNA, or a wild-type mature mRNA. In some
embodiments, the target protein produced is full-length protein, or
wild-type protein. In some embodiments, the retained intron is a
rate-limiting intron. In some embodiments, the retained intron is
the most abundant retained intron in the RIC pre-mRNA. In some
embodiments, the retained intron is the second most abundant
retained intron in the RIC pre-mRNA.
[0112] In some embodiments of any of the aforementioned
compositions, the antisense oligomer comprises a backbone
modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. In some embodiments, the antisense
oligomer is an antisense oligonucleotide. In some embodiments, the
antisense oligomer comprises a phosphorodiamidate morpholino, a
locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a
2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the
antisense oligomer comprises at least one modified sugar moiety. In
some embodiments, each sugar moiety is a modified sugar moiety. In
some embodiments, the antisense oligomer consists of from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
the targeted portion of the RIC pre-mRNA encoding the protein. In
some embodiments, the antisense oligomer binds to a targeted
portion of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA.
[0113] Disclosed herein, in some embodiments, are pharmaceutical
compositions comprising the antisense oligomer of any of the
aforementioned compositions and an excipient.
[0114] Disclosed herein, in some embodiments, are methods of
treating a subject in need thereof by administering any of the
aforementioned pharmaceutical compositions by intravitreal
injection, subretinal injection, topical application, implantation,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0115] Disclosed herein, in some embodiments, are pharmaceutical
compositions comprising: an antisense oligomer that hybridizes to a
target sequence of a deficient ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA mRNA transcript,
wherein the deficient ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA mRNA transcript
comprises a retained intron, wherein the antisense oligomer induces
splicing out of the retained intron from the deficient ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA mRNA transcript; and a pharmaceutical acceptable
excipient. In some embodiments, the deficient ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA RIC pre-mRNA transcript. In some embodiments, the targeted
portion of the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA transcript is
in the retained intron within the region +500 relative to the 5'
splice site of the retained intron to -500 relative to the 3'
spliced site of the retained intron. In some embodiments, the ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA RIC pre-mRNA transcript is encoded by a genetic
sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%
or 100% sequence identity to any one of SEQ ID NOs: 34790-34812. In
some embodiments, the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA
transcript comprises a sequence with at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ
ID NOs: 34813-34872. In some embodiments, the antisense oligomer
comprises a backbone modification comprising a phosphorothioate
linkage or a phosphorodiamidate linkage. In some embodiments, the
antisense oligomer is an antisense oligonucleotide. In some
embodiments, the antisense oligomer comprises a phosphorodiamidate
morpholino, a locked nucleic acid, a peptide nucleic acid, a
2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety. In some
embodiments, the antisense oligomer comprises at least one modified
sugar moiety. In some embodiments, the antisense oligomer comprises
from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases,
8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to
15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases. In some embodiments, the
antisense oligomer is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or is 100% complementary to
a targeted portion of the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA
transcript. In some embodiments, the targeted portion of the ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA RIC pre-mRNA transcript is within a sequence
selected from SEQ ID NOs: 61443-61503. In some embodiments, the
antisense oligomer comprises a nucleotide sequence that is at least
about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to any one of SEQ ID NOs: 34873-61443. In some
embodiments, the antisense oligomer comprises a nucleotide sequence
selected from SEQ ID NOs: 34873-61443. In some embodiments, the
pharmaceutical composition is formulated for intrathecal injection,
intracerebroventricular injection, intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection.
[0116] Disclosed herein, in some embodiments, are methods of
inducing processing of a deficient ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA mRNA
transcript to facilitate removal of a retained intron to produce a
fully processed ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA mRNA transcript that encodes
a functional form of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, the method
comprising: (a) contacting an antisense oligomer to a target cell
of a subject; (b) hybridizing the antisense oligomer to the
deficient ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA mRNA transcript, wherein the
deficient ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA mRNA transcript is capable of
encoding the functional form of a ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein
and comprises at least one retained intron; (c) removing the at
least one retained intron from the deficient ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA mRNA transcript to produce the fully processed ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA mRNA transcript that encodes the functional form of
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein; and (d) translating the
functional form of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein from the fully
processed ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA mRNA transcript. In some
embodiments, the retained intron is an entire retained intron. In
some embodiments, the deficient ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA mRNA
transcript is a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA transcript.
[0117] Disclosed herein, in some embodiments, are methods of
treating a subject having a condition caused by a deficient amount
or activity of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein comprising
administering to the subject an antisense oligomer comprising a
nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one
of SEQ ID NOs: 34873-61443.
[0118] In one aspect, provided herein is a method of treating a
liver disease in a subject in need thereof by increasing the
expression of a target protein or functional RNA by cells of the
subject, wherein the cells have a retained-intron-containing
pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained
intron, an exon flanking the 5' splice site, an exon flanking the
3' splice site, and wherein the RIC pre-mRNA encodes the target
protein or functional RNA, the method comprising contacting the
cells of the subject with an antisense oligomer (ASO) complementary
to a targeted portion of the RIC pre-mRNA encoding the target
protein or functional RNA, whereby the retained intron is
constitutively spliced from the RIC pre-mRNA encoding the target
protein or functional RNA, thereby increasing the level of mRNA
encoding the target protein or functional RNA, and increasing the
expression of the target protein or functional RNA in the cells of
the subject.
[0119] In some embodiments, the liver disease is glycine
encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine
Deaminase deficiency, porphyria variegate, porphyria cutanea tarda,
acute intermittent porphyria, very long chain acyl-CoA
dehydrogenase deficiency, pyruvate carboxylase deficiency,
isovaleric academia, hyperchylomicronemia, hypertriglyceridemia,
galactosemia, hypercholesterolemia, maturity-onset diabetes of the
young type 1, maturity-onset diabetes of the young type 2,
maturity-onset diabetes of the young type 3, noninsulin-dependent
diabetes mellitus, insulin-dependent diabetes mellitus 1,
insulin-dependent diabetes mellitus 20, Falconi renotubular
syndrome 4 with maturity-onset diabetes of the young,
hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes
mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome,
immunodeficiency 36, agammaglobulinemia 7, lipid metabolism
deficiency, liver inflammation, hemochromatosis type 2B,
thrombocytopenia, non-alcoholic fatty liver disease, Wilson
disease, tyrosinemia type I, argininosuccinate lyase deficiency,
hemochromatosis type I, Alstrom syndrome, congenital bile acid
synthesis defect 1, steatohepatitis, insulin resistance, glucose
intolerance, type II diabetes or liver cancer.
[0120] In one aspect, provided herein is a method of increasing
expression of a target protein by cells having a
retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC
pre-mRNA comprising a retained intron, an exon flanking the 5'
splice site of the retained intron, an exon flanking the 3' splice
site of the retained intron, and wherein the RIC pre-mRNA encodes
the target protein, the method comprising contacting the cells with
an antisense oligomer (ASO) complementary to a targeted portion of
the RIC pre-mRNA encoding the target protein, whereby the retained
intron is constitutively spliced from the RIC pre-mRNA encoding the
target protein, thereby increasing the level of mRNA encoding the
target protein, and increasing the expression of the target protein
in the cells, wherein the target protein is aminomethyltransferase,
adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen
decarboxylase, hydroxymethylbilane synthase, very long chain
acyl-CoA dehydrogenase, pyruvate carboxylase isovaleryl-CoA
dehydrogenase, apolipoprotein A-V, galactose-1-phosphate
uridylyltransferase, low density lipoprotein receptor adaptor
protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic
nuclear factor-1-alpha albumin proximal factor,
O-glucosyltransferase 1, phosphatidylinositol 3-kinase regulatory
subunit 1, Tribbles-1, transforming growth factor beta-1,
hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase
domain-containing protein 3, copper-transporting ATPase 2,
fumarylacetoacetase, argininosuccinate lyase, hereditary
hemochromatosis protein, alstrom syndrome protein 1, 3
beta-hydroxysteroid dehydrogenase type 7, peroxisome proliferator
activated receptor delta, interleukin 6, ceramide synthase 2 or
nuclear receptor coactivator 5.
[0121] In some embodiments, the target protein is
aminomethyltransferase, adenosine deaminase, protoporphyrinogen
oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane
synthase, very long chain acyl-CoA dehydrogenase, pyruvate
carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V,
galactose-1-phosphate uridylyltransferase, low density lipoprotein
receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha,
glucokinase, hepatic nuclear factor-1-alpha albumin proximal
factor, protein O-glucosyltransferase 1, phosphatidylinositol
3-kinase regulatory subunit 1, Tribbles-1, transforming growth
factor beta-1, hemochromatosis type 2B, thrombopoietin,
patatin-like phospholipase domain-containing protein 3,
copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5.
[0122] In some embodiments, the target protein or the functional
RNA is a compensating protein or a compensating functional RNA that
functionally augments or replaces a target protein or functional
RNA that is deficient in amount or activity in the subject.
[0123] In some embodiments, the cells are in or from a subject
having a condition caused by a deficient amount or activity of the
target protein.
[0124] In some embodiments, the deficient amount of the target
protein is caused by haploinsufficiency of the target protein,
wherein the subject has a first allele encoding a functional target
protein, and a second allele from which the target protein is not
produced, or a second allele encoding a nonfunctional target
protein, and wherein the antisense oligomer binds to a targeted
portion of a RIC pre-mRNA transcribed from the first allele.
[0125] In some embodiments, the subject has a condition caused by a
disorder resulting from a deficiency in the amount or function of
the target protein, wherein the subject has a first mutant allele
from which the target protein is produced at a reduced level
compared to production from a wild-type allele, the target protein
is produced in a form having reduced function compared to an
equivalent wild-type protein, or the target protein is not
produced, and a second mutant allele from which the target protein
is produced at a reduced level compared to production from a
wild-type allele, the target protein is produced in a form having
reduced function compared to an equivalent wild-type protein, or
the target protein is not produced, and wherein when the subject
has a first mutant allele a(iii), the second mutant allele is b(i)
or b(ii), and wherein when the subject has a second mutant allele
b(iii), the first mutant allele is a(i) or a(ii), and wherein the
RIC pre-mRNA is transcribed from either the first mutant allele
that is a(i) or a(ii), and/or the second allele that is b(i) or
b(ii)
[0126] In some embodiments, the target protein is produced in a
form having reduced function compared to the equivalent wild-type
protein.
[0127] In some embodiments, the target protein is produced in a
form that is fully-functional compared to the equivalent wild-type
protein.
[0128] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +6 relative to
the 5' splice site of the retained intron to -16 relative to the 3'
splice site of the retained intron.
[0129] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +69 relative
to the 5' splice site of the retained intron to -79 relative to the
3' splice site of the retained intron.
[0130] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within: the region +6 to +100
relative to the 5' splice site of the retained intron; or the
region -16 to -100 relative to the 3' splice site of the retained
intron.
[0131] In some embodiments, the targeted portion of the RIC
pre-mRNA is within: the region +2e to -4e in the exon flanking the
5' splice site of the retained intron; or the region +2e to -4e in
the exon flanking the 3' splice site of the retained intron.
[0132] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is within the region about 500
nucleotides downstream of the 5' splice site of the at least one
retained intron, to about 500 nucleotides upstream of the 3' splice
site of the at least one retained intron.
[0133] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is within the region about 100
nucleotides downstream of the 5' splice site of the at least one
retained intron, to about 100 nucleotides upstream of the 3' splice
site of the at least one retained intron.
[0134] In some embodiments, the target protein is (a) AMT, (b) ADA,
(c) PPOX, (d) UROD, (e) HMBS, (f) ACADVL, (g) PC, (h) IVD, (i)
APOA5, (j) GALT, (k) LDLRAP1, (l) HNF4A, (m) GCK, (n) POGLUT1, (o)
PIK3R1, (p) HNF1A, (q) TRIB1, (r) TGFB1, (s) HAMP, (t) THPO, (u)
PNPLA3, (v) ATP7B, (w) FAH, (x) ASL, (y) HFE, (z) ALMS1, (aa)
PPARD, (bb) IL6, (cc) HSD3B7, (dd) CERS2, or (ee) NCOA5.
[0135] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 97%, or 100% complimentary to (a) any one of SEQ ID NOs
64316-65413, (b) any one of SEQ ID NOs 127350-128784, (c) any one
of SEQ ID NOs 63403-64102, (d) any one of SEQ ID NOs 62429-63282,
(e) any one of SEQ ID NOs 98986-99547, (f) any one of SEQ ID NOs
110926-111472, (g) any one of SEQ ID NOs 97403-97795, (h) any one
of SEQ ID NOs 106129-108516, (i) any one of SEQ ID NOs 97796-98985,
(j) any one of SEQ ID NOs 96024-97402, (k) any one of SEQ ID NOs
61634-62428, (l) any one of SEQ ID NOs 120461-127349 or
129035-138877, (m) any one of SEQ ID NOs 86561-92479, (n) any one
of SEQ ID NOs 65414-66767, (o) any one of SEQ ID NOs 75976-76379,
(p) any one of SEQ ID NOs 99548-103608, (q) any one of SEQ ID NOs
93972-96023, (r) any one of SEQ ID NOs 113475-113528, (s) any one
of SEQ ID NOs 113173-113474, (t) any one of SEQ ID NOs 66768-75975,
(u) any one of SEQ ID NOs 138878-139851, (v) any one of SEQ ID NOs
103609-105873, (w) any one of SEQ ID NOs 105874-106128, (x) any one
of SEQ ID NOs 92480-93971, (y) any one of SEQ ID NOs 83313-84988,
(z) any one of SEQ ID NOs 64103-64315, (aa) any one of SEQ ID NOs
76380-83312, (bb) any one of SEQ ID NOs 84989-86560, (cc) any one
of SEQ ID NOs 108517-110925, (dd) any one of SEQ ID NOs
63283-63402, or any one of SEQ ID NOs 128785-129034.
[0136] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
97%, or 100% sequence identity to a region comprising at least 8
contiguous nucleic acids of (a) SEQ ID NO 139986, 139968 or 139937;
(b) SEQ ID NO 139888, 139985 or 139872; (c) SEQ ID NO 139964,
139976, 139983 or 139924; (d) SEQ ID NO 139913, 139889, 139914,
139963 or 139966; (e) SEQ ID NO 139858, 139970 or 139957; (0 SEQ ID
NO 139870, 139879, 139943, 139951, 139980, 139988, 139999, 139925
or 139915; (g) SEQ ID NO 139998, (h) SEQ ID NO 139967, 139878 or
139883; (i) SEQ ID NO 139910, 140002, 139923, 139875 or 139900; (j)
SEQ ID NO 139992, 139919, 139919, 139855, 139938, 139996, 139926,
139940 or 139952; (k) SEQ ID NO 139857 or 139962; (l) SEQ ID NO
139944, 139895, 139959, 139931, 139994, 140004, 139863, 139932,
139861, 139867, 139978, 139894, 139982, 139904, 139876 or 139953;
(m) SEQ ID NO 139948, 139984, 139882, 139934, 139972, 139911,
139880, 139920, 139890, 139958, 139987 or 139873; (n) SEQ ID NO
61503, 139853, 139935, 139942 or 139892; (o) SEQ ID NO 139893 or
SEQ ID NO 139921, (p) SEQ ID NO 139965, 139971, 139956, 139864,
139866, 139936, 139941, 139933, 139991, 139908, 139995 or 139930;
(q) SEQ ID NO 139990, 139989 or 139989; (r) SEQ ID NO 139961, (s)
SEQ ID NO 140000 or 139929; (t) SEQ ID NO 139928, 139903, 139896,
139854, 139884, 139869, 139960, 139946, 139865 or 139949; (u) SEQ
ID NO 139891, 139905, 139974 or 139859; (v) SEQ ID NO 139930,
139886, 139947 or 139897; (w) SEQ ID NO 139885; (x) SEQ ID NO
139997, 139907, 139874, 139868 or 139856; (y) SEQ ID NO 139922,
139887, 140003, 139927 or 139969; (z) SEQ ID NO 139902, (aa) SEQ ID
NO 139917, 139981, 139975, 139862, 139898, 139860, 139877 or
139993; (bb) SEQ ID NO 139939, 139916, 139918, or 139906; (cc) SEQ
ID NO 139852, 139973, 140001, 139909, 139945, 139954, 139899,
139912 or 139881; (dd) SEQ ID NO 139950; or SEQ ID NO 139955.
[0137] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to
(a) any one of SEQ ID NOs 64316-65413, (b) any one of SEQ ID NOs
127350-128784, (c) any one of SEQ ID NOs 63403-64102, (d) any one
of SEQ ID NOs 62429-63282, (e) any one of SEQ ID NOs 98986-99547,
(f) any one of SEQ ID NOs 110926-111472, (g) any one of SEQ ID NOs
97403-97795, (h) any one of SEQ ID NOs 106129-108516, (i) any one
of SEQ ID NOs 97796-98985, (j) any one of SEQ ID NOs 96024-97402,
(k) any one of SEQ ID NOs 61634-62428, (l) any one of SEQ ID NOs
120461-127349 or 129035-138877, (m) any one of SEQ ID NOs
86561-92479, (n) any one of SEQ ID NOs 65414-66767, (o) any one of
SEQ ID NOs 75976-76379, (p) any one of SEQ ID NOs 99548-103608, (q)
any one of SEQ ID NOs 93972-96023, (r) any one of SEQ ID NOs
113475-113528, (s) any one of SEQ ID NOs 113173-113474, (t) any one
of SEQ ID NOs 66768-75975, (u) any one of SEQ ID NOs 138878-139851,
(v) any one of SEQ ID NOs 103609-105873, (w) any one of SEQ ID NOs
105874-106128, (x) any one of SEQ ID NOs 92480-93971, (y) any one
of SEQ ID NOs 83313-84988, (z) any one of SEQ ID NOs 64103-64315,
(aa) any one of SEQ ID NOs 76380-83312, (bb) any one of SEQ ID NOs
84989-86560, (cc) any one of SEQ ID NOs 108517-110925, (dd) any one
of SEQ ID NOs 63283-63402, or any one of SEQ ID NOs
128785-129034.
[0138] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of (a) SEQ ID NOs 61544-61547, (b) SEQ
ID NOs 61531, 61624-61627, (c) SEQ ID NOs 61540 or 61541, (d) SEQ
ID NOs 61536 or 61537, (e) SEQ ID NOs 61595-61598, (f) SEQ ID NOs
61612-61615, (g) SEQ ID NOs 61590-61592, (h) SEQ ID NOs 61607 or
61608, (i) SEQ ID NOs 61593 or 61594, (j) SEQ ID NOs 61588 or
61589, (k) SEQ ID NO 61535, (l) SEQ ID NOs 61618-61623 or
61629-61632, (m) SEQ ID NOs 61579-61581, (n) SEQ ID NOs 61548 or
61549, (o) SEQ ID NOs 61560-61563, (p) SEQ ID NOs 61599 or 61600,
(q) SEQ ID NOs 61586 or 61587, (r) SEQ ID NO 61617, (s) SEQ ID NO
61726, (t) SEQ ID NOs 61550-61559, (u) SEQ ID NO 61633, (v) SEQ ID
NOs 61601-61605, (w) SEQ ID NO 61606, (x) SEQ ID NOs 61583-61585,
(y) SEQ ID NOs 61569-61576, (z) SEQ ID NO 61542, (aa) SEQ ID NOs
61564-61568, (bb) SEQ ID NOs 61577 or 61578, (cc) SEQ ID NOs
61609-61611, (dd) SEQ ID NOs 61538 or 61539, or SEQ ID NO
61628.
[0139] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to (a) SEQ ID NO 61509, (b) SEQ
ID NO 61531, (c) SEQ ID NO 61507, (d) SEQ ID NO 61505, (e) SEQ ID
NO 61522, (f) SEQ ID NO 61528, (g) SEQ ID NO 61520, (h) SEQ ID NO
61526, (i) SEQ ID NO 61521, (j) SEQ ID NO 61519, (k) SEQ ID NO
61504, (l) SEQ ID NO 61533, (m) SEQ ID NO 61516, (n) SEQ ID NO
61510, (o) SEQ ID NO 61512, (p) SEQ ID NO 61523, (q) SEQ ID NO
61518, (r) SEQ ID NO 61530, (s) SEQ ID NO 61529, (t) SEQ ID NO
61511, (u) SEQ ID NO 61534, (v) SEQ ID NO 61524, (w) SEQ ID NO
61525, (x) SEQ ID NO 61517, (y) SEQ ID NO 61514, (z) SEQ ID NO
61508, (aa) SEQ ID NO 61513, (bb) SEQ ID NO 61515, (cc) SEQ ID NO
61527, (dd) SEQ ID NO 61506, or (ee) SEQ ID NO 61532.
[0140] In some embodiments, the antisense oligomer does not
increase the amount of the target protein or the functional RNA by
modulating alternative splicing of pre-mRNA transcribed from a gene
encoding the functional RNA or target protein.
[0141] In some embodiments, the antisense oligomer does not
increase the amount of the target protein or the functional RNA by
modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or the functional RNA.
[0142] In some embodiments, the RIC pre-mRNA was produced by
partial splicing of a full-length pre-mRNA or partial splicing of a
wild-type pre-mRNA.
[0143] In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA.
[0144] In some embodiments, the target protein produced is
full-length protein, or wild-type protein.
[0145] In some embodiments, the total amount of the mRNA encoding
the target protein or functional RNA produced in the cell contacted
with the antisense oligomer is increased about 1.1 to about
10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold,
about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to
about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold,
about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to
about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold,
about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about
6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3
to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold,
about 4 to about 9-fold, at least about 1.1-fold, at least about
1.5-fold, at least about 2-fold, at least about 2.5-fold, at least
about 3-fold, at least about 3.5-fold, at least about 4-fold, at
least about 5-fold, or at least about 10-fold, compared to the
total amount of the mRNA encoding the target protein or functional
RNA produced in a control cell.
[0146] In some embodiments, the total amount of target protein
produced by the cell contacted with the antisense oligomer is
increased about 1.1 to about 10-fold, about 1.5 to about 10-fold,
about 2 to about 10-fold, about 3 to about 10-fold, about 4 to
about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the total amount of target protein
produced by a control cell.
[0147] In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage.
[0148] In some embodiments, the antisense oligomer comprises a
phosphorodiamidate morpholino, a locked nucleic acid, a peptide
nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxy ethyl
moiety.
[0149] In some embodiments, the antisense oligomer comprises at
least one modified sugar moiety.
[0150] In some embodiments, each sugar moiety is a modified sugar
moiety.
[0151] In some embodiments, the antisense oligomer consists of from
8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to
30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases.
[0152] In some embodiments, the antisense oligomer is at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or 100%, complementary to the targeted portion of the RIC
pre-mRNA encoding the protein.
[0153] In some embodiments, the cell comprises a population of RIC
pre-mRNAs transcribed from the gene encoding the target protein or
functional RNA, wherein the population of RIC pre-mRNAs comprises
two or more retained introns, and wherein the antisense oligomer
binds to the most abundant retained intron in the population of RIC
pre-mRNAs.
[0154] In some embodiments, the binding of the antisense oligomer
to the most abundant retained intron induces splicing out of the
two or more retained introns from the population of RIC pre-mRNAs
to produce mRNA encoding the target protein or functional RNA.
[0155] In some embodiments, the cell comprises a population of RIC
pre-mRNAs transcribed from the gene encoding the target protein or
functional RNA, wherein the population of RIC pre-mRNAs comprises
two or more retained introns, and wherein the antisense oligomer
binds to the second most abundant retained intron in the population
of RIC pre-mRNAs.
[0156] In some embodiments, the binding of the antisense oligomer
to the second most abundant retained intron induces splicing out of
the two or more retained introns from the population of RIC
pre-mRNAs to produce mRNA encoding the target protein or functional
RNA.
[0157] In some embodiments, the condition is a disease or
disorder.
[0158] In some embodiments, the disease or disorder is a liver
disease.
[0159] In some embodiments, the liver disease is glycine
encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine
Deaminase deficiency, porphyria variegate, porphyria cutanea tarda,
acute intermittent porphyria, very long chain acyl-CoA
dehydrogenase deficiency, pyruvate carboxylase deficiency,
isovaleric academia, hyperchylomicronemia, hypertriglyceridemia,
galactosemia, hypercholesterolemia, maturity-onset diabetes of the
young type 1, maturity-onset diabetes of the young type 2,
maturity-onset diabetes of the young type 3, noninsulin-dependent
diabetes mellitus, insulin-dependent diabetes mellitus 1,
insulin-dependent diabetes mellitus 20, Falconi renotubular
syndrome 4 with maturity-onset diabetes of the young,
hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes
mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome,
immunodeficiency 36, agammaglobulinemia 7, lipid metabolism
deficiency, liver inflammation, hemochromatosis type 2B,
thrombocytopenia, non-alcoholic fatty liver disease, Wilson
disease, tyrosinemia type I, argininosuccinate lyase deficiency,
hemochromatosis type I, Alstrom syndrome, congenital bile acid
synthesis defect 1, steatohepatitis, insulin resistance, glucose
intolerance, type II diabetes or liver cancer.
[0160] In some embodiments, the target protein and the RIC pre-mRNA
are encoded by a gene, wherein the gene is AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5.
[0161] In some embodiments, the method further comprises assessing
protein expression.
[0162] In some embodiments, the subject is a human.
[0163] In some embodiments, the subject is a non-human animal.
[0164] In some embodiments, the subject is a fetus, an embryo, or a
child.
[0165] In some embodiments, the cells are ex vivo.
[0166] In some embodiments, the antisense oligomer is administered
by intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection of the subject.
[0167] In some embodiments, the 9 nucleotides at -3e to -1e of the
exon flanking the 5' splice site and +1 to +6 of the retained
intron, are identical to the corresponding wild-type sequence.
[0168] In some embodiments, the 16 nucleotides at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site are
identical to the corresponding wild-type sequence.
[0169] In one aspect, provided herein is an antisense oligomer as
used in a method described herein.
[0170] In one aspect, provided herein is an antisense oligomer
comprising a sequence with at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs
61634-139851.
[0171] In one aspect, provided herein is a pharmaceutical
composition comprising an antisense oligomer described herein and
an excipient.
[0172] In one aspect, provided herein is a method of treating a
subject in need thereof by administering a pharmaceutical
composition described herein by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection.
[0173] In one aspect, provided herein is a composition comprising
an antisense oligomer for use in a method of increasing expression
of a target protein or a functional RNA by cells to treat a liver
disease in a subject in need thereof associated with a deficient
protein or deficient functional RNA, wherein the deficient protein
or deficient functional RNA is deficient in amount or activity in
the subject, wherein the antisense oligomer enhances constitutive
splicing of a retained intron-containing pre-mRNA (RIC pre-mRNA)
encoding the target protein or the functional RNA, wherein the
target protein is: the deficient protein; or a compensating protein
which functionally augments or replaces the deficient protein or in
the subject; and wherein the functional RNA is: the deficient RNA;
or a compensating functional RNA which functionally augments or
replaces the deficient functional RNA in the subject; wherein the
RIC pre-mRNA comprises a retained intron, an exon flanking the 5'
splice site and an exon flanking the 3' splice site, and wherein
the retained intron is spliced from the RIC pre-mRNA encoding the
target protein or the functional RNA, thereby increasing production
or activity of the target protein or the functional RNA in the
subject.
[0174] In some embodiments, the liver disease is glycine
encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine
Deaminase deficiency, porphyria variegate, porphyria cutanea tarda,
acute intermittent porphyria, very long chain acyl-CoA
dehydrogenase deficiency, pyruvate carboxylase deficiency,
isovaleric academia, hyperchylomicronemia, hypertriglyceridemia,
galactosemia, hypercholesterolemia, maturity-onset diabetes of the
young type 1, maturity-onset diabetes of the young type 2,
maturity-onset diabetes of the young type 3, noninsulin-dependent
diabetes mellitus, insulin-dependent diabetes mellitus 1,
insulin-dependent diabetes mellitus 20, Falconi renotubular
syndrome 4 with maturity-onset diabetes of the young,
hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes
mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome,
immunodeficiency 36, agammaglobulinemia 7, lipid metabolism
deficiency, liver inflammation, hemochromatosis type 2B,
thrombocytopenia, non-alcoholic fatty liver disease, Wilson
disease, tyrosinemia type I, argininosuccinate lyase deficiency,
hemochromatosis type I, Alstrom syndrome, congenital bile acid
synthesis defect 1, steatohepatitis, insulin resistance, glucose
intolerance, type II diabetes or liver cancer.
[0175] In one aspect, provided herein is a composition comprising
an antisense oligomer for use in a method of treating a condition
associated with a target protein in a subject in need thereof, the
method comprising the step of increasing expression of the target
protein by cells of the subject, wherein the cells have a
retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a
retained intron, an exon flanking the 5' splice site of the
retained intron, an exon flanking the 3' splice site of the
retained intron, and wherein the RIC pre-mRNA encodes the target
protein, the method comprising contacting the cells with the
antisense oligomer, whereby the retained intron is constitutively
spliced from the RIC pre-mRNA transcripts encoding the target
protein, thereby increasing the level of mRNA encoding the target
protein or functional RNA, and increasing the expression of the
target protein, in the cells of the subject.
[0176] In some embodiments, the target protein is
aminomethyltransferase, adenosine deaminase, protoporphyrinogen
oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane
synthase, very long chain acyl-CoA dehydrogenase, pyruvate
carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V,
galactose-1-phosphate uridylyltransferase, low density lipoprotein
receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha,
glucokinase, hepatic nuclear factor-1-alpha albumin proximal
factor, protein O-glucosyltransferase 1, phosphatidylinositol
3-kinase regulatory subunit 1, Tribbles-1, transforming growth
factor beta-1, hemochromatosis type 2B, thrombopoietin,
patatin-like phospholipase domain-containing protein 3,
copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5.
[0177] In some embodiments, the condition is a disease or
disorder.
[0178] In some embodiments, the disease or disorder is a liver
disease.
[0179] In some embodiments, the liver disease is glycine
encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine
Deaminase deficiency, porphyria variegate, porphyria cutanea tarda,
acute intermittent porphyria, very long chain acyl-CoA
dehydrogenase deficiency, pyruvate carboxylase deficiency,
isovaleric academia, hyperchylomicronemia, hypertriglyceridemia,
galactosemia, hypercholesterolemia, maturity-onset diabetes of the
young type 1, maturity-onset diabetes of the young type 2,
maturity-onset diabetes of the young type 3, noninsulin-dependent
diabetes mellitus, insulin-dependent diabetes mellitus 1,
insulin-dependent diabetes mellitus 20, Falconi renotubular
syndrome 4 with maturity-onset diabetes of the young,
hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes
mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome,
immunodeficiency 36, agammaglobulinemia 7, lipid metabolism
deficiency, liver inflammation, hemochromatosis type 2B,
thrombocytopenia, non-alcoholic fatty liver disease, Wilson
disease, tyrosinemia type I, argininosuccinate lyase deficiency,
hemochromatosis type I, Alstrom syndrome, congenital bile acid
synthesis defect 1, steatohepatitis, insulin resistance, glucose
intolerance, type II diabetes or liver cancer.
[0180] In some embodiments, the target protein and RIC pre-mRNA are
encoded by a gene, wherein the gene is AMT, ADA, PPOX, UROD, HMBS,
ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1,
HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE,
ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5.
[0181] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is in the retained intron within
the region +6 relative to the 5' splice site of the retained intron
to -16 relative to the 3' splice site of the retained intron.
[0182] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is in the retained intron within:
the region +6 to +100 relative to the 5' splice site of the
retained intron; or the region -16 to -100 relative to the 3'
splice site of the retained intron.
[0183] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is within the region about 500
nucleotides downstream of the 5' splice site of the at least one
retained intron, to about 500 nucleotides upstream of the 3' splice
site of the at least one retained intron
[0184] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is within the region about 100
nucleotides downstream of the 5' splice site of the at least one
retained intron, to about 100 nucleotides upstream of the 3' splice
site of the at least one retained intron.
[0185] In some embodiments, the targeted portion of the RIC
pre-mRNA is within: the region +2e to -4e in the exon flanking the
5' splice site of the retained intron; or the region +2e to -4e in
the exon flanking the 3' splice site of the retained intron.
[0186] In some embodiments, the target protein is (a) AMT, (b) ADA,
(c) PPOX, (d) UROD, (e) HMBS, (f) ACADVL, (g) PC, (h) IVD, (i)
APOA5, (j) GALT, (k) LDLRAP1, (l) HNF4A, (m) GCK, (n) POGLUT1, (o)
PIK3R1, (p) HNF1A, (q) TRIB1, (r) TGFB1, (s) HAMP, (t) THPO, (u)
PNPLA3, (v) ATP7B, (w) FAH, (x) ASL, (y) HFE, (z) ALMS1, (aa)
PPARD, (bb) IL6, (cc) HSD3B7, (dd) CERS2, or (ee) NCOA5.
[0187] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to (a) any one of SEQ
ID NOs 64316-65413, (b) any one of SEQ ID NOs 127350-128784, (c)
any one of SEQ ID NOs 63403-64102, (d) any one of SEQ ID NOs
62429-63282, (e) any one of SEQ ID NOs 98986-99547, (f) any one of
SEQ ID NOs 110926-111472, (g) any one of SEQ ID NOs 97403-97795,
(h) any one of SEQ ID NOs 106129-108516, (i) any one of SEQ ID NOs
97796-98985, (j) any one of SEQ ID NOs 96024-97402, (k) any one of
SEQ ID NOs 61634-62428, (l) any one of SEQ ID NOs 120461-127349 or
129035-138877, (m) any one of SEQ ID NOs 86561-92479, (n) any one
of SEQ ID NOs 65414-66767, (o) any one of SEQ ID NOs 75976-76379,
(p) any one of SEQ ID NOs 99548-103608, (q) any one of SEQ ID NOs
93972-96023, (r) any one of SEQ ID NOs 113475-113528, (s) any one
of SEQ ID NOs 113173-113474, (t) any one of SEQ ID NOs 66768-75975,
(u) any one of SEQ ID NOs 138878-139851, (v) any one of SEQ ID NOs
103609-105873, (w) any one of SEQ ID NOs 105874-106128, (x) any one
of SEQ ID NOs 92480-93971, (y) any one of SEQ ID NOs 83313-84988,
(z) any one of SEQ ID NOs 64103-64315, (aa) any one of SEQ ID NOs
76380-83312, (bb) any one of SEQ ID NOs 84989-86560, (cc) any one
of SEQ ID NOs 108517-110925, (dd) any one of SEQ ID NOs
63283-63402, or any one of SEQ ID NOs 128785-129034.
[0188] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of (a) SEQ ID NO 139986, 139968
or 139937; (b) SEQ ID NO 139888, 139985 or 139872; (c) SEQ ID NO
139964, 139976, 139983 or 139924; (d) SEQ ID NO 139913, 139889,
139914, 139963 or 139966; (e) SEQ ID NO 139858, 139970 or 139957;
(f) SEQ ID NO 139870, 139879, 139943, 139951, 139980, 139988,
139999, 139925 or 139915; (g) SEQ ID NO 139998, (h) SEQ ID NO
139967, 139878 or 139883; (i) SEQ ID NO 139910, 140002, 139923,
139875 or 139900; (j) SEQ ID NO 139992, 139919, 139919, 139855,
139938, 139996, 139926, 139940 or 139952; (k) SEQ ID NO 139857 or
139962; (l) SEQ ID NO 139944, 139895, 139959, 139931, 139994,
140004, 139863, 139932, 139861, 139867, 139978, 139894, 139982,
139904, 139876 or 139953; (m) SEQ ID NO 139948, 139984, 139882,
139934, 139972, 139911, 139880, 139920, 139890, 139958, 139987 or
139873; (n) SEQ ID NO 61503, 139853, 139935, 139942 or 139892; (o)
SEQ ID NO 139893 or SEQ ID NO 139921, (p) SEQ ID NO 139965, 139971,
139956, 139864, 139866, 139936, 139941, 139933, 139991, 139908,
139995 or 139930; (q) SEQ ID NO 139990, 139989 or 139989; (r) SEQ
ID NO 139961, (s) SEQ ID NO 140000 or 139929; (t) SEQ ID NO 139928,
139903, 139896, 139854, 139884, 139869, 139960, 139946, 139865 or
139949; (u) SEQ ID NO 139891, 139905, 139974 or 139859; (v) SEQ ID
NO 139930, 139886, 139947 or 139897; (w) SEQ ID NO 139885; (x) SEQ
ID NO 139997, 139907, 139874, 139868 or 139856; (y) SEQ ID NO
139922, 139887, 140003, 139927 or 139969; (z) SEQ ID NO 139902,
(aa) SEQ ID NO 139917, 139981, 139975, 139862, 139898, 139860,
139877 or 139993; (bb) SEQ ID NO 139939, 139916, 139918, 139901 or
139906; (cc) SEQ ID NO 139852, 139973, 140001, 139909, 139945,
139954, 139899, 139912 or 139881; (dd) SEQ ID NO 139950; or SEQ ID
NO 139955.
[0189] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to (a) any one of SEQ ID NOs 64316-65413, (b) any one of
SEQ ID NOs 127350-128784, (c) any one of SEQ ID NOs 63403-64102,
(d) any one of SEQ ID NOs 62429-63282, (e) any one of SEQ ID NOs
98986-99547, (f) any one of SEQ ID NOs 110926-111472, (g) any one
of SEQ ID NOs 97403-97795, (h) any one of SEQ ID NOs 106129-108516,
(i) any one of SEQ ID NOs 97796-98985, (j) any one of SEQ ID NOs
96024-97402, (k) any one of SEQ ID NOs 61634-62428, (l) any one of
SEQ ID NOs 120461-127349 or 129035-138877, (m) any one of SEQ ID
NOs 86561-92479, (n) any one of SEQ ID NOs 65414-66767, (o) any one
of SEQ ID NOs 75976-76379, (p) any one of SEQ ID NOs 99548-103608,
(q) any one of SEQ ID NOs 93972-96023, (r) any one of SEQ ID NOs
113475-113528, (s) any one of SEQ ID NOs 113173-113474, (t) any one
of SEQ ID NOs 66768-75975, (u) any one of SEQ ID NOs 138878-139851,
(v) any one of SEQ ID NOs 103609-105873, (w) any one of SEQ ID NOs
105874-106128, (x) any one of SEQ ID NOs 92480-93971, (y) any one
of SEQ ID NOs 83313-84988, (z) any one of SEQ ID NOs 64103-64315,
(aa) any one of SEQ ID NOs 76380-83312, (bb) any one of SEQ ID NOs
84989-86560, (cc) any one of SEQ ID NOs 108517-110925, (dd) any one
of SEQ ID NOs 63283-63402, or any one of SEQ ID NOs
128785-129034.
[0190] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of (a) SEQ ID NOs 6154441-6154744, (b)
SEQ ID NOs 6153128, 61624121-61627124, (c) SEQ ID NOs 6154037 or
6154138, (d) SEQ ID NOs 6153633 or 6153734, (e) SEQ ID NOs
6159592-6159895, (f) SEQ ID NOs 61612109-61615112, (g) SEQ ID NOs
6159087-6159289, (h) SEQ ID NOs 61607104 or 61608105, (i) SEQ ID
NOs 6159390 or 6159491, (j) SEQ ID NOs 6158885 or 6158986, (k) SEQ
ID NO 6153532, (l) SEQ ID NOs 61618115-61623120 or
61629126-61632129, (m) SEQ ID NOs 6157976-6158178, (n) SEQ ID NOs
6154845 or 6154946, (o) SEQ ID NOs 6156057-6156360, (p) SEQ ID NOs
6159996 or 6160097, (q) SEQ ID NOs 6158683 or 6158784, (r) SEQ ID
NO 61617114, (s) SEQ ID NO 61726223, (t) SEQ ID NOs
6155047-6155956, (u) SEQ ID NO 61633130, (v) SEQ ID NOs
6160198-61605102, (w) SEQ ID NO 61606103, (x) SEQ ID NOs
6158380-6158582, (y) SEQ ID NOs 6156966-6157673, (z) SEQ ID NO
6154239, (aa) SEQ ID NOs 6156461-6156865, (bb) SEQ ID NOs 6157774
or 6157875, (cc) SEQ ID NOs 61609106-61611108, (dd) SEQ ID NOs
6153835 or 6153936, or SEQ ID NO 61628125.
[0191] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to (a) SEQ ID NO 61509, (b) SEQ
ID NO 61531, (c) SEQ ID NO 61507, (d) SEQ ID NO 61505, (e) SEQ ID
NO 61522, (f) SEQ ID NO 61528, (g) SEQ ID NO 61520, (h) SEQ ID NO
61526, (i) SEQ ID NO 61521, (j) SEQ ID NO 61519, (k) SEQ ID NO
61504, (l) SEQ ID NO 61533, (m) SEQ ID NO 61516, (n) SEQ ID NO
61510, (o) SEQ ID NO 61512, (p) SEQ ID NO 61523, (q) SEQ ID NO
61518, (r) SEQ ID NO 61530, (s) SEQ ID NO 61529, (t) SEQ ID NO
61511, (u) SEQ ID NO 61534, (v) SEQ ID NO 61524, (w) SEQ ID NO
61525, (x) SEQ ID NO 61517, (y) SEQ ID NO 61514, (z) SEQ ID NO
61508, (aa) SEQ ID NO 61513, (bb) SEQ ID NO 61515, (cc) SEQ ID NO
61527, (dd) SEQ ID NO 61506, or (ee) SEQ ID NO 61532.
[0192] In some embodiments, the antisense oligomer does not
increase the amount of target protein or functional RNA by
modulating alternative splicing of the pre-mRNA transcribed from a
gene encoding the target protein or functional RNA.
[0193] In some embodiments, the antisense oligomer does not
increase the amount of the functional RNA or functional protein by
modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or functional RNA.
[0194] In some embodiments, the RIC pre-mRNA was produced by
partial splicing from a full-length pre-mRNA or a wild-type
pre-mRNA.
[0195] In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA.
[0196] In some embodiments, the target protein produced is
full-length protein, or wild-type protein.
[0197] In some embodiments, the retained intron is a rate-limiting
intron.
[0198] In some embodiments, said retained intron is the most
abundant retained intron in said RIC pre-mRNA.
[0199] In some embodiments, the retained intron is the second most
abundant retained intron in said RIC pre-mRNA.
[0200] In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage.
[0201] In some embodiments, said antisense oligomer is an antisense
oligonucleotide.
[0202] In some embodiments, the antisense oligomer comprises a
phosphorodiamidate morpholino, a locked nucleic acid, a peptide
nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxy ethyl
moiety.
[0203] In some embodiments, the antisense oligomer comprises at
least one modified sugar moiety.
[0204] In some embodiments, each sugar moiety is a modified sugar
moiety.
[0205] In some embodiments, the antisense oligomer consists of from
8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to
30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases.
[0206] In some embodiments, the antisense oligomer is at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or is 100% complementary to the targeted portion of the RIC
pre-mRNA encoding the protein.
[0207] In one aspect, provided herein is a pharmaceutical
composition comprising an antisense oligomer of any of the
compositions described herein, and an excipient.
[0208] In one aspect, provided herein is a method of treating a
subject in need thereof by administering a pharmaceutical
composition described herein by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection.
[0209] In one aspect, provided herein is a pharmaceutical
composition comprising: an antisense oligomer that hybridizes to a
target sequence of a deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL,
PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript, wherein the
deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP,
THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7,
CERS2 or NCOA5 mRNA transcript comprises a retained intron, wherein
the antisense oligomer induces splicing out of the retained intron
from the deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD,
APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1,
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 mRNA transcript; and a pharmaceutical
acceptable excipient.
[0210] In some embodiments, the deficient AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is a
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA transcript.
[0211] In some embodiments, the targeted portion of the AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA transcript is in the retained intron within the region
+500 relative to the 5' splice site of the retained intron to -500
relative to the 3' spliced site of the retained intron.
[0212] In some embodiments, the AMT, ADA, PPOX, UROD, HMBS, ACADVL,
PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript is
encoded by a genetic sequence with at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ
ID NOs: 61504-61534.
[0213] In some embodiments, the AMT, ADA, PPOX, UROD, HMBS, ACADVL,
PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript
comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:
61535-61633.
[0214] In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage.
[0215] In some embodiments, the antisense oligomer is an antisense
oligonucleotide.
[0216] In some embodiments, the antisense oligomer comprises a
phosphorodiamidate morpholino, a locked nucleic acid, a peptide
nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxy ethyl
moiety.
[0217] In some embodiments, the antisense oligomer comprises at
least one modified sugar moiety.
[0218] In some embodiments, the antisense oligomer comprises from 8
to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to
30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases.
[0219] In some embodiments, the antisense oligomer is at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or is 100% complementary to a targeted portion of the AMT,
ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA transcript.
[0220] In some embodiments, the targeted portion of the AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA transcript is within a sequence selected from SEQ ID NOs:
139852-140004.
[0221] In some embodiments, the antisense oligomer comprises a
nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one
of SEQ ID NOs: 61634-139851.
[0222] In some embodiments, the antisense oligomer comprises a
nucleotide sequence selected from SEQ ID NOs: 61634-139851.
[0223] In some embodiments, the pharmaceutical composition is
formulated for intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection.
[0224] In one aspect, provided herein is a method of inducing
processing of a deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript to facilitate
removal of a retained intron to produce a fully processed AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5
mRNA transcript that encodes a functional form of a AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein,
the method comprising: contacting an antisense oligomer to a target
cell of a subject; hybridizing the antisense oligomer to the
deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP,
THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7,
CERS2 or NCOA5 mRNA transcript, wherein the deficient AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5
mRNA transcript is capable of encoding the functional form of a
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 protein and comprises at least one retained intron; removing
the at least one retained intron from the deficient AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript to produce the fully processed AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript that
encodes the functional form of AMT, ADA, PPOX, UROD, HMBS, ACADVL,
PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein; and translating the
functional form of AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD,
APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1,
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 protein from the fully processed AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5
mRNA transcript.
[0225] In some embodiments, the retained intron is an entire
retained intron.
[0226] In some embodiments, the deficient AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is a
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA transcript.
[0227] In one aspect, provided herein is a method of treating a
subject having a condition caused by a deficient amount or activity
of AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP,
THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7,
CERS2 or NCOA5 protein comprising administering to the subject an
antisense oligomer comprising a nucleotide sequence with at least
about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to any one of SEQ ID NOs: 61634-139851.
[0228] In one aspect, provided herein is a method of treating a
kidney disease in a subject in need thereof by increasing the
expression of a target protein or functional RNA by cells of the
subject, wherein the cells have a retained-intron-containing
pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained
intron, an exon flanking the 5' splice site, an exon flanking the
3' splice site, and wherein the RIC pre-mRNA encodes the target
protein or functional RNA, the method comprising contacting the
cells of the subject with an antisense oligomer (ASO) complementary
to a targeted portion of the RIC pre-mRNA encoding the target
protein or functional RNA, whereby the retained intron is
constitutively spliced from the RIC pre-mRNA encoding the target
protein or functional RNA, thereby increasing the level of mRNA
encoding the target protein or functional RNA, and increasing the
expression of the target protein or functional RNA in the cells of
the subject.
[0229] In some embodiments, the kidney disease is infantile
nephropathic cystinosis, late-onset cystinosis, focal segmental
glomerulosclerosis 7, Papillorenal syndrome, or infantile
hypercalcemia.
[0230] In one aspect, provided herein is a method of increasing
expression of a target protein by cells having a
retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC
pre-mRNA comprising a retained intron, an exon flanking the 5'
splice site of the retained intron, an exon flanking the 3' splice
site of the retained intron, and wherein the RIC pre-mRNA encodes
the target protein, the method comprising contacting the cells with
an antisense oligomer (ASO) complementary to a targeted portion of
the RIC pre-mRNA encoding the target protein, whereby the retained
intron is constitutively spliced from the RIC pre-mRNA encoding the
target protein, thereby increasing the level of mRNA encoding the
target protein, and increasing the expression of the target protein
in the cells, wherein the target protein is cystinosin, protein
paired box gene 2 protein, protein cytochrome P450 family 24,
subfamily A, polypeptide 1, or peroxisome proliferator activated
receptor delta.
[0231] In some embodiments, the target protein is cystinosin,
protein paired box gene 2 protein, protein cytochrome P450 family
24, subfamily A, polypeptide 1, or peroxisome proliferator
activated receptor delta.
[0232] In some embodiments, the target protein or the functional
RNA is a compensating protein or a compensating functional RNA that
functionally augments or replaces a target protein or functional
RNA that is deficient in amount or activity in the subject.
[0233] In some embodiments, the cells are in or from a subject
having a condition caused by a deficient amount or activity of the
target protein.
[0234] In some embodiments, the deficient amount of the target
protein is caused by haploinsufficiency of the target protein,
wherein the subject has a first allele encoding a functional target
protein, and a second allele from which the target protein is not
produced, or a second allele encoding a nonfunctional target
protein, and wherein the antisense oligomer binds to a targeted
portion of a RIC pre-mRNA transcribed from the first allele.
[0235] In some embodiments, the subject has a condition caused by a
disorder resulting from a deficiency in the amount or function of
the target protein, wherein the subject has a first mutant allele
from which the target protein is produced at a reduced level
compared to production from a wild-type allele, the target protein
is produced in a form having reduced function compared to an
equivalent wild-type protein, or the target protein is not
produced, and a second mutant allele from which the target protein
is produced at a reduced level compared to production from a
wild-type allele, the target protein is produced in a form having
reduced function compared to an equivalent wild-type protein, or
the target protein is not produced, and wherein when the subject
has a first mutant allele a(iii), the second mutant allele is b(i)
or b(ii), and wherein when the subject has a second mutant allele
b(iii), the first mutant allele is a(i) or a(ii), and wherein the
RIC pre-mRNA is transcribed from either the first mutant allele
that is a(i) or a(ii), and/or the second allele that is b(i)(ii) or
b(ii).
[0236] In some embodiments, the target protein is produced in a
form having reduced function compared to the equivalent wild-type
protein.
[0237] In some embodiments, the target protein is produced in a
form that is fully-functional compared to the equivalent wild-type
protein.
[0238] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +6 relative to
the 5' splice site of the retained intron to -16 relative to the 3'
splice site of the retained intron.
[0239] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +69 relative
to the 5' splice site of the retained intron to -79 relative to the
3' splice site of the retained intron.
[0240] In some embodiments, the target protein is cystinosin.
[0241] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' splice site of the retained intron.
[0242] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs 148628-150072.
[0243] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150752 or SEQ ID
NO 150738.
[0244] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 148628-150072.
[0245] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO 140020 or SEQ ID NO 140021.
[0246] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140007.
[0247] In some embodiments, the target protein is paired box gene 2
protein.
[0248] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' splice site of the retained intron.
[0249] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs. 146957-148627.
[0250] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150742 or SEQ ID
NO 150744.
[0251] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 146957-148627.
[0252] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs 140014-140019.
[0253] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140006.
[0254] In some embodiments, the target protein is protein
cytochrome P450 family 24, subfamily A, polypeptide 1.
[0255] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' splice site of the retained intron.
[0256] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs. 149524-150737.
[0257] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150752, SEQ ID NO
150738, SEQ ID NO 150750, SEQ ID NO 150749 or SEQ ID NO 150745.
[0258] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 149524-150737.
[0259] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs 140020-140023.
[0260] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140008.
[0261] In some embodiments, the target protein is peroxisome
proliferator activated receptor delta.
[0262] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' splice site of the retained intron.
[0263] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs 140024-146956.
[0264] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150740, SEQ ID NO
150751, SEQ ID NO 150743, SEQ ID NO 150747, SEQ ID NO 150746, SEQ
ID NO 150741, SEQ ID NO 150739, or SEQ ID NO 150748.
[0265] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 140024-146956.
[0266] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs 140009-140013.
[0267] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140005.
[0268] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within: the region +6 to +100
relative to the 5' splice site of the retained intron; or the
region -16 to -100 relative to the 3' splice site of the retained
intron.
[0269] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is within the region about 100
nucleotides downstream of the 5' splice site of the at least one
retained intron, to about 100 nucleotides upstream of the 3' splice
site of the at least one retained intron
[0270] In some embodiments, the targeted portion of the RIC
pre-mRNA is within: the region +2e to -4e in the exon flanking the
5' splice site of the retained intron; or the region +2e to -4e in
the exon flanking the 3' splice site of the retained intron.
[0271] In some embodiments, the antisense oligomer does not
increase the amount of the target protein or the functional RNA by
modulating alternative splicing of pre-mRNA transcribed from a gene
encoding the functional RNA or target protein.
[0272] In some embodiments, the antisense oligomer does not
increase the amount of the target protein or the functional RNA by
modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or the functional RNA.
[0273] In some embodiments, the RIC pre-mRNA was produced by
partial splicing of a full-length pre-mRNA or partial splicing of a
wild-type pre-mRNA.
[0274] In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA.
[0275] In some embodiments, the target protein produced is
full-length protein, or wild-type protein.
[0276] In some embodiments, the total amount of the mRNA encoding
the target protein or functional RNA produced in the cell contacted
with the antisense oligomer is increased about 1.1 to about
10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold,
about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to
about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold,
about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to
about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold,
about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about
6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3
to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold,
about 4 to about 9-fold, at least about 1.1-fold, at least about
1.5-fold, at least about 2-fold, at least about 2.5-fold, at least
about 3-fold, at least about 3.5-fold, at least about 4-fold, at
least about 5-fold, or at least about 10-fold, compared to the
total amount of the mRNA encoding the target protein or functional
RNA produced in a control cell.
[0277] In some embodiments, the total amount of target protein
produced by the cell contacted with the antisense oligomer is
increased about 1.1 to about 10-fold, about 1.5 to about 10-fold,
about 2 to about 10-fold, about 3 to about 10-fold, about 4 to
about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the total amount of target protein
produced by a control cell.
[0278] In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage.
[0279] In some embodiments, the antisense oligomer comprises a
phosphorodiamidate morpholino, a locked nucleic acid, a peptide
nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxy ethyl
moiety.
[0280] In some embodiments, the antisense oligomer comprises at
least one modified sugar moiety.
[0281] In some embodiments, each sugar moiety is a modified sugar
moiety.
[0282] In some embodiments, the antisense oligomer consists of from
8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to
30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases.
[0283] In some embodiments, the antisense oligomer is at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or 100%, complementary to the targeted portion of the RIC
pre-mRNA encoding the protein.
[0284] In some embodiments, the cell comprises a population of RIC
pre-mRNAs transcribed from the gene encoding the target protein or
functional RNA, wherein the population of RIC pre-mRNAs comprises
two or more retained introns, and wherein the antisense oligomer
binds to the most abundant retained intron in the population of RIC
pre-mRNAs.
[0285] In some embodiments, the binding of the antisense oligomer
to the most abundant retained intron induces splicing out of the
two or more retained introns from the population of RIC pre-mRNAs
to produce mRNA encoding the target protein or functional RNA.
[0286] In some embodiments, the cell comprises a population of RIC
pre-mRNAs transcribed from the gene encoding the target protein or
functional RNA, wherein the population of RIC pre-mRNAs comprises
two or more retained introns, and wherein the antisense oligomer
binds to the second most abundant retained intron in the population
of RIC pre-mRNAs.
[0287] In some embodiments, the binding of the antisense oligomer
to the second most abundant retained intron induces splicing out of
the two or more retained introns from the population of RIC
pre-mRNAs to produce mRNA encoding the target protein or functional
RNA.
[0288] In some embodiments, the condition is a disease or
disorder.
[0289] In some embodiments, the disease or disorder is a kidney
disease.
[0290] In some embodiments, the kidney disease is infantile
nephropathic cystinosis, late-onset cystinosis, focal segmental
glomerulosclerosis 7, Papillorenal syndrome, or infantile
hypercalcemia.
[0291] In some embodiments, the target protein and the RIC pre-mRNA
are encoded by a gene, wherein the gene is CTNS, PAX2, CYP24A1 or
PPARD.
[0292] In some embodiments, the method further comprises assessing
protein expression.
[0293] In some embodiments, the subject is a human.
[0294] In some embodiments, the subject is a non-human animal.
[0295] In some embodiments, the subject is a fetus, an embryo, or a
child.
[0296] In some embodiments, the cells are ex vivo.
[0297] In some embodiments, the antisense oligomer is administered
by intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection of the subject.
[0298] In some embodiments, the 9 nucleotides at -3e to -1e of the
exon flanking the 5' splice site and +1 to +6 of the retained
intron, are identical to the corresponding wild-type sequence.
[0299] In some embodiments, the 16 nucleotides at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site are
identical to the corresponding wild-type sequence.
[0300] In one aspect, provided herein is an antisense oligomer as
used in a method described herein.
[0301] In one aspect, provided herein is an antisense oligomer
comprising a sequence with at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs
140024-150737.
[0302] In one aspect, provided herein is a pharmaceutical
composition comprising an antisense oligomer described herein and
an excipient.
[0303] In one aspect, provided herein is a method of treating a
subject in need thereof by administering a pharmaceutical
composition disclosed herein by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection.
[0304] In one aspect, provided herein is a composition comprising
an antisense oligomer for use in a method of increasing expression
of a target protein or a functional RNA by cells to treat a kidney
disease in a subject in need thereof associated with a deficient
protein or deficient functional RNA, wherein the deficient protein
or deficient functional RNA is deficient in amount or activity in
the subject, wherein the antisense oligomer enhances constitutive
splicing of a retained intron-containing pre-mRNA (RIC pre-mRNA)
encoding the target protein or the functional RNA, wherein the
target protein is: the deficient protein; or a compensating protein
which functionally augments or replaces the deficient protein or in
the subject; and wherein the functional RNA is: the deficient RNA;
or a compensating functional RNA which functionally augments or
replaces the deficient functional RNA in the subject; wherein the
RIC pre-mRNA comprises a retained intron, an exon flanking the 5'
splice site and an exon flanking the 3' splice site, and wherein
the retained intron is spliced from the RIC pre-mRNA encoding the
target protein or the functional RNA, thereby increasing production
or activity of the target protein or the functional RNA in the
subject.
[0305] In some embodiments, the kidney disease is infantile
nephropathic cystinosis, late-onset cystinosis, focal segmental
glomerulosclerosis 7, Papillorenal syndrome, or infantile
hypercalcemia.
[0306] In one aspect, provided herein is a composition comprising
an antisense oligomer for use in a method of treating a condition
associated with a target protein in a subject in need thereof, the
method comprising the step of increasing expression of the target
protein by cells of the subject, wherein the cells have a
retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a
retained intron, an exon flanking the 5' splice site of the
retained intron, an exon flanking the 3' splice site of the
retained intron, and wherein the RIC pre-mRNA encodes the target
protein, the method comprising contacting the cells with the
antisense oligomer, whereby the retained intron is constitutively
spliced from the RIC pre-mRNA transcripts encoding the target
protein, thereby increasing the level of mRNA encoding the target
protein or functional RNA, and increasing the expression of the
target protein, in the cells of the subject.
[0307] In some embodiments, the target protein is cystinosin,
protein paired box gene 2 protein, protein cytochrome P450 family
24, subfamily A, polypeptide 1, or peroxisome proliferator
activated receptor delta.
[0308] In some embodiments, the condition is a disease or
disorder.
[0309] In some embodiments, the disease or disorder is a kidney
disease.
[0310] In some embodiments, the kidney disease is infantile
nephropathic cystinosis, late-onset cystinosis, focal segmental
glomerulosclerosis 7, Papillorenal syndrome, or infantile
hypercalcemia.
[0311] In some embodiments, the target protein and RIC pre-mRNA are
encoded by a gene, wherein the gene is CTNS, PAX2, CYP24A1 or
PPARD.
[0312] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is in the retained intron within
the region +6 relative to the 5' splice site of the retained intron
to -16 relative to the 3' splice site of the retained intron.
[0313] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +69 relative
to the 5' splice site of the retained intron to -79 relative to the
3' splice site of the retained intron.
[0314] In some embodiments, the target protein is cystinosin.
[0315] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' of the retained intron.
[0316] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs 148628-150072.
[0317] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150752 or SEQ ID
NO 150738.
[0318] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 148628-150072.
[0319] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO 140020 or SEQ ID NO 140021.
[0320] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140007.
[0321] In some embodiments, the target protein is paired box gene 2
protein.
[0322] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' splice site of the retained intron.
[0323] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs. 146957-148627.
[0324] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150742 or SEQ ID
NO 150744.
[0325] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 146957-148627.
[0326] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs 140014-140019.
[0327] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140006.
[0328] In some embodiments, the target protein is protein
cytochrome P450 family 24, subfamily A, polypeptide 1.
[0329] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' splice site of the retained intron.
[0330] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs. 149524-150737.
[0331] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150752, SEQ ID NO
150738, SEQ ID NO 150750, SEQ ID NO 150749 or SEQ ID NO 150745.
[0332] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 149524-150737.
[0333] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs 140020-19.
[0334] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140008.
[0335] In some embodiments, the target protein is peroxisome
proliferator activated receptor delta.
[0336] In some embodiments, the targeted portion of the RIC
pre-mRNA is in the retained intron within the region +500 relative
to the 5' splice site of the retained intron to -500 relative to
the 3' splice site of the retained intron.
[0337] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID
NOs 140024-146956.
[0338] In some embodiments, the targeted portion of the RIC
pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to a region comprising
at least 8 contiguous nucleic acids of SEQ ID NO 150740, SEQ ID NO
150751, SEQ ID NO 150743, SEQ ID NO 150747, SEQ ID NO 150746, SEQ
ID NO 150741, SEQ ID NO 150739, or SEQ ID NO 150748.
[0339] In some embodiments, the ASO comprises a sequence with at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs 140024-146956.
[0340] In some embodiments, the RIC pre-mRNA comprises a sequence
with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to any one of SEQ ID NOs 140009-140013.
[0341] In some embodiments, the RIC pre-mRNA is encoded by a
genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99% or 100% sequence identity to SEQ ID NO 140005.
[0342] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is in the retained intron within:
the region +6 to +100 relative to the 5' splice site of the
retained intron; or the region -16 to -100 relative to the 3'
splice site of the retained intron.
[0343] In some embodiments, the antisense oligomer targets a
portion of the RIC pre-mRNA that is within the region about 100
nucleotides downstream of the 5' splice site of the at least one
retained intron, to about 100 nucleotides upstream of the 3' splice
site of the at least one retained intron.
[0344] In some embodiments, the targeted portion of the RIC
pre-mRNA is within: the region +2e to -4e in the exon flanking the
5' splice site of the retained intron; or the region +2e to -4e in
the exon flanking the 3' splice site of the retained intron.
[0345] In some embodiments, the antisense oligomer does not
increase the amount of target protein or functional RNA by
modulating alternative splicing of the pre-mRNA transcribed from a
gene encoding the target protein or functional RNA.
[0346] In some embodiments, the antisense oligomer does not
increase the amount of the functional RNA or functional protein by
modulating aberrant splicing resulting from mutation of the gene
encoding the target protein or functional RNA.
[0347] In some embodiments, the RIC pre-mRNA was produced by
partial splicing from a full-length pre-mRNA or a wild-type
pre-mRNA.
[0348] In some embodiments, the mRNA encoding the target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature
mRNA.
[0349] In some embodiments, the target protein produced is
full-length protein, or wild-type protein.
[0350] In some embodiments, the retained intron is a rate-limiting
intron.
[0351] In some embodiments, said retained intron is the most
abundant retained intron in said RIC pre-mRNA.
[0352] In some embodiments, the retained intron is the second most
abundant retained intron in said RIC pre-mRNA.
[0353] In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage.
[0354] In some embodiments, said antisense oligomer is an antisense
oligonucleotide.
[0355] In some embodiments, the antisense oligomer comprises a
phosphorodiamidate morpholino, a locked nucleic acid, a peptide
nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxy ethyl
moiety.
[0356] In some embodiments, the antisense oligomer comprises at
least one modified sugar moiety.
[0357] In some embodiments, each sugar moiety is a modified sugar
moiety.
[0358] In some embodiments, the antisense oligomer consists of from
8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to
30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases.
[0359] In some embodiments, the antisense oligomer is at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or is 100% complementary to the targeted portion of the RIC
pre-mRNA encoding the protein.
[0360] In one aspect, provided herein is a pharmaceutical
composition comprising an antisense oligomer described herein, and
an excipient.
[0361] In one aspect, provided herein is a method of treating a
subject in need thereof by administering a pharmaceutical
composition described herein by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection.
[0362] In one aspect, provided herein is a pharmaceutical
composition comprising: an antisense oligomer that hybridizes to a
target sequence of a deficient CTNS, PAX2, CYP24A1 or PPARD mRNA
transcript, wherein the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA
transcript comprises a retained intron, wherein the antisense
oligomer induces splicing out of the retained intron from the
deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript; and a
pharmaceutical acceptable excipient.
[0363] In some embodiments, the deficient CTNS, PAX2, CYP24A1 or
PPARD mRNA transcript is a CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA transcript.
[0364] In some embodiments, the targeted portion of the CTNS, PAX2,
CYP24A1 or PPARD RIC pre-mRNA transcript is in the retained intron
within the region +500 relative to the 5' splice site of the
retained intron to -500 relative to the 3' spliced site of the
retained intron.
[0365] In some embodiments, the CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA transcript is encoded by a genetic sequence with at least
about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to any one of SEQ ID NOs: 140005-140008.
[0366] In some embodiments, the CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA transcript comprises a sequence with at least about 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any
one of SEQ ID NOs: 140009-140023.
[0367] In some embodiments, the antisense oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage.
[0368] In some embodiments, the antisense oligomer is an antisense
oligonucleotide.
[0369] In some embodiments, the antisense oligomer comprises a
phosphorodiamidate morpholino, a locked nucleic acid, a peptide
nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxy ethyl
moiety.
[0370] In some embodiments, the antisense oligomer comprises at
least one modified sugar moiety.
[0371] In some embodiments, the antisense oligomer comprises from 8
to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to
30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, or 12 to 15 nucleobases.
[0372] In some embodiments, the antisense oligomer is at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or is 100% complementary to a targeted portion of the CTNS,
PAX2, CYP24A1 or PPARD RIC pre-mRNA transcript.
[0373] In some embodiments, the targeted portion of the CTNS, PAX2,
CYP24A1 or PPARD RIC pre-mRNA transcript is within a sequence
selected from SEQ ID NOs: 150038-150752.
[0374] In some embodiments, the antisense oligomer comprises a
nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one
of SEQ ID NOs: 140024-150737.
[0375] In some embodiments, the antisense oligomer comprises a
nucleotide sequence selected from SEQ ID NOs: 140024-150737.
[0376] In some embodiments, the pharmaceutical composition is
formulated for intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection.
[0377] In one aspect, provided herein is a method of inducing
processing of a deficient CTNS, PAX2, CYP24A1 or PPARD mRNA
transcript to facilitate removal of a retained intron to produce a
fully processed CTNS, PAX2, CYP24A1 or PPARD mRNA transcript that
encodes a functional form of a CTNS, PAX2, CYP24A1 or PPARD
protein, the method comprising: contacting an antisense oligomer to
a target cell of a subject; hybridizing the antisense oligomer to
the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript, wherein
the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is
capable of encoding the functional form of a CTNS, PAX2, CYP24A1 or
PPARD protein and comprises at least one retained intron; removing
the at least one retained intron from the deficient CTNS, PAX2,
CYP24A1 or PPARD mRNA transcript to produce the fully processed
CTNS, PAX2, CYP24A1 or PPARD mRNA transcript that encodes the
functional form of CTNS, PAX2, CYP24A1 or PPARD protein; and
translating the functional form of CTNS, PAX2, CYP24A1 or PPARD
protein from the fully processed CTNS, PAX2, CYP24A1 or PPARD mRNA
transcript.
[0378] In some embodiments, the retained intron is an entire
retained intron.
[0379] In some embodiments, the deficient CTNS, PAX2, CYP24A1 or
PPARD mRNA transcript is a CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA transcript.
[0380] In one aspect, provided herein is a method of treating a
subject having a condition caused by a deficient amount or activity
of CTNS, PAX2, CYP24A1 or PPARD protein comprising administering to
the subject an antisense oligomer comprising a nucleotide sequence
with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:
140024-150737.
INCORPORATION BY REFERENCE
[0381] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0382] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings.
[0383] FIG. 1 illustrates a schematic representation of an
exemplary retained-intron-containing (RIC) pre-mRNA transcript. The
5' splice site consensus sequence is indicated with underlined
letters (letters are nucleotides; upper case: exonic portion and
lower case: intronic portion) from -3e to -1e and +1 to +6 (numbers
labeled "e" are exonic and unlabeled numbers are intronic). The 3'
splice site consensus sequence is indicated with underlined letters
(letters are nucleotides; upper case: exonic portion and lower
case: intronic portion) from -15 to -1 and +1e (numbers labeled "e"
are exonic and unlabeled numbers are intronic). Intronic target
regions for ASO screening comprise nucleotides +6 relative to the
5' splice site of the retained intron (arrow at left) to -16
relative to the 3' splice site of the retained intron (arrow at
right). In embodiments, intronic target regions for ASO screening
comprise nucleotides +6 to +100 relative to the 5' splice site of
the retained intron and -16 to -100 relative to the 3' splice site
of the retained intron. Exonic target regions comprise nucleotides
+2e to -4e in the exon flanking the 5' splice site of the retained
intron and +2e to -4e in the exon flanking the 3' splice site of
the retained intron. "n" or "N" denote any nucleotide, "y" denotes
pyrimidine. The sequences shown represent consensus sequences for
mammalian splice sites and individual introns and exons need not
match the consensus sequences at every position.
[0384] FIG. 2A-FIG. 2B illustrates schematic representations of the
Targeted Augmentation of Nuclear Gene Output (TANGO) approach. FIG.
2A shows a cell divided into nuclear and cytoplasmic compartments.
In the nucleus, a pre-mRNA transcript of a target gene consisting
of exons (rectangles) and introns (connecting lines) undergoes
splicing to generate an mRNA, and this mRNA is exported to the
cytoplasm and translated into target protein. For this target gene,
the splicing of intron 1 is inefficient and a retained
intron-containing (RIC) pre-mRNA accumulates primarily in the
nucleus, and if exported to the cytoplasm, is degraded, leading to
no target protein production. FIG. 2B shows an example of the same
cell divided into nuclear and cytoplasmic compartments. Treatment
with an antisense oligomer (ASO) promotes the splicing of intron 1
and results in an increase in mRNA, which is in turn translated
into higher levels of target protein
[0385] FIG. 3 shows intron-retention in the JAG1 gene with intron
13 detail. The identification of intron-retention events in the
JAG1 gene using RNA sequencing (RNAseq) is shown, visualized in the
UCSC genome browser. The upper panel shows the read density
corresponding to the JAG1 transcript expressed in THLE-3 (human
liver epithelial) cells and localized in either the cytoplasmic
(top) or nuclear fraction (bottom). At the bottom of this panel, a
graphic representation of the JAG1 gene is shown to scale. The read
density is shown as peaks. The highest read density corresponds to
exons (black boxes), while no reads are observed for the majority
of the introns (lines with arrow heads) in either cellular
fraction. Higher read density is detected for introns 13, 18, and
23 (pointed by the arrows) in the nuclear fraction compared to the
cytoplasmic fraction indicating that splicing efficiency of introns
13, 18, and 23 is low, resulting in intron retention. The
retained-intron containing pre-mRNA transcripts accumulate
primarily in the nucleus and are not translated into the JAG1
protein. The read density for intron 13 in THLE-3 cells is shown in
detail in the lower panel indicating 12% intron retention as
calculated by bioinformatic analysis.
[0386] FIG. 4 shows JAG1 gene IVS 13 ASO walk. A graphic
representation of the ASO walk performed for JAG1 IVS 13 targeting
sequences immediately downstream of the 5' splice site or upstream
of the 3' splice site using 2'-O-Me ASOs, PS backbone, is shown.
ASOs were designed to cover these regions by shifting 5 nucleotides
at a time. The JAG1 exon-intron structure is drawn to scale.
[0387] FIG. 5 shows JAG1 intron 13 ASO walk evaluated by
radioactive RT-PCR. At the top, a schematic drawing (not to scale)
of exon 13-intron 13-exon 14 shows the primers Forward 1 (F1) and
Reverse 1 (R1) used for the RT-PCR assay. In the middle panel, a
representative gel shows radioactive RT-PCR products of JAG1
mock-treated (neg, RNAiMAX only), SMN-control ASO treated, or
treated with a 2'-O-Me ASO targeting intron 13 as described in FIG.
2, at 60 nM concentration in ARPE-19 cells. In the lower panel,
quantification of the bands corresponding to JAG1 radioactive
RT-PCR products normalized to Beta actin from two independent
experiments is plotted in the bar graph as fold-change with respect
to the mock-treated products. The black line labeled 1 on the
Y-axis indicates a ratio of 1 (no change).
[0388] FIG. 6 shows JAG1 intron 13 ASO walk evaluated by RT-qPCR.
At the top left, a schematic representation of the RT-qPCR assay
shows the primer recognition sites. RT-qPCR amplification results,
obtained using the same ASO transfection experiment that were
evaluated by radioactive RT-PCR as shown in FIG. 3, are plotted
relative to mock-treated products normalized to Beta actin (upper
bar graph) or normalized to RPL32 (lower bar graph) confirming the
radioactive RT-PCR results. The black line labeled 1 on the Y-axis
indicates a ratio of 1 (no change).
[0389] FIG. 7 shows intron-retention in the JAG1 gene with intron
18 detail. Intron retention in the JAG1 gene was identified by RNA
sequencing (RNAseq), visualized in the UCSC genome browser, as
described herein in the Examples. The read density for intron 18 in
THLE-3 cells is shown in detail in the lower panel, indicating 14%
intron retention as calculated by bioinformatic analysis.
[0390] FIG. 8 shows JAG1 gene IVS 18 ASO walk. A graphic
representation of the ASO walk performed for JAG1 IVS 18 targeting
sequences immediately downstream of the 5' splice site or upstream
of the 3' splice site using 2'-O-Me ASOs, PS backbone, is shown.
ASOs were designed to cover these regions by shifting 5 nucleotides
at a time. The JAG1 exon-intron structure is drawn to scale.
[0391] FIG. 9 shows JAG1 intron 18 ASO walk evaluated by
radioactive RT-PCR. At the top, a schematic drawing (not to scale)
of exon 18 to exon 20 shows the primers Forward 1 (F1) and Reverse
1 (R1) used for the RT-PCR assay. In the middle panel, a
representative gel shows radioactive RT-PCR products of JAG1
mock-treated (neg, RNAiMAX only), SMN-control ASO treated, or
treated with a 2'-O-Me ASO targeting intron 18 as described herein
in the Examples and in the description of FIG. 6, at 60 nM
concentration in ARPE-19 cells. Quantification of the bands
corresponding to JAG1 radioactive RT-PCR products normalized to
Beta actin from 2 independent experiments is plotted in the bar
graph below as fold change with respect to the mock-treated
products. The black line labeled 1 on the Y-axis indicates a ratio
of 1 (no change).
[0392] FIG. 10 shows JAG1 intron 18 ASO walk evaluated by RT-qPCR.
At the top left, a schematic representation of the RT-qPCR assay
shows the primer recognition sites. RT-qPCR amplification results,
obtained using the same ASO transfection experiment that were
evaluated by radioactive RT-PCR as shown in FIG. 7, are plotted
relative to mock-treated products normalized to Beta actin (upper
bar graph) or normalized to RPL32 (lower bar graph) confirming the
radioactive RT-PCR results. The black line labeled 1 on the Y-axis
indicates a ratio of 1 (no change).
[0393] FIG. 11 shows intron-retention in the JAG1 gene with intron
23 detail. Intron retention in the JAG1 gene was identified by RNA
sequencing (RNAseq), visualized in the UCSC genome browser, as
described herein in the Examples and in the description of FIG. 1.
The read density for intron 23 in THLE-3 cells is shown in detail
in the lower panel, indicating 17% intron retention as calculated
by bioinformatic analysis.
[0394] FIG. 12 shows JAG1 gene IVS 23 ASO walk. A graphic
representation of the ASO walk performed for JAG1 IVS 23 targeting
sequences immediately downstream of the 5' splice site or upstream
of the 3' splice site using 2'-O-Me ASOs, PS backbone, is shown.
ASOs were designed to cover these regions by shifting 5 nucleotides
at a time. The JAG1 exon-intron structure is drawn to scale.
[0395] FIG. 13 shows intron-retention in the TSC2 gene with intron
4 detail. The identification of intron-retention events in the TSC2
gene using RNA sequencing (RNAseq) is shown, visualized in the UCSC
genome browser. The upper panel shows the read density
corresponding to the TSC2 transcript expressed in HCN (human
cortical neurons) and localized in either the cytoplasmic (top) or
nuclear fraction (bottom). At the bottom of this panel, a graphic
representation of the TSC2 gene is shown to scale. The read density
is shown as peaks. The highest read density corresponds to exons
(black boxes), while no reads are observed for the majority of the
introns (lines with arrow heads) in either cellular fraction.
Higher read density is detected for introns 4, 25/26, and 31/32
(indicated by the arrows) in the nuclear fraction compared to the
cytoplasmic fraction indicating that splicing efficiency of introns
4, 25/26, and 31/32 is low, resulting in intron retention. The
retained-intron containing pre-mRNA transcripts are retained in the
nucleus and are not exported out to the cytoplasm. The read density
for intron 4 in HCN is shown in detail in the lower panel.
[0396] FIG. 14 shows TSC2 gene IVS 4 ASO walk. A graphic
representation of the ASO walk performed for TSC2 IVS 4 targeting
sequences immediately downstream of the 5' splice site or upstream
of the 3' splice site using 2'-O-Me ASOs, PS backbone, is shown.
ASOs were designed to cover these regions by shifting 5 nucleotides
at a time. The TSC2 exon-intron structure is drawn to scale.
[0397] FIG. 15 shows intron-retention in the TSC2 gene with introns
25 and 26 detail. Intron retention in the TSC2 gene was identified
by RNA sequencing (RNAseq), visualized in the UCSC genome browser,
as described herein in the Examples. The read density for introns
25 and 26 in HCN is shown in detail in the lower panel. Introns 25
and 26 flank exon 26, an alternatively spliced exon. This is
evidenced in the graphic representations of the TSC2 transcripts
and the RNAseq data, such that the rectangle depicting exon 26 is
present in some transcripts while absent in others, and the read
density corresponding to exon 26 in the cytoplasmic fraction is
significantly lower than that of the constitutively spliced exons
in TSC2. The read density for intron 26 is shown in detail in the
lower panel indicating 51% intron retention as calculated by
bioinformatic analysis.
[0398] FIG. 16 shows TSC2 gene IVS 25 and 26 ASO walk. A graphic
representation of the ASO walk performed for TSC2 IVS 25 and 26
targeting sequences immediately downstream of the 5' splice site of
intron 25 or upstream of the 3' splice site of intron 26 using
2'-O-Me ASOs, PS backbone, is shown. The splice site intronic
regions flanking alternative exon 26 are not targeted to avoid
affecting the inclusion level of exon 26. ASOs were designed to
cover these regions by shifting 5 nucleotides at a time. The TSC2
exon-intron structure is drawn to scale.
[0399] FIG. 17 shows intron-retention in the TSC2 gene with introns
31 and 32 detail. Intron retention in the TSC2 gene was identified
by RNA sequencing (RNAseq), visualized in the UCSC genome browser,
as described herein in the Examples. The read density for introns
31 and 32 is shown in detail in the lower panel. Introns 31 and 32
flank exon 32, an alternatively spliced exon. This is evidenced in
the graphic representations of the TSC2 transcripts and the RNAseq
data, such that the rectangle depicting exon 32 is present in some
transcripts while absent in others, and the read density
corresponding to exon 32 in the cytoplasmic fraction is
significantly lower than that of the constitutively spliced exons
in TSC2. The read density for intron 31 is shown in detail in the
lower panel indicating 43% intron retention as calculated by
bioinformatic analysis.
[0400] FIG. 18 shows TSC2 gene IVS 31 and 32 ASO walk. A graphic
representation of the ASO walk performed for TSC2 IVS 31 and 32
targeting sequences immediately downstream of the 5' splice site of
intron 31 or upstream of the 3' splice site of intron 32 using
2'-O-Me ASOs, PS backbone, is shown. The splice site intronic
regions flanking alternative exon 32 are not targeted to avoid
affecting the inclusion level of exon 32. ASOs were designed to
cover these regions by shifting 5 nucleotides at a time with the
exception of TSC2-IVS32-33 and TSC2-IVS32-51. The TSC2 exon-intron
structure is drawn to scale.
[0401] FIG. 19 depicts a schematic of the RefSeq Genes for TSC2
intron 4 corresponding to NM_000548. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0402] FIG. 20 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of TSC2 mRNA without intron 4 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0403] FIG. 21 depicts a schematic of the RefSeq Genes for TSC2
intron 25 corresponding to NM_000548. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0404] FIG. 22 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of TSC2 mRNA without intron 25 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0405] FIG. 23 depicts a schematic of the RefSeq Genes for TSC2
intron 26 corresponding to NM_000548. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0406] FIG. 24 depicts a schematic of the RefSeq Genes for TSC2
intron 31 corresponding to NM_000548. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0407] FIG. 25 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of TSC2 mRNA without intron 31 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0408] FIG. 26 depicts a schematic of the RefSeq Genes for TSC2
intron 32 corresponding to NM_000548. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0409] FIG. 27 depicts intron-retention in the PKD2 gene with
intron 5 shown in detail. The identification of intron-retention
events in the PKD2 gene using RNA sequencing (RNAseq) is shown,
visualized in the UCSC genome browser. The upper panel shows the
read density corresponding to the PKD2 transcript expressed in
renal epithelial cells and localized in either the cytoplasmic
(top) or nuclear fraction (bottom). At the bottom of this panel, a
graphic representation of the PKD2 gene is shown to scale. The read
density is shown as peaks. The highest read density corresponds to
exons (black boxes), while no reads are observed for the majority
of the introns in either cellular fraction. Higher read density is
detected for intron 5 (pointed by the arrow) in the nuclear
fraction compared to the cytoplasmic fraction indicating that
splicing efficiency of intron 5 is low, resulting in intron
retention. The retained-intron containing pre-mRNA transcripts are
retained in the nucleus and are not exported out to the cytoplasm.
The read density for intron 5 in renal epithelial cells is shown in
detail in the lower panel.
[0410] FIG. 28 depicts an exemplary PKD2 gene intron 5 (IVS 5) ASO
walk. A graphic representation of the ASO walk performed for PKD2
IVS 5 targeting sequences immediately downstream of the 5' splice
site or upstream of the 3' splice site using 2'-O-Me ASOs, PS
backbone, is shown. ASOs were designed to cover these regions by
shifting 5 nucleotides at a time. The PKD2 exon-intron structure is
drawn to scale.
[0411] FIG. 29 depicts a schematic of the RefSeq Gene for PKD2
corresponding to NM_000297. The Percent Intron Retention (PIR) of
intron 5 is detailed.
[0412] FIG. 30 depicts intron-retention in the PRPF3 gene with
intron 12 detail. The identification of intron-retention events in
the PRPF3 gene using RNA sequencing (RNAseq) is shown, visualized
in the UCSC genome browser. The upper panel shows the read density
corresponding to the PRPF3 transcript expressed in ARPE-19 (retina
epithelial) cells and localized in either the cytoplasmic (top) or
nuclear fraction (bottom). At the bottom of this panel, a graphic
representation of the PRPF3 gene is shown to scale. The read
density is shown as peaks. The highest read density corresponds to
exons (black boxes), while no reads are observed for the majority
of the introns (lines with arrow heads) in either cellular
fraction. Higher read density is detected for intron 12 (indicated
by the arrow) in the nuclear fraction compared to the cytoplasmic
fraction indicating that splicing efficiency of intron 12 is low,
resulting in intron retention. The retained-intron containing
pre-mRNA transcripts are retained in the nucleus and are not
exported out to the cytoplasm. The read density for intron 12 in
renal epithelial cells is shown in detail in the lower panel.
[0413] FIG. 31 illustrates a graphic representation of the ASO walk
performed for PRPF3 IVS 12 targeting sequences immediately
downstream of the 5' splice site or upstream of the 3' splice site
using 2'-O-Me ASOs, PS backbone, is shown. ASOs were designed to
cover these regions by shifting 5 nucleotides at a time (with the
exception of ASO P3-IVS12+28). The PRPF3 exon-intron structure is
drawn to scale.
[0414] FIG. 32 depicts intron-retention in the PRPF3 gene with
intron 13. Intron retention in the PRPF3 gene was identified by RNA
sequencing (RNAseq), visualized in the UCSC genome browser, as
described herein in the Examples. The read density for intron 13 in
ARPE-19 cells is shown in detail in the lower panel.
[0415] FIG. 33 illustrates a graphic representation of the ASO walk
performed for PRPF3 IVS 13 targeting sequences immediately
downstream of the 5' splice site or upstream of the 3' splice site
using 2'-O-Me ASOs, PS backbone, is shown. ASOs were designed to
cover these regions by shifting 5 nucleotides at a time (with the
exception of ASO P3-IVS13+15, P3-IVS13+31, and P3-IVS13-47). The
PRPF3 exon-intron structure is drawn to scale.
[0416] FIG. 34 depicts intron-retention in the PRPF8 gene with
intron 31 detail. The identification of intron-retention events in
the PRPF8 gene using RNA sequencing (RNAseq) is shown, visualized
in the UCSC genome browser. The upper panel shows the read density
corresponding to the PRPF8 transcript expressed in ARPE-19 (retina
epithelial) cells and localized in either the cytoplasmic (top) or
nuclear fraction (bottom). At the bottom of this panel, a graphic
representation of the PRPF8 gene is shown to scale. The read
density is shown as peaks. The highest read density corresponds to
exons (black boxes), while no reads are observed for the majority
of the introns (lines with arrow heads) in either cellular
fraction. Higher read density is detected for intron 31 (indicated
by the arrow) in the nuclear fraction compared to the cytoplasmic
fraction indicating that splicing efficiency of intron 31 is low,
resulting in intron retention. The retained-intron containing
pre-mRNA transcripts are retained in the nucleus and are not
exported out to the cytoplasm. The read density for intron 31 in
renal epithelial cells is shown in detail in the lower panel.
[0417] FIG. 35 illustrates a graphic representation of the ASO walk
performed for PRPF8 IVS 31 targeting sequences immediately
downstream of the 5' splice site or upstream of the 3' splice site
using 2'-O-Me ASOs, PS backbone, is shown. ASOs were designed to
cover these regions by shifting 5 nucleotides at a time. The PRPF8
exon-intron structure is drawn to scale.
[0418] FIG. 36 depicts a schematic of the RefSeq Genes for PRPF3
corresponding to NM_004698. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0419] FIG. 37 depicts an exemplary graph is depicted showing the
average (n=3) fold change in expression levels of PRPF3 mRNA
without intron 12 in ARPE-19 cells treated for 24 hrs with 80 nM of
the indicated ASOs over mock treated cells. Data is normalized to
RPL32 expression.
[0420] FIG. 38 depicts a schematic of the RefSeq Genes for PRPF3
corresponding to NM_004698. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0421] FIG. 39 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of PRPF3 mRNA without intron 13 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0422] FIG. 40 depicts a schematic of the RefSeq Genes for PRPF8
corresponding to NM_006445. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0423] FIG. 41 depicts an exemplary graph is depicted showing the
average (n=3) fold change in expression levels of PRPF8 mRNA
without intron 31 in ARPE-19 cells treated for 24 hrs with 80 nM of
the indicated ASOs over mock treated cells. Data is normalized to
RPL32 expression.
[0424] FIG. 42 depicts a schematic of the RefSeq Genes for ADAR
intron 2 corresponding to NM_001111. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0425] FIG. 43 depicts a schematic of the RefSeq Genes for ARSA
intron 3 corresponding to NM_001085425. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0426] FIG. 44 depicts a schematic of the RefSeq Genes for ARSA
intron 4 corresponding to NM_001085425. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0427] FIG. 45 depicts a schematic of the RefSeq Genes for DNMT1
intron 30 corresponding to NM_001130823. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0428] FIG. 46 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of DNMT1 mRNA without intron 30 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0429] FIG. 47 depicts a schematic of the RefSeq Genes for EIF2B2
intron 1 corresponding to NM_014239. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0430] FIG. 48 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of EIF2B2 mRNA without intron 1 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0431] FIG. 49 depicts a schematic of the RefSeq Genes for EIF2B5
intron 12 corresponding to NM_003907. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0432] FIG. 50 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of EIF2B5 mRNA without intron 12
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0433] FIG. 51 depicts a schematic of the RefSeq Genes for EIF2B5
intron 13 corresponding to NM_003907. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0434] FIG. 52 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of EIF2B5 mRNA without intron 13
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0435] FIG. 53 depicts a schematic of the RefSeq Genes for PEX1
intron 10 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0436] FIG. 54 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of PEX1 mRNA without intron 10 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0437] FIG. 55 depicts a schematic of the RefSeq Genes for PEX1
intron 14 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0438] FIG. 56 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of PEX1 mRNA without intron 14 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0439] FIG. 57 depicts a schematic of the RefSeq Genes for PRICKLE2
intron 4 corresponding to NM_198859. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0440] FIG. 58 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of PRICKLE2 mRNA without intron 4
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0441] FIG. 59 depicts a schematic of the RefSeq Genes for PRRT2
intron 1 corresponding to NM_145239. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0442] FIG. 60 depicts a schematic of the RefSeq Genes for RAI1
intron 4 corresponding to NM_030665. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0443] FIG. 61 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of RAI1 mRNA without intron 4 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0444] FIG. 62 depicts a schematic of the RefSeq Genes for SHANK3
intron 16 corresponding to NM_033517. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0445] FIG. 63 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of SHANK3 mRNA without intron 16
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0446] FIG. 64 depicts a schematic of the RefSeq Genes for STXBP1
intron 18 corresponding to NM_001032221. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0447] FIG. 65 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of STXBP1 mRNA without intron 18
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0448] FIG. 66 depicts a schematic of the RefSeq Genes for SETD5
intron 4 corresponding to NM_001080517. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0449] FIG. 67 depicts a schematic of the RefSeq Genes for ATP1A2
intron 22 corresponding to NM_000702. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0450] FIG. 68 depicts a schematic of the RefSeq Genes for CACNA1A
intron 36 corresponding to NM_023035. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0451] FIG. 69 depicts a schematic of the RefSeq Genes for CACNA1A
intron 37 corresponding to NM_023035. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0452] FIG. 70 depicts a schematic of the RefSeq Genes for NF2
intron 16 corresponding to NM_181832. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0453] FIG. 71 depicts a schematic of the RefSeq Genes for PEX1
intron 19 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0454] FIG. 72 depicts a schematic of the RefSeq Genes for PEX1
intron 21 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0455] FIG. 73 depicts a schematic of the RefSeq Genes for STX1B
intron 6 corresponding to NM_052874. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0456] FIG. 74 depicts a schematic of the RefSeq Genes for STX1B
intron 7 corresponding to NM_052874. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0457] FIG. 75 depicts a schematic of the RefSeq Genes for NF2
intron 16 corresponding to NM_181832. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0458] FIG. 76 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of NF2 mRNA without intron 16 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0459] FIG. 77 depicts a schematic of the RefSeq Genes for NPC1
intron 15 corresponding to NM_000271. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0460] FIG. 78 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of NPC1 mRNA without intron 15 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[0461] FIG. 79 depicts a schematic of the RefSeq Genes for ATP1A2
intron 22 corresponding to NM_000702. The Percent Intron Retention
(PIR) of the circled intron is shown.
[0462] FIG. 80 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of ATP1A2 mRNA without intron 22
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0463] FIG. 81 depicts a schematic of the RefSeq Genes for TCF4
intron 18 corresponding to NM_001243226. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0464] FIG. 82 depicts a schematic of the RefSeq Genes for FSCN2
corresponding to NM_012418 and NM_001077182. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0465] FIG. 83 depicts an exemplary graph showing the fold change
in expression levels of FSCN2 mRNA without intron 1 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0466] FIG. 84 depicts a schematic of the RefSeq Genes for FSCN2
corresponding to NM_012418 and NM_001077182. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0467] FIG. 85 depicts an exemplary graph showing the fold change
in expression levels of FSCN2 mRNA without intron 3 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0468] FIG. 86 depicts an exemplary graph showing the fold change
in expression levels of FSCN2 mRNA without intron 3 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0469] FIG. 87 depicts a schematic of the RefSeq Genes for MFSD8
corresponding to NM_152778. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0470] FIG. 88 depicts a schematic of the RefSeq Genes for MFSD8
corresponding to NM_152778. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0471] FIG. 89 depicts an exemplary graph showing the fold change
in expression levels of MFSD8 mRNA without intron 12 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0472] FIG. 90 depicts a schematic of the RefSeq Genes for OPTN
corresponding to NM_001008211, NM_00100212, NM_001008213, and
NM_021980. The Percent Intron Retention (PIR) of the circled intron
is shown.
[0473] FIG. 91 depicts an exemplary graph showing the fold change
in expression levels of OPTN mRNA without intron 7 in ARPE-19 cells
treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0474] FIG. 92 depicts a schematic of the RefSeq Genes for RDH5
corresponding to NM_002905 and NM_001199771. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0475] FIG. 93 depicts an exemplary graph showing the fold change
in expression levels of RDH5 mRNA without intron 1 in ARPE-19 cells
treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0476] FIG. 94 depicts a schematic of the RefSeq Genes for RDH5
corresponding to NM_002905 and NM_001199771. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0477] FIG. 95 depicts a schematic of the RefSeq Genes for RLBP1
corresponding to NM_000326. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0478] FIG. 96 depicts an exemplary graph showing the fold change
in expression levels of RLBP1 mRNA without intron 2 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0479] FIG. 97 depicts a schematic of the RefSeq Genes for TCF4
corresponding to NM_001243236, NM_00123235, NM_001243234,
NM_001243233, NM_001243232, NM_001243231, NM_003199, NM_001306207,
NM_001306208, NM_001243227, NM_001243228, NM_001243230,
NM_001243226, NM_001083962, NM_001330605, and NM_001330604. The
Percent Intron Retention (PIR) of the circled intron is shown.
[0480] FIG. 98 depicts a schematic of the RefSeq Genes for ROM1
corresponding to NM_000327. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0481] FIG. 99 depicts a schematic of the RefSeq Genes for CNGA3
corresponding to NM_001298 and NM_001079878. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0482] FIG. 100 depicts a schematic of the RefSeq Genes for RGR
corresponding to NM_002921, NM_001012722, and NM_001012720. The
Percent Intron Retention (PIR) of the circled intron is shown.
[0483] FIG. 101 depicts a schematic of the RefSeq Genes for RGR
corresponding to NM_002921, NM_001012722, and NM_001012720. The
Percent Intron Retention (PIR) of the circled intron is shown.
[0484] FIG. 102 depicts a schematic of the RefSeq Genes for RPE65
corresponding to NM_000329. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0485] FIG. 103 depicts a schematic of the RefSeq Genes for RPE65
corresponding to NM_000329. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0486] FIG. 104 depicts a schematic of the RefSeq Genes for RDH8
corresponding to NM_015725. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0487] FIG. 105 depicts a schematic of the RefSeq Genes for NXNL1
corresponding to NM_138454. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0488] FIG. 106 depicts a schematic of the RefSeq Genes for ABCA4
corresponding to NM_000350. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0489] FIG. 107 depicts a schematic of the RefSeq Genes for ABCA4
corresponding to NM_000350. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0490] FIG. 108 depicts an exemplary graph showing the fold change
in expression levels of ABCA4 mRNA without intron 38 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0491] FIG. 109 depicts a schematic of the RefSeq Genes for ABCA4
corresponding to NM_000350. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0492] FIG. 110 depicts a schematic of the RefSeq Genes for ABCA4
corresponding to NM_000350. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0493] FIG. 111 depicts an exemplary graph showing the fold change
in expression levels of ABCA4 mRNA without intron 40 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0494] FIG. 112 depicts a schematic of the RefSeq Genes for ABCA4
corresponding to NM_000350. The Percent Intron Retention (PIR) of
the circled intron is shown
[0495] FIG. 113 depicts a schematic of the RefSeq Genes for RDH12
corresponding to NM_152443. The Percent Intron Retention (PIR) of
the circled intron is shown.
[0496] FIG. 114 depicts a schematic of the RefSeq Genes for IDUA
corresponding to NM_000203 and NR_110313. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0497] FIG. 115 depicts an exemplary graph showing the fold change
in expression levels of IDUA mRNA without intron 3 in ARPE-19 cells
treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0498] FIG. 116 depicts a schematic of the RefSeq Genes for IDUA
corresponding to NM_000203 and NR_110313. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0499] FIG. 117 depicts a schematic of the RefSeq Genes for IDUA
corresponding to NM_000203 and NR_110313. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0500] FIG. 118 depicts a schematic of the RefSeq Genes for IDUA
corresponding to NM_000203 and NR_110313. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0501] FIG. 119 depicts a schematic of the RefSeq Genes for IDUA
corresponding to NM_000203 and NR_110313. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0502] FIG. 120 depicts a schematic of the RefSeq Genes for CTNS
corresponding to NM_004937 and NM_001031681. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0503] FIG. 121 depicts a schematic of the RefSeq Genes for CTNS
corresponding to NM_004937 and NM_001031681. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0504] FIG. 122 depicts an exemplary graph showing the fold change
in expression levels of CTNS mRNA without intron 10 in ARPE-19
cells treated for 24 hrs with 80 nM of the indicated ASOs over mock
treated cells. Data is normalized to RPL32 expression.
[0505] FIG. 123A depicts a schematic of the RefSeq Genes for AMT
corresponding to NM_001164710, NM_00116471, 1 NM_001164712,
NM_000481 and NR_028435. The Percent Intron Retention (PIR) of the
circled intron is shown.
[0506] FIG. 123B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of AMT mRNA without intron 4
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 4-exon 5 splice junction) over mock treated
cells. Data is normalized to RPL32 expression.
[0507] FIG. 123C depicts a schematic of the RefSeq Genes for AMT
corresponding to NM_001164710, NM_00116471, 1 NM_001164712,
NM_000481 and NR_028435. The Percent Intron Retention (PIR) of the
circled intron is shown.
[0508] FIG. 124A depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
2, NM_000155).
[0509] FIG. 124B depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
3, NM_000155).
[0510] FIG. 124C depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
4, NM_000155).
[0511] FIG. 124D depicts an exemplary graph showing the average
(n=3) fold change in expression levels of GALT mRNA without intron
4 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 4-exon 5 splice junction) over mock treated
cells. Data is normalized to RPL32 expression.
[0512] FIG. 124E depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
5, NM_000155).
[0513] FIG. 124F depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
7, NM_000155).
[0514] FIG. 124G depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
8, NM_000155).
[0515] FIG. 124H depicts an exemplary graph showing the average
(n=3) fold change in expression levels of GALT mRNA without intron
8 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 8-exon 9 splice junction) over mock treated
cells. Data is normalized to RPL32 expression.
[0516] FIG. 124I depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
9, NM_000155).
[0517] FIG. 124J depicts a schematic of the RefSeq Genes for GALT
corresponding to GALT: NM_000155 and NM_001258332. The Percent
Intron Retention (PIR) of the circled intron is shown (GALT intron
10, NM_000155).
[0518] FIG. 125A depicts a schematic of the RefSeq Genes for PC
corresponding to PC: NM_000920, NM_022172 and NM_001040716. The
Percent Intron Retention (PIR) of the circled intron is shown (PC
intron 16, NM_022172).
[0519] FIG. 125B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of PC mRNA without intron 16
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 16-exon 17 splice junction) over mock
treated cells. Data is normalized to RPL32 expression.
[0520] FIG. 126A depicts a schematic of the RefSeq Genes for FAH
corresponding to FAH: NM_000137. The Percent Intron Retention (PIR)
of the circled intron is shown (FAH intron 1, NM_000137).
[0521] FIG. 126B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of FAH mRNA without intron 1
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 1-exon 2 splice junction) over mock treated
cells. Data is normalized to RPL32 expression.
[0522] FIG. 127A depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0523] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 3, NM_006238).
[0524] FIG. 127B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of PPARD mRNA without intron
3 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0525] FIG. 127C depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0526] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 4, NM_006238).
[0527] FIG. 127D depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0528] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 5, NM_006238).
[0529] FIG. 127E depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0530] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 6, NM_006238).
[0531] FIG. 127F depicts an exemplary graph showing the average
(n=3) fold change in expression levels of PPARD mRNA without intron
6 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0532] FIG. 127G depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0533] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 7, NM_006238).
[0534] FIG. 128A depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 3, NM_000018).
[0535] FIG. 128B depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 5, NM_000018).
[0536] FIG. 128C depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 8, NM_000018).
[0537] FIG. 128D depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 9, NM_000018).
[0538] FIG. 128E depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 10, NM_000018).
[0539] FIG. 128F depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 13, NM_000018).
[0540] FIG. 128G depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 18, NM_000018).
[0541] FIG. 128H depicts a schematic of the RefSeq Genes for ACADVL
corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and
NM_001033859. The Percent Intron Retention (PIR) of the circled
intron is shown (ACADVL intron 19, NM_000018).
[0542] FIG. 129A depicts a schematic of the RefSeq Genes for HMBS
corresponding to HMBS: NM_000190, NM_001258208, NM_001024382 and
NM_001258209. The Percent Intron Retention (PIR) of the circled
intron is shown (HMBS intron 10, NM_000190).
[0543] FIG. 129B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of HMBS mRNA without intron
10 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 10-exon 11 splice junction) over mock
treated cells. Data is normalized to RPL32 expression.
[0544] FIG. 129C depicts a schematic of the RefSeq Genes for HMBS
corresponding to HMBS: NM_000190, NM_001258208, NM_001024382 and
NM_001258209. The Percent Intron Retention (PIR) of the circled
intron is shown (HMBS intron 11, NM_000190).
[0545] FIG. 130A depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD: NM_000374 and NR_036510. The Percent Intron
Retention (PIR) of the circled intron is shown (UROD intron 3,
NM_000374).
[0546] FIG. 130B depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD: NM_000374 and NR_036510. The Percent Intron
Retention (PIR) of the circled intron is shown (UROD intron 4,
NM_000374).
[0547] FIG. 130C depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD: NM_000374 and NR_036510. The Percent Intron
Retention (PIR) of the circled intron is shown (UROD intron 5
NM_000374).
[0548] FIG. 130D depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD: NM_000374 and NR_036510. The Percent Intron
Retention (PIR) of the circled intron is shown (UROD intron 6,
NM_000374).
[0549] FIG. 130E depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD: NM_000374 and NR_036510. The Percent Intron
Retention (PIR) of the circled intron is shown (UROD intron 7,
NM_000374).
[0550] FIG. 131A depicts a schematic of the RefSeq Genes for ALMS1
corresponding to ALMS1: NM_015120. The Percent Intron Retention
(PIR) of the circled intron is shown (ALMS1 intron 21,
NM_015120).
[0551] FIG. 131B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of ALMS1 mRNA without intron
21 in Huh7 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 21-exon 22 splice junction) over mock
treated cells. Data is normalized to RPL32 expression.
[0552] FIG. 132A depicts a schematic of the RefSeq Genes for ASL
corresponding to ASL: NM_000048, NM_001024943, NM_001024944 and
NM_001024946. The Percent Intron Retention (PIR) of the circled
intron is shown (ASL intron 7, NM_000048).
[0553] FIG. 132B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of ASL mRNA without intron 7
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 7-exon 8 splice junction) over mock treated
cells. Data is normalized to RPL32 expression.
[0554] FIG. 132C depicts a schematic of the RefSeq Genes for ASL
corresponding to ASL: NM_000048, NM_001024943, NM_001024944 and
NM_001024946. The Percent Intron Retention (PIR) of the circled
intron is shown (ASL intron 8, NM_000048).
[0555] FIG. 132D depicts an exemplary graph showing the average
(n=3) fold change in expression levels of ASL mRNA without intron 8
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 8-exon 9 splice junction) over mock treated
cells. Data is normalized to RPL32 expression.
[0556] FIG. 132E depicts a schematic of the RefSeq Genes for ASL
corresponding to ASL: NM_000048, NM_001024943, NM_001024944 and
NM_001024946. The Percent Intron Retention (PIR) of the circled
intron is shown (ASL intron 9, NM_000048).
[0557] FIG. 132F depicts an exemplary graph showing the average
(n=3) fold change in expression levels of ASL mRNA without intron 7
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs (spanning the exon 9-exon 10 splice junction) over mock
treated cells. Data is normalized to RPL32 expression.
[0558] FIG. 132G depicts a schematic of the RefSeq Genes for ASL
corresponding to ASL: NM_000048, NM_001024943, NM_001024944 and
NM_001024946. The Percent Intron Retention (PIR) of the circled
intron is shown (ASL intron 16, NM_000048).
[0559] FIG. 133A depicts a schematic of the RefSeq Genes for ATP7B1
corresponding to ATP7B1: NM_001243182, NM_000053, NM_001005918,
NM_001330579 and NM_001330578. The Percent Intron Retention (PIR)
of the circled intron is shown (ATP7B1 intron 7, NM_000053).
[0560] FIG. 133B depicts a schematic of the RefSeq Genes for ATP7B1
corresponding to ATP7B1: NM_001243182, NM_000053, NM_001005918,
NM_001330579 and
[0561] NM_001330578. The Percent Intron Retention (PIR) of the
circled intron is shown (ATP7B1 intron 13, NM_000053).
[0562] FIG. 133C depicts an exemplary graph showing the average
(n=3) fold change in expression levels of ATP7B1 mRNA without
intron 13 in ARPE-19 cells treated for 24 hrs with 80 nM of the
indicated ASOs (spanning the exon 13-exon 14 splice junction) over
mock treated cells. Data is normalized to RPL32 expression.
[0563] FIG. 134 depicts a schematic of the RefSeq Genes for HFE
corresponding to HFE: NM_139007, NM_139006, NM_139004, NM_139003,
NM_001300749, NM_139009, NM_139008, and NM_000410. The Percent
Intron Retention (PIR) of the circled intron is shown (HFE intron
2, NM_000410).
[0564] FIG. 135A depicts a schematic of the RefSeq Genes for HSD3B7
corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
The Percent Intron Retention (PIR) of the circled intron is shown
(HSD3B7 intron 1, NM_001142777).
[0565] FIG. 135B depicts a schematic of the RefSeq Genes for HSD3B7
corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
The Percent Intron Retention (PIR) of the circled intron is shown
(HSD3B7 intron 1, NM_001142777).
[0566] FIG. 135C depicts an exemplary graph showing the average
(n=3) fold change in expression levels of HSD3B7 mRNA without
intron 2 in ARPE-19 cells treated for 24 hrs with 80 nM of the
indicated ASOs (spanning the exon 2-exon 3 splice junction) over
mock treated cells. Data is normalized to RPL32 expression.
[0567] FIG. 135D depicts a schematic of the RefSeq Genes for HSD3B7
corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
The Percent Intron Retention (PIR) of the circled intron is shown
(HSD3B7 intron 3, NM_001142777).
[0568] FIG. 135E depicts a schematic of the RefSeq Genes for HSD3B7
corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
The Percent Intron Retention (PIR) of the circled intron is shown
(HSD3B7 intron 4, NM_001142777).
[0569] FIG. 135F depicts an exemplary graph showing the average
(n=3) fold change in expression levels of HSD3B7 mRNA without
intron 4 in ARPE-19 cells treated for 24 hrs with 80 nM of the
indicated ASOs (spanning the exon 4-exon 5 splice junction) over
mock treated cells. Data is normalized to RPL32 expression.
[0570] FIG. 135G depicts a schematic of the RefSeq Genes for HSD3B7
corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
The Percent Intron Retention (PIR) of the circled intron is shown
(HSD3B7 intron 5, NM_025193).
[0571] FIG. 135H depicts a schematic of the RefSeq Genes for HSD3B7
corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
The Percent Intron Retention (PIR) of the circled intron is shown
(HSD3B7 intron 6, NM_025193).
[0572] FIG. 136 depicts a schematic of the RefSeq Genes for NCOA5
corresponding to NCOA5: NM_020967. The Percent Intron Retention
(PIR) of the circled intron is shown (NCOA5 intron2,
NM_020967).
[0573] FIG. 137A depicts a schematic of the RefSeq Genes for PPOX
corresponding to PPOX: NM_000309 and NM_001122764. The Percent
Intron Retention (PIR) of the circled intron is shown (PPOX intron
3, 000309).
[0574] FIG. 137B depicts a schematic of the RefSeq Genes for PPOX
corresponding to PPOX: NM_000309 and NM_001122764. The Percent
Intron Retention (PIR) of the circled intron is shown (PPOX intron
4, 000309).
[0575] FIG. 137C depicts a schematic of the RefSeq Genes for PPOX
corresponding to PPOX: NM_000309 and NM_001122764. The Percent
Intron Retention (PIR) of the circled intron is shown (PPOX intron
5, 000309).
[0576] FIG. 137D depicts a schematic of the RefSeq Genes for PPOX
corresponding to PPOX: NM_000309 and NM_001122764. The Percent
Intron Retention (PIR) of the circled intron is shown (PPOX intron
7, 000309).
[0577] FIG. 137E depicts a schematic of the RefSeq Genes for PPOX
corresponding to PPOX: NM_000309 and NM_001122764. The Percent
Intron Retention (PIR) of the circled intron is shown (PPOX intron
8, 000309).
[0578] FIG. 137F depicts a schematic of the RefSeq Genes for PPOX
corresponding to PPOX: NM_000309 and NM_001122764. The Percent
Intron Retention (PIR) of the circled intron is shown (PPOX intron
10, 000309).
[0579] FIG. 137G depicts a schematic of the RefSeq Genes for PPOX
corresponding to PPOX: NM_000309 and NM_001122764. The Percent
Intron Retention (PIR) of the circled intron is shown (PPOX intron
12, 000309).
[0580] FIG. 138A depicts a schematic of the RefSeq Genes for CTNS
corresponding to NM_004937 and NM_001031681. The Percent Intron
Retention (PIR) of the circled intron is shown.
[0581] FIG. 138B depicts a schematic of the RefSeq Genes for CTNS
corresponding to NM_004937 and NM_001031681. The PIR of the circled
intron is shown.
[0582] FIG. 138C depicts an exemplary graph showing the average
(n=3) fold change in expression levels of CTNS mRNA without intron
10 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0583] FIG. 139A depicts a schematic of the RefSeq Genes for
CYP24A1 corresponding to NM_000782 and NM_001128915. The PIR of the
circled intron is shown.
[0584] FIG. 139B depicts a schematic of the RefSeq Genes for
CYP24A1 corresponding to NM_000782 and NM_001128915. The PIR of the
circled intron is shown.
[0585] FIG. 139C depicts an exemplary graph showing the average
(n=3) fold change in expression levels of CYP24A1 mRNA without
intron 10 in ARPE-19 cells treated for 24 hrs with 80 nM of the
indicated ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0586] FIG. 140 depicts a schematic of the RefSeq Genes for PAX2
corresponding to NM_001304569, NM_000278, NM_003990, NM_003988,
NM_003987 and NM_003989.
[0587] FIG. 141A depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0588] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 3, NM_006238).
[0589] FIG. 141B depicts an exemplary graph showing the average
(n=3) fold change in expression levels of PPARD mRNA without intron
3 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0590] FIG. 141C depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0591] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 4, NM_006238).
[0592] FIG. 141D depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0593] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 5, NM_006238).
[0594] FIG. 141E depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820.
[0595] The Percent Intron Retention (PIR) of the circled intron is
shown (PPARD intron 6, NM_006238).
[0596] FIG. 141F depicts an exemplary graph showing the average
(n=3) fold change in expression levels of PPARD mRNA without intron
6 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[0597] FIG. 141G depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD: NM_006238, NM_177435, NM_001171818,
NM_001171819 and NM_001171820. The Percent Intron Retention (PIR)
of the circled intron is shown (PPARD intron 7, NM_006238).
DETAILED DESCRIPTION OF THE INVENTION
Compositions and Methods for Treatment of Alagille Syndrome
[0598] Individual introns in primary transcripts of protein-coding
genes having more than one intron are spliced from the primary
transcript with different efficiencies. In most cases only the
fully spliced mRNA is exported through nuclear pores for subsequent
translation in the cytoplasm. Unspliced and partially spliced
transcripts are detectable in the nucleus. It is generally thought
that nuclear accumulation of transcripts that are not fully spliced
is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[0599] Substantial levels of partially-spliced transcripts of the
JAG1 gene, which encodes the JAG1 protein that is deficient in the
debilitating genetic diseases, Alagille Syndrome, have been
discovered in the nucleus of human cells. These JAG1 pre-mRNA
species comprise at least one retained intron. The present
invention provides compositions and methods for upregulating
splicing of one or more retained JAG1 introns that are
rate-limiting for the nuclear stages of gene expression to increase
steady-state production of fully-spliced, mature mRNA, and thus,
translated JAG1 protein levels. These compositions and methods
utilize antisense oligomers (ASOs) that promote constitutive
splicing at an intron splice site of a retained-intron-containing
JAG1 pre-mRNA that accumulates in the nucleus. Thus, in
embodiments, JAG1 protein is increased using the methods of the
invention to treat a condition caused by JAG1 deficiency.
[0600] In other embodiments, the methods of the invention are used
to increase JAG1 production to treat a condition in a subject in
need thereof. In embodiments, the subject has a condition in which
JAG1 is not necessarily deficient relative to wild-type, but where
an increase in JAG1 mitigates the condition nonetheless. In
embodiments, the subject has a muscular dystrophy. In embodiments,
the subject has a dystrophin deficiency. For example, Vieira, et
al., 2015, "Jagged 1 Rescues the Duchenne Muscular Dystrophy
Phenotype," Cell 163:1204-1213, describe findings suggesting that
increasing JAG1 expression in muscle can rescue
dystrophin-deficient phenotypes in two different animal models for
the disease.
Alagille Syndrome
[0601] Alagille syndrome (ALGS), also known as arteriohepatic
dysplasia, is an autosomal dominant, multisystem disorder
(Turnpenny and Ellard, 2012). The main clinical and pathological
findings are liver disease due to paucity of intrahepatic bile
ducts, heart disease, vascular anomalies, skeletal anomalies,
ophthalmic features, facial features, renal anomalies, growth
retardation, and pancreatic insufficiency. Clinical findings of
ALGS are variable, with clinical diagnosis of "classic" ALGS based
on the presence of 3 of 5 clinical features including anomalies of
the liver, heart, vertebrae, eye, or face along with bile duct
paucity (Lu et al., Am. J. Hum. Gen. 2003, 72, 1065-1070; Penton et
al., Seminars Cell & Dev. Biol. 2012, 23, 450-457). The
reported ALGS prevalence of 1:70,000 is thought to be an
underestimate because of the variability and reduced penetrance of
the condition.
[0602] Mutations of genes involved in Notch signaling have been
reported to cause ALGS. JAG1 encodes JAG1 protein, a cell surface
ligand for the Notch transmembrane receptors. The human genomic
sequence of the JAG1 gene is set forth at NCBI Gene ID 182,
described by, e.g., Jurkiewicz, D., et al., 2014, "Spectrum of JAG1
gene mutations in Polish patients with Alagille syndrome," J. Appl.
Genetics 55:329-336, and the amino acid sequence at UniProtKB:
P78504-1, Oda, T., et al., 1997, "Identification and cloning of the
human homolog (JAG1) of the rat Jagged1 gene from the Alagille
syndrome critical region at 20p12," Genomics 43 (3): 376-379, all
incorporated by reference herein. The JAG1 canonical mRNA sequence
is set forth at NCBI Reference Sequence: NM_000214.2, described by
Duryagina R, et al., 2013, "Overexpression of Jagged-1 and its
intracellular domain in human mesenchymal stromal cells
differentially affect the interaction with hematopoietic stem and
progenitor cells," Stem Cells Dev. 22 (20), 2736-2750, both
incorporated by reference herein. Binding of JAG1 ligand to Notch
triggers a signaling cascade that results in transcription of genes
involved in cell fate determination and differentiation. Mutations
in JAG1 cause ALGS type 1, the most prevalent disease type, while
mutations in NOTCH2 cause ALGS type 2.
[0603] The JAG1 gene consists of 26 exons and is located on
chromosome 20p11.2-20p12. JAG1 mutations in ALGS are spread across
the entire protein, and there is a high de novo mutation rate of
approximately 60%. 80% of mutations include frameshift, nonsense,
and splice site mutations; whole gene deletions account for 7% of
mutations, and missense mutations represent 12% (Lu et al., 2003).
Because whole gene deletion results in a phenotype similar to that
seen for truncating and missense mutations, haploinsufficiency is
the likely mechanism of disease causation in the majority of cases
(Lu et al., 2003; Turnpenny and Ellard, 2012).
[0604] Mutations associated with the classic ALGS phenotype that
includes liver disease displayed functional haploinsufficiency,
resulting in cell surface concentrations of JAG1 that were 50% of
wild-type levels (Lu et al., 2003). By contrast, a JAG1 missense
mutation that results in expression of the JAG1-G274D protein has
been reported to produce two protein populations, with one
population exhibiting a glycosylation defect, thus preventing
transport to the cell surface. The JAG1-G274D mutation was
associated with a cardiac-specific phenotype in the absence of
liver disease, with carriers of the mutation having more than 50%
but less than 100% of the normal concentration of JAG1 on the cell
surface. Thus, the developing liver may require less JAG1 than the
developing heart (Lu et al., 2003).
Muscle Repair
[0605] As noted above, the potential for JAG1 to rescue
dystrophin-deficient phenotypes in two animal models has been
described (Vieira, et al., 2015). Deficiency of muscle dystrophin
in muscular dystrophies, including Duchenne Muscular Dystrophy
(DMD) and Becker Muscular Dystrophy (BMD), causes progressive
muscle degeneration and wasting. In DMD, death eventually results
due to respiratory or cardiac failure. Although JAG1 is not
deficient in these conditions, increased levels of JAG1 may
stimulate muscle cell proliferation and repair. In embodiments, the
methods and compositions of the present invention are used to
increase JAG1 production to stimulate muscle repair in a subject in
need thereof. In embodiments, the subject has a muscular dystrophy.
In embodiments, the muscular dystrophy is DMD, BMD, or limb girdle,
congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal,
or Emery-Dreifuss muscular dystrophy. In embodiments, the subject
has a dystrophin deficiency. In embodiments, the dystrophin
deficiency is DMD or BMD. In embodiments, the subject has a
muscular dystrophy caused by a deficiency in dysferlin (encoded by
the DYSF gene), emerin (encoded by the EMD gene), DUX4,
myotonin-protein kinase (MT-PK) also known as myotonic dystrophy
protein kinase (MDPK) or dystrophia myotonica protein kinase (DMK)
(encoded by the DMPK gene), Cellular nucleic acid-binding protein
(encoded by the CNBP gene), or polyadenylate-binding protein 2
(PABP-2) also known as polyadenylate-binding nuclear protein 1
(PABPN1) (encoded by the PABPN1 gene). Retained Intron Containing
Pre-mRNA (RIC Pre-mRNA)
[0606] In embodiments, the methods of the present invention exploit
the presence of retained-intron-containing pre-mRNA (RIC pre-mRNA)
transcribed from the JAG1 gene and encoding JAG1 protein, in the
cell nucleus. Splicing of the identified JAG1 RIC pre-mRNA species
to produce mature, fully-spliced, JAG1 mRNA, is induced using ASOs
that stimulate splicing out of the retained introns. The resulting
mature JAG1 mRNA can be exported to the cytoplasm and translated,
thereby increasing the amount of JAG1 protein in the patient's
cells and alleviating symptoms of Alagille syndrome. This method,
described further below, is known as Targeted Augmentation of
Nuclear Gene Output (TANGO).
JAG1 Nuclear Transcripts
[0607] As described herein in the Examples, the JAG1 gene was
analyzed for intron-retention events and retention of introns 13,
18, and 23 was observed. RNA sequencing (RNAseq), visualized in the
UCSC genome browser, showed JAG1 transcripts expressed in THLE-3
(human liver epithelial) cells and localized in either the
cytoplasmic or nuclear fraction. In both fractions, reads were not
observed for the majority of the introns. However, higher read
density was detected for introns 13, 18, and 23 in the nuclear
fraction compared to the cytoplasmic fraction indicating that
splicing efficiency of introns 13, 18, and 23 is low, resulting in
intron retention. The retained-intron containing pre-mRNA
transcripts accumulate primarily in the nucleus and are not
translated into the JAG1 protein. The read density for introns 13,
18, and 23, indicated 12%, 14%, and 17% intron retention,
respectively.
[0608] Table 1 provides a non-limiting list of target sequences of
a JAG1 RIC pre-mRNA transcript by sequence ID, and ASOs by sequence
ID, useful for increasing production of JAG1 protein by targeting a
region of a JAG1 RIC pre-mRNA. In embodiments, other ASOs useful
for this purpose are identified, using, e.g., methods described
herein.
TABLE-US-00001 TABLE 1 List of targets and ASOs targeting the JAG1
gene Gene Target SEQ ID Pre-mRNA Retained Sequence NO: SEQ ID NO:
ASOs Intron SEQ ID NO: JAG1 JAG1:NM_ SEQ ID NOs: 3-111 18 439 SEQ
ID 000214 SEQ ID NOs: 112- 13 438 NO: SEQ ID NO: 2 331 1 SEQ ID
NOs: 332- 23 437 436
[0609] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a JAG1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a JAG1
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 1 comprising a retained intron. In some embodiments, the ASOs
disclosed herein target a JAG1 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a JAG1 RIC pre-mRNA transcript
comprising a retained intron at 13, 18, 23 or a combination
thereof, wherein the intron numbering correspond to the mRNA
sequence at NM_000214.
[0610] In some embodiments, the ASO targets a JAG1 RIC pre-mRNA
sequence according to SEQ ID NO: 2. In some embodiments, the ASO
targets a JAG1 RIC pre-mRNA sequence according to SEQ ID NO: 2
comprising a retained intron 13, a retained intron 18, a retained
intron 23, or a combination thereof. In some embodiments, the ASOs
disclosed herein target SEQ ID NOs: 437-439. In some embodiments,
the ASO has a sequence according to any one of SEQ ID NOs:
3-436.
[0611] In some embodiments, the ASO targets exon 18 or exon 19 of a
JAG1 RIC pre-mRNA comprising a retained intron 18, wherein the
intron numbering correspond to the mRNA sequence at NM_000214. In
some embodiments, the ASO targets an exon 18 sequence upstream (or
5') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an exon 18
sequence about 4 to about 99 nucleotides upstream (or 5') from the
5' splice site of a JAG1 RIC pre-mRNA comprising the retained
intron 18. In some embodiments, the ASO targets an exon 19 sequence
downstream (or 3') from the 3' splice site of a JAG1 RIC pre-mRNA
comprising the retained intron 18. In some embodiments, the ASO
targets an exon 19 sequence about 2 to about 7 nucleotides
downstream (or 3') from the 3' splice site of a JAG1 RIC pre-mRNA
comprising the retained intron 18.
[0612] In some embodiments, the ASO targets intron 18 in a JAG1 RIC
pre-mRNA comprising a retained intron 18, wherein the intron
numbering correspond to the mRNA sequence at NM_000214. In some
embodiments, the ASO targets an intron 18 sequence downstream (or
3') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence about 16 to about 239 nucleotides downstream (or 3')
from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence upstream (or 5') from the 3' splice site of a JAG1 RIC
pre-mRNA comprising the retained intron 18. In some embodiments,
the ASO targets an intron 18 sequence about 16 to about 253
nucleotides upstream (or 5') from the 3' splice site of a JAG1 RIC
pre-mRNA comprising the retained intron 18.
[0613] In some embodiments, the ASO targets exon 13 or exon 14 of a
JAG1 RIC pre-mRNA comprising a retained intron 13, wherein the
intron numbering correspond to the mRNA sequence at NM_000214. In
some embodiments, the ASO targets an exon 13 sequence upstream (or
5') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an exon 13
sequence about 14 to about 131 nucleotides upstream (or 5') from
the 5' splice site of a JAG/RIC pre-mRNA comprising the retained
intron 13. In some embodiments, the ASO targets an exon 14 sequence
downstream (or 3') from the 3' splice site of a JAG1 RIC pre-mRNA
comprising the retained intron 13. In some embodiments, the ASO
targets an exon 14 sequence about 2 to about 147 nucleotides
downstream (or 3') from the 3' splice site of a JAG1 RIC pre-mRNA
comprising the retained intron 13.
[0614] In some embodiments, the ASO targets intron 13 in a JAG1 RIC
pre-mRNA comprising a retained intron 13, wherein the intron
numbering correspond to the mRNA sequence at NM_000214. In some
embodiments, the ASO targets an intron 13 sequence downstream (or
3') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence about 16 to about 440 nucleotides downstream (or 3')
from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence upstream (or 5') from the 3' splice site of a JAG1 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an intron 13 sequence about 16 to about 441
nucleotides upstream (or 5') from the 3' splice site of a JAG1 RIC
pre-mRNA comprising the retained intron 13.
[0615] In some embodiments, the ASO targets exon 23 or exon 24 of a
JAG1 RIC pre-mRNA comprising a retained intron 23, wherein the
intron numbering correspond to the mRNA sequence at NM_000214. In
some embodiments, the ASO targets an exon 23 sequence upstream (or
5') from the 5' splice site of a JAG/RIC pre-mRNA comprising the
retained intron 23. In some embodiments, the ASO targets an exon 23
sequence about 4 to about 216 nucleotides upstream (or 5') from the
5' splice site of a JAG1 RIC pre-mRNA comprising the retained
intron 23. In some embodiments, the ASO targets an exon 24 sequence
downstream (or 3') from the 3' splice site of a JAG1 RIC pre-mRNA
comprising the retained intron 23. In some embodiments, the ASO
targets an exon 24 sequence about 2 to about 114 nucleotides
downstream (or 3') from the 3' splice site of a JAG1 RIC pre-mRNA
comprising the retained intron 23.
[0616] In some embodiments, the ASO targets intron 23 in a JAG1 RIC
pre-mRNA comprising a retained intron 23, wherein the intron
numbering correspond to the mRNA sequence at NM_000214. In some
embodiments, the ASO targets an intron 23 sequence downstream (or
3') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 23. In some embodiments, the ASO targets an intron
23 sequence about 6 to about 121 nucleotides downstream (or 3')
from the 5' splice site of a JAG1 RIC pre-mRNA comprising the
retained intron 23. In some embodiments, the ASO targets an intron
23 sequence upstream (or 5') from the 3' splice site of a JAG1 RIC
pre-mRNA comprising the retained intron 23. In some embodiments,
the ASO targets an intron 23 sequence about 16 to about 121
nucleotides upstream (or 5') from the 3' splice site of a JAG/RIC
pre-mRNA comprising the retained intron 23.
[0617] It is understood that the intron numbering may change in
reference to a different JAG1 isoform sequence. One of skill in the
art can determine the corresponding intron number in any JAG1
isoform based on an intron sequence provided herein or using the
intron number provided in reference to the mRNA sequence at
NM_000214.2. One of skill in the art also can determine the
sequences of flanking exons in any JAG1 isoform for targeting using
the methods of the invention, based on an intron sequence provided
herein or using the intron number provided in reference to the mRNA
sequence at NM_000214.2. In embodiments, the compositions and
methods of the present invention are used to increase the
expression of any known JAG1 isoform.
JAG1 Protein Expression
[0618] As described above, JAG1 mutations in ALGS are spread across
the entire protein, and there is a high de novo mutation rate of
approximately 60%. Of the mutations characterized, 80% include
frameshift, nonsense, and splice site mutations; whole gene
deletions account for 7% of mutations, and missense mutations
represent 12% (Lu et al., 2003).
[0619] In embodiments, the methods described herein are used to
increase the production of a functional JAG1 protein. As used
herein, the term "functional" refers to the amount of activity or
function of a JAG1 protein that is necessary to eliminate any one
or more symptoms of a treated condition, e.g., Alagille syndrome or
a muscular dystrophy. In embodiments, the methods are used to
increase the production of a partially functional JAG1 protein. As
used herein, the term "partially functional" refers to any amount
of activity or function of the JAG1 protein that is less than the
amount of activity or function that is necessary to eliminate or
prevent any one or more symptoms of a disease or condition. In some
embodiments, a partially functional protein or RNA will have at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 75%, at least 80%, 85%, at
least 90%, or at least 95% less activity relative to the fully
functional protein or RNA.
[0620] In embodiments, the method is a method of increasing the
expression of the JAG1 protein by cells of a subject having a RIC
pre-mRNA encoding the JAG1 protein, wherein the subject has
Alagille syndrome caused by a deficient amount of activity of JAG1
protein, and wherein the deficient amount of the JAG1 protein is
caused by haploinsufficiency of the JAG1 protein. In such an
embodiment, the subject has a first allele encoding a functional
JAG1 protein, and a second allele from which the JAG1 protein is
not produced. In another such embodiment, the subject has a first
allele encoding a functional JAG1 protein, and a second allele
encoding a nonfunctional JAG1 protein. In another such embodiment,
the subject has a first allele encoding a functional JAG1 protein,
and a second allele encoding a partially functional JAG1 protein.
In any of these embodiments, the antisense oligomer binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele (encoding functional JAG1 protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding
functional JAG1 protein, and an increase in the expression of the
JAG1 protein in the cells of the subject.
[0621] In embodiments, the subject has a first allele encoding a
functional JAG1 protein, and a second allele encoding a partially
functional JAG1 protein, and the antisense oligomer binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele (encoding functional JAG1 protein) or a targeted portion of
the RIC pre-mRNA transcribed from the second allele (encoding
partially functional JAG1 protein), thereby inducing constitutive
splicing of the retained intron from the RIC pre-mRNA, and causing
an increase in the level of mature mRNA encoding the JAG1 protein,
and an increase in the expression of functional or partially
functional JAG1 protein in the cells of the subject.
[0622] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of JAG1
protein in cells of a subject having a RIC pre-mRNA encoding JAG1
protein, wherein the subject has a deficiency, e.g., Alagille
syndrome, in the amount or function of a JAG1 protein.
[0623] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease or condition is targeted
by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the
disease is targeted by the ASOs. For example, a disease that is the
result of a mutation or deficiency of a first protein in a
particular pathway may be ameliorated by targeting a RIC pre-mRNA
that encodes a second protein, thereby increasing production of the
second protein. In some embodiments, the function of the second
protein is able to compensate for the mutation or deficiency of the
first protein (which is causative of the disease or condition). In
embodiments, the first protein is a protein deficient in a muscular
dystrophy and the second protein is JAG1. In embodiments, the first
protein is a dystrophin and the second protein is JAG1.
[0624] In embodiments, the subject has:
[0625] a. a first mutant allele from which [0626] i) the JAG1
protein is produced at a reduced level compared to production from
a wild-type allele, [0627] ii) the JAG1 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0628] iii) the JAG1 protein or functional RNA is not
produced; and
[0629] b. a second mutant allele from which [0630] i) the JAG1
protein is produced at a reduced level compared to production from
a wild-type allele, [0631] ii) the JAG1 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0632] iii) the JAG1 protein is not produced, and
wherein the RIC pre-mRNA is transcribed from the first allele
and/or the second allele. In these embodiments, the ASO binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele or the second allele, thereby inducing constitutive splicing
of the retained intron from the RIC pre-mRNA, and causing an
increase in the level of mRNA encoding JAG1 protein and an increase
in the expression of the target protein or functional RNA in the
cells of the subject. In these embodiments, the target protein or
functional RNA having an increase in expression level resulting
from the constitutive splicing of the retained intron from the RIC
pre-mRNA is either in a form having reduced function compared to
the equivalent wild-type protein (partially-functional), or having
full function compared to the equivalent wild-type protein
(fully-functional).
[0633] In embodiments, the level of mRNA encoding JAG1 protein is
increased 1.1 to 10-fold, when compared to the amount of mRNA
encoding JAG1 that is produced in a control cell, e.g., one that is
not treated with the antisense oligomer or one that is treated with
an antisense oligomer that does not bind to the targeted portion of
the JAG1 RIC pre-mRNA.
[0634] In embodiments, the condition caused by a deficient amount
or activity of JAG1 protein is not a condition caused by
alternative or aberrant splicing of the retained intron to which
the ASO is targeted. In embodiments, the condition caused by a
deficient amount or activity of the JAG1 protein is not a condition
caused by alternative or aberrant splicing of any retained intron
in a RIC pre-mRNA encoding the JAG1 protein. In embodiments,
alternative or aberrant splicing may occur in a pre-mRNA
transcribed from the gene, however the compositions and methods of
the invention do not prevent or correct this alternative or
aberrant splicing.
[0635] In embodiments, a subject treated using the methods of the
invention expresses a partially functional JAG1 protein from one
allele, wherein the partially functional JAG1 protein is caused by
a frameshift mutation, a nonsense mutation, a missense mutation, or
a partial gene deletion. In embodiments, a subject treated using
the methods of the invention expresses a nonfunctional JAG1 protein
from one allele, wherein the nonfunctional JAG1 protein is caused
by a frameshift mutation, a nonsense mutation, a missense mutation,
a partial gene deletion, in one allele. In embodiments, a subject
treated using the methods of the invention has a JAG1 whole gene
deletion, in one allele.
[0636] In embodiments, the subject has a JAG1 missense mutation
selected from G274D, L37S, R184H, P163L, P871R, C234Y, C664S,
P810L, or R937Q. In embodiments, the subject has JAG1 deletion
mutation 1485-1486delCT. In embodiments, the subject has JAG1
duplication mutation 414-415dupGT. In embodiments, a subject having
any JAG1 mutation known in the art and described in the literature,
e.g., by Lu, et al., 2003, Penton, et al., 2012, referenced above,
is treated using the methods and compositions of the present
invention.
Use of TANGO for Increasing JAG1 Protein Expression
[0637] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) is used in the methods of the invention
to increase expression of a JAG1 protein. In these embodiments, a
retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding JAG1
protein is present in the nucleus of a cell. Cells having a JAG1
RIC pre-mRNA that comprises a retained intron, an exon flanking the
5' splice site, and an exon flanking the 3' splice site, encoding
the JAG1 protein, are contacted with antisense oligomers (ASOs)
that are complementary to a targeted portion of the RIC pre-mRNA.
Hybridization of the ASOs to the targeted portion of the RIC
pre-mRNA results in enhanced splicing at the splice site (5' splice
site or 3' splice site) of the retained intron and subsequently
increases target protein production.
[0638] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[0639] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a population of RIC pre-mRNA encoding the JAG1 protein.
In embodiments, the most retained intron in a population of RIC
pre-mRNA encoding the JAG1 protein is intron 23.
[0640] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 65%, about 5% to about 60%, about 5%
to about 55%, about 5% to about 50%, about 5% to about 45%, about
5% to about 40%, about 5% to about 35%, about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 10% to about 100%, about 10% to about 95%, about 10% to
about 90%, about 10% to about 85%, about 10% to about 80%, about
10% to about 75%, about 10% to about 70%, about 10% to about 65%,
about 10% to about 60%, about 10% to about 65%, about 10% to about
60%, about 10% to about 55%, about 10% to about 50%, about 10% to
about 45%, about 10% to about 40%, about 10% to about 35%, about
10% to about 30%, about 10% to about 25%, about 10% to about 20%,
about 15% to about 100%, about 15% to about 95%, about 15% to about
90%, about 15% to about 85%, about 15% to about 80%, about 15% to
about 75%, about 15% to about 70%, about 15% to about 65%, about
15% to about 60%, about 15% to about 65%, about 15% to about 60%,
about 15% to about 55%, about 15% to about 50%, about 15% to about
45%, about 15% to about 40%, about 15% to about 35%, about 15% to
about 30%, about 15% to about 25%, about 20% to about 100%, about
20% to about 95%, about 20% to about 90%, about 20% to about 85%,
about 20% to about 80%, about 20% to about 75%, about 20% to about
70%, about 20% to about 65%, about 20% to about 60%, about 20% to
about 65%, about 20% to about 60%, about 20% to about 55%, about
20% to about 50%, about 20% to about 45%, about 20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about
100%, about 25% to about 95%, about 25% to about 90%, about 25% to
about 85%, about 25% to about 80%, about 25% to about 75%, about
25% to about 70%, about 25% to about 65%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 60%, about 25% to about
55%, about 25% to about 50%, about 25% to about 45%, about 25% to
about 40%, or about 25% to about 35%, retention. ENCODE data
(described by, e.g., Tilgner, et al., 2012, "Deep sequencing of
subcellular RNA fractions shows splicing to be predominantly
co-transcriptional in the human genome but inefficient for
lncRNAs," Genome Research 22(9):1616-25) can be used to aid in
identifying retained introns.
[0641] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g. in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features (e.g.
in the case of an antisense oligomer, the presence of additional,
unrecited nucleobases).
[0642] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g. nucleobase sequence) as well as those which do not materially
affect the character or function of the claimed invention. As used
herein, the term "consisting" is used to indicate the presence of
the recited feature (e.g. nucleobase sequence) alone (so that in
the case of an antisense oligomer consisting of a specified
nucleobase sequence, the presence of additional, unrecited
nucleobases is excluded).
[0643] In embodiments, the targeted region is in a retained intron
that is the second most abundant intron in a population of RIC
pre-mRNA encoding the JAG1 protein. For example, the second most
abundant retained intron may be targeted rather than the most
abundant retained intron due to the uniqueness of the nucleotide
sequence of the second most abundant retained intron, ease of ASO
design to target a particular nucleotide sequence, and/or amount of
increase in protein production resulting from targeting the intron
with an ASO. In embodiments, the retained intron is the second most
abundant intron in a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein in a cell, wherein the
population of RIC pre-mRNAs comprises two or more retained introns.
In embodiments, an antisense oligomer targeted to the second most
abundant intron in the population of RIC pre-mRNAs encoding the
target protein induces splicing out of two or more retained introns
in the population, including the retained intron to which the
antisense oligomer is targeted or binds. In embodiments,
fully-spliced (mature) RNA encoding the target protein is thereby
produced. In embodiments, the second-most retained intron in a
population of RIC pre-mRNA encoding the JAG1 protein is intron 18.
In embodiments, the second-most retained intron in a population of
RIC pre-mRNA encoding the JAG1 protein is intron 13.
[0644] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[0645] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[0646] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for the
JAG1 gene in the published reference genome deposited in the NCBI
repository of biological and scientific information (operated by
National Center for Biotechnology Information, National Library of
Medicine, 8600 Rockville Pike, Bethesda, Md. USA 20894). As used
herein, the "wild-type sequence" refers to the canonical sequence
available at NCBI Gene ID 182. Also used herein, a nucleotide
position denoted with an "e" indicates the nucleotide is present in
the sequence of an exon (e.g., the exon flanking the 5' splice site
or the exon flanking the 3' splice site).
[0647] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding JAG1 protein,
resulting in increased expression of JAG1. As used herein,
"contacting" or administering to cells refers to any method of
providing an ASO in immediate proximity with the cells such that
the ASO and the cells interact. A cell that is contacted with an
ASO will take up or transport the ASO into the cell. The method
involves contacting a condition or disease-associated or condition
or disease-relevant cell with any of the ASOs described herein. In
some embodiments, the ASO may be further modified or attached
(e.g., covalently attached) to another molecule to target the ASO
to a cell type, enhance contact between the ASO and the condition
or disease-associated or condition or disease-relevant cell, or
enhance uptake of the ASO.
[0648] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[0649] In embodiments, contacting a cell that expresses a JAG1 RIC
pre-mRNA with an ASO that is complementary to a targeted portion of
the JAG1 RIC pre-mRNA transcript results in a measurable increase
in the amount of the JAG1 protein (e.g., a target protein) encoded
by the pre-mRNA. Methods of measuring or detecting production of a
protein will be evident to one of skill in the art and include any
known method, for example, Western blotting, flow cytometry,
immunofluorescence microscopy, and ELISA.
[0650] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a JAG1 RIC pre-mRNA
transcript results in an increase in the amount of JAG1 protein
produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200,
250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of
the protein produced by a cell in the absence of the ASO/absence of
treatment. In embodiments, the total amount of JAG1 protein
produced by the cell to which the antisense oligomer was contacted
is increased about 1.1 to about 10-fold, about 1.5 to about
10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about
4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the amount of target protein produced by
an control compound. A control compound can be, for example, an
oligonucleotide that is not complementary to the targeted portion
of the RIC pre-mRNA.
[0651] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a JAG1 RIC pre-mRNA
transcript results in an increase in the amount of mRNA encoding
JAG1, including the mature mRNA encoding the target protein. In
some embodiments, the amount of mRNA encoding JAG1 protein, or the
mature mRNA encoding the JAG1 protein, is increased by at least 10,
20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450,
500, or 1000%, compared to the amount of the protein produced by a
cell in the absence of the ASO/absence of treatment. In
embodiments, the total amount of the mRNA encoding JAG1 protein, or
the mature mRNA encoding JAG1 protein produced in the cell to which
the antisense oligomer was contacted is increased about 1.1 to
about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold compared to the
amount of mature RNA produced in an untreated cell, e.g., an
untreated cell or a cell treated with a control compound. A control
compound can be, for example, an oligonucleotide that is not
complementary to the targeted portion of the JAG1 RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[0652] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of JAG1
protein in cells, for example, in a subject having Alagille
syndrome caused by a deficiency in the amount or activity of JAG1
protein, by increasing the level of mRNA encoding JAG1 protein, or
the mature mRNA encoding JAG1 protein. In particular, the methods
and compositions as described herein induce the constitutive
splicing of a retained intron from a JAG1 RIC pre-mRNA transcript
encoding JAG1 protein, thereby increasing the level of mRNA
encoding JAG1 protein, or the mature mRNA encoding JAG1 protein and
increasing the expression of JAG1 protein.
[0653] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[0654] In embodiments, constitutive splicing correctly removes a
retained intron from a JAG1 RIC pre-mRNA, wherein the JAG1 RIC
pre-mRNA is transcribed from a wild-type gene or allele, or a
polymorphic gene or allele, that encodes a fully-functional target
protein or functional RNA, and wherein the gene or allele does not
have a mutation that causes alternative splicing or aberrant
splicing of the retained intron.
[0655] In some embodiments, constitutive splicing of a retained
intron from a JAG1 RIC pre-mRNA encoding JAG1 protein correctly
removes a retained intron from a JAG1 RIC pre-mRNA encoding JAG1
protein, wherein the JAG1 RIC pre-mRNA is transcribed from a gene
or allele from which the target gene or functional RNA is produced
at a reduced level compared to production from a wild-type allele,
and wherein the gene or allele does not have a mutation that causes
alternative splicing or aberrant splicing of the retained intron.
In these embodiments, the correct removal of the constitutively
spliced retained intron results in production of target protein or
functional RNA that is functional when compared to an equivalent
wild-type protein or functional RNA.
[0656] In other embodiments, constitutive splicing correctly
removes a retained intron from a JAG1 RIC pre-mRNA, wherein the
JAG1 RIC pre-mRNA is transcribed from a gene or allele that encodes
a target protein or functional RNA produced in a form having
reduced function compared to an equivalent wild-type protein or
functional RNA, and wherein the gene or allele does not have a
mutation that causes alternative splicing or aberrant splicing of
the retained intron. In these embodiments, the correct removal of
the constitutively spliced retained intron results in production of
partially functional target protein, or functional RNA that is
partially functional when compared to an equivalent wild-type
protein or functional RNA.
[0657] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[0658] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding JAG1 protein or the amount of JAG1 protein by
modulating alternative splicing or aberrant splicing of a pre-mRNA
transcribed from the JAG1 gene. Modulation of alternative splicing
or aberrant splicing can be measured using any known method for
analyzing the sequence and length of RNA species, e.g., by RT-PCR
and using methods described elsewhere herein and in the literature.
In embodiments, modulation of alternative or aberrant splicing is
determined based on an increase or decrease in the amount of the
spliced species of interest of at least 10% or 1.1-fold. In
embodiments, modulation is determined based on an increase or
decrease at a level that is at least 10% to 100% or 1.1 to 10-fold,
as described herein regarding determining an increase in mRNA
encoding JAG1 protein in the methods of the invention.
[0659] In embodiments, the method is a method wherein the JAG1 RIC
pre-mRNA was produced by partial splicing of a wild-type JAG1
pre-mRNA. In embodiments, the method is a method wherein the JAG1
RIC pre-mRNA was produced by partial splicing of a full-length
wild-type JAG1 pre-mRNA. In embodiments, the JAG1 RIC pre-mRNA was
produced by partial splicing of a full-length JAG1 pre-mRNA. In
these embodiments, a full-length JAG1 pre-mRNA may have a
polymorphism in a splice site of the retained intron that does not
impair correct splicing of the retained intron as compared to
splicing of the retained intron having the wild-type splice site
sequence.
[0660] In embodiments, the mRNA encoding JAG1 protein is a
full-length mature mRNA, or a wild-type mature mRNA. In these
embodiments, a full-length mature mRNA may have a polymorphism that
does not affect the activity of the target protein or the
functional RNA encoded by the mature mRNA, as compared to the
activity of JAG1 protein encoded by the wild-type mature mRNA.
Antisense Oligomers
[0661] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of a JAG1 RIC pre-mRNA. As used herein, the
terms "ASO" and "antisense oligomer" are used interchangeably and
refer to an oligomer such as a polynucleotide, comprising
nucleobases that hybridizes to a target nucleic acid (e.g., a JAG1
RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base
pairing (G-U). The ASO may have exact sequence complementary to the
target sequence or near complementarity (e.g., sufficient
complementarity to bind the target sequence and enhancing splicing
at a splice site). ASOs are designed so that they bind (hybridize)
to a target nucleic acid (e.g., a targeted portion of a pre-mRNA
transcript) and remain hybridized under physiological conditions.
Typically, if they hybridize to a site other than the intended
(targeted) nucleic acid sequence, they hybridize to a limited
number of sequences that are not a target nucleic acid (to a few
sites other than a target nucleic acid). Design of an ASO can take
into consideration the occurrence of the nucleic acid sequence of
the targeted portion of the pre-mRNA transcript or a sufficiently
similar nucleic acid sequence in other locations in the genome or
cellular pre-mRNA or transcriptome, such that the likelihood the
ASO will bind other sites and cause "off-target" effects is
limited. Any antisense oligomers known in the art, for example in
PCT Application No. PCT/US2014/054151, published as WO 2015/035091,
titled "Reducing Nonsense-Mediated mRNA Decay," can be used to
practice the methods described herein.
[0662] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[0663] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0664] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[0665] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and
Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by
reference in their entirety.
[0666] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine,
5,6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[0667] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081
(1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et
al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer
Drug Design 6:539 (1991); Zon et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphorothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[0668] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the invention, including, but not limited
to, any of the ASOs set forth herein in Table 1, comprises an ASO
having phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[0669] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention, including, but not limited to, any of the ASOs set
forth herein in Table 1, comprises about 5-100% Rp, at least about
5% Rp, at least about 10% Rp, at least about 15% Rp, at least about
20% Rp, at least about 25% Rp, at least about 30% Rp, at least
about 35% Rp, at least about 40% Rp, at least about 45% Rp, at
least about 50% Rp, at least about 55% Rp, at least about 60% Rp,
at least about 65% Rp, at least about 70% Rp, at least about 75%
Rp, at least about 80% Rp, at least about 85% Rp, at least about
90% Rp, or at least about 95% Rp, with the remainder Sp, or about
100% Rp. In embodiments, an ASO used in the methods of the
invention, including, but not limited to, any of the ASOs set forth
herein in Table 1, comprises about 10% to about 100% Rp, about 15%
to about 100% Rp, about 20% to about 100% Rp, about 25% to about
100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp,
about 40% to about 100% Rp, about 45% to about 100% Rp, about 50%
to about 100% Rp, about 55% to about 100% Rp, about 60% to about
100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp,
about 75% to about 100% Rp, about 80% to about 100% Rp, about 85%
to about 100% Rp, about 90% to about 100% Rp, or about 95% to about
100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp,
about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45%
to about 55% Rp, with the remainder Sp.
[0670] In embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 1, comprises about 5-100% Sp, at least about 5% Sp, at least
about 10% Sp, at least about 15% Sp, at least about 20% Sp, at
least about 25% Sp, at least about 30% Sp, at least about 35% Sp,
at least about 40% Sp, at least about 45% Sp, at least about 50%
Sp, at least about 55% Sp, at least about 60% Sp, at least about
65% Sp, at least about 70% Sp, at least about 75% Sp, at least
about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at
least about 95% Sp, with the remainder Rp, or about 100% Sp. In
embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 1, comprises about 10% to about 100% Sp, about 15% to about
100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp,
about 30% to about 100% Sp, about 35% to about 100% Sp, about 40%
to about 100% Sp, about 45% to about 100% Sp, about 50% to about
100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp,
about 65% to about 100% Sp, about 70% to about 100% Sp, about 75%
to about 100% Sp, about 80% to about 100% Sp, about 85% to about
100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp,
about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to
about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55%
Sp, with the remainder Rp.
[0671] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[0672] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[0673] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[0674] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[0675] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[0676] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[0677] In other embodiments, the ASOs are complementary to (and
bind to) a targeted portion of a JAG1 RIC pre-mRNA that is
downstream (in the 3' direction) of the 5' splice site of the
retained intron in a JAG1 RIC pre-mRNA (e.g., the direction
designated by positive numbers relative to the 5' splice site)
(FIG. 1). In some embodiments, the ASOs are complementary to a
targeted portion of the JAG1 RIC pre-mRNA that is within the region
+6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100
relative to the 5' splice site of the retained intron. In some
embodiments, the ASO is not complementary to nucleotides +1 to +5
relative to the 5' splice site (the first five nucleotides located
downstream of the 5' splice site). In some embodiments, the ASOs
may be complementary to a targeted portion of a JAG1 RIC pre-mRNA
that is within the region between nucleotides +6 and +50 relative
to the 5' splice site of the retained intron. In some aspects, the
ASOs are complementary to a targeted portion that is within the
region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to
+40, +6 to +30, or +6 to +20 relative to 5' splice site of the
retained intron.
[0678] In some embodiments, the ASOs are complementary to a
targeted region of a JAG1 RIC pre-mRNA that is upstream (5'
relative) of the 3' splice site of the retained intron in a JAG1
RIC pre-mRNA (e.g., in the direction designated by negative
numbers) (FIG. 1). In some embodiments, the ASOs are complementary
to a targeted portion of the JAG1 RIC pre-mRNA that is within the
region -16 to -500, -16 to -400, -16 to -300, -6 to -200, or -16 to
-100 relative to the 3' splice site of the retained intron. In some
embodiments, the ASO is not complementary to nucleotides -1 to -15
relative to the 3' splice site (the first 15 nucleotides located
upstream of the 3' splice site). In some embodiments, the ASOs are
complementary to a targeted portion of the JAG1 RIC pre-mRNA that
is within the region -16 to -50 relative to the 3' splice site of
the retained intron. In some aspects, the ASOs are complementary to
a targeted portion that is within the region -16 to -90, -16 to
-80, -16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30
relative to 3' splice site of the retained intron.
[0679] In embodiments, the targeted portion of the JAG1 RIC
pre-mRNA is within the region +6 relative to the 5' splice site of
the retained intron to -16 relative to the 3' splice site of the
retained intron.
[0680] In some embodiments, the ASOs are complementary to a
targeted portion of a JAG1 RIC pre-mRNA that is within the exon
flanking the 5' splice site (upstream) of the retained intron (FIG.
1). In some embodiments, the ASOs are complementary to a targeted
portion of the JAG1 RIC pre-mRNA that is within the region +2e to
-4e in the exon flanking the 5' splice site of the retained intron.
In some embodiments, the ASOs are not complementary to nucleotides
-1e to -3e relative to the 5' splice site of the retained intron.
In some embodiments, the ASOs are complementary to a targeted
portion of the JAG1 RIC pre-mRNA that is within the region -4e to
-100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to
-50e, -4 to -40e, -4e to -30e, or -4e to -20e relative to the 5'
splice site of the retained intron.
[0681] In some embodiments, the ASOs are complementary to a
targeted portion of a JAG1 RIC pre-mRNA that is within the exon
flanking the 3' splice site (downstream) of the retained intron
(FIG. 1). In some embodiments, the ASOs are complementary to a
targeted portion to the JAG1 RIC pre-mRNA that is within the region
+2e to -4e in the exon flanking the 3' splice site of the retained
intron. In some embodiments, the ASOs are not complementary to
nucleotide +1e relative to the 3' splice site of the retained
intron. In some embodiments, the ASOs are complementary to a
targeted portion of the JAG1 RIC pre-mRNA that is within the region
+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e,
+2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative to
the 3' splice site of the retained intron. The ASOs may be of any
length suitable for specific binding and effective enhancement of
splicing. In some embodiments, the ASOs consist of 8 to 50
nucleobases. For example, the ASO may be 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some
embodiments, the ASOs consist of more than 50 nucleobases. In some
embodiments, the ASO is from 8 to 50 nucleobases, 8 to 40
nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25
nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50
nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30
nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15
nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35
nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20
nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40
nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25
nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50
nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30
nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15
nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35
nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20
nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35
nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20
nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35
nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20
nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35
nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50
nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to
30 nucleobases in length. In some embodiments, the ASOs are 30
nucleotides in length. In some embodiments, the ASOs are 29
nucleotides in length. In some embodiments, the ASOs are 28
nucleotides in length. In some embodiments, the ASOs are 27
nucleotides in length. In some embodiments, the ASOs are 26
nucleotides in length. In some embodiments, the ASOs are 25
nucleotides in length. In some embodiments, the ASOs are 24
nucleotides in length. In some embodiments, the ASOs are 23
nucleotides in length. In some embodiments, the ASOs are 22
nucleotides in length. In some embodiments, the ASOs are 21
nucleotides in length. In some embodiments, the ASOs are 20
nucleotides in length. In some embodiments, the ASOs are 19
nucleotides in length. In some embodiments, the ASOs are 18
nucleotides in length. In some embodiments, the ASOs are 17
nucleotides in length. In some embodiments, the ASOs are 16
nucleotides in length. In some embodiments, the ASOs are 15
nucleotides in length. In some embodiments, the ASOs are 14
nucleotides in length. In some embodiments, the ASOs are 13
nucleotides in length. In some embodiments, the ASOs are 12
nucleotides in length. In some embodiments, the ASOs are 11
nucleotides in length. In some embodiments, the ASOs are 10
nucleotides in length.
[0682] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[0683] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[0684] In some embodiments, the nucleic acid to be targeted by an
ASO is a JAG1 RIC pre-mRNA expressed in a cell, such as a
eukaryotic cell. In some embodiments, the term "cell" may refer to
a population of cells. In some embodiments, the cell is in a
subject. In some embodiments, the cell is isolated from a subject.
In some embodiments, the cell is ex vivo. In some embodiments, the
cell is a condition or disease-relevant cell or a cell line. In
some embodiments, the cell is in vitro (e.g., in cell culture).
Pharmaceutical Compositions
[0685] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent
or carrier.
[0686] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose. The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[0687] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[0688] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancers is a surfactant, fatty acid,
bile salt, chelating agent, or non-chelating nonsurfactant.
[0689] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[0690] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[0691] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[0692] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[0693] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[0694] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. Multiple tissues and organs are affected by
Alagille syndrome, with the liver being the most significantly
affected tissue. The ASOs of the present invention may be
administered to patients parenterally. In embodiments, the ASOs of
the present invention are administered to patients by
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection. In embodiments, delivery is to
the heart or liver. In embodiments, a fetus is treated in utero,
e.g., by administering the ASO composition to the fetus directly or
indirectly (e.g., via the mother).
[0695] In the treatment of subjects having muscular dystrophy, the
compositions of the present invention may be provided to muscle
cells by any suitable means, including direct administration (e.g.,
locally by injection or topical administration at a treatment site)
or systemically (e.g., parenterally or orally), intranasally,
orally, or by inhalational, enteral, topical, intrauterine,
vaginal, sublingual, rectal, intramuscular, intrapleural,
intraventricular, intraperitoneal, ophthalmic, intravenous, or
subcutaneous means.
Methods of Identifying Additional ASOs that Enhance Splicing
[0696] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of a JAG1 RIC pre-mRNA, specifically at the target intron. ASOs
that specifically hybridize to different nucleotides within the
target region of the pre-mRNA may be screened to identify
(determine) ASOs that improve the rate and/or extent of splicing of
the target intron. In some embodiments, the ASO may block or
interfere with the binding site(s) of a splicing
repressor(s)/silencer. Any method known in the art may be used to
identify (determine) an ASO that when hybridized to the target
region of the intron results in the desired effect (e.g., enhanced
splicing, protein or functional RNA production). These methods also
can be used for identifying ASOs that enhance splicing of the
retained intron by binding to a targeted region in an exon flanking
the retained intron, or in a non-retained intron. An example of a
method that may be used is provided below.
[0697] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site.
[0698] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[0699] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[0700] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[0701] As described for the ASO walk above, the ASO micro-walk is
performed by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[0702] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. Following administration, the cells, tissues, and/or
organs of the model animals may be assessed to determine the effect
of the ASO treatment by for example evaluating splicing
(efficiency, rate, extent) and protein production by methods known
in the art and described herein. The animal models may also be any
phenotypic or behavioral indication of the disease or disease
severity.
Compositions and Methods for Treatment of Tuberous Sclerosis
[0703] Individual introns in primary transcripts of protein-coding
genes having more than one intron are spliced from the primary
transcript with different efficiencies. In most cases only the
fully spliced mRNA is exported through nuclear pores for subsequent
translation in the cytoplasm. Unspliced and partially spliced
transcripts are detectable in the nucleus. It is generally thought
that nuclear accumulation of transcripts that are not fully spliced
is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[0704] The present invention provides compositions and methods for
upregulating splicing of one or more retained TSC2 introns that are
rate-limiting for the nuclear stages of gene expression to increase
steady-state production of fully-spliced, mature mRNA, and thus,
translated tuberin protein levels. These compositions and methods
utilize antisense oligomers (ASOs) that promote constitutive
splicing at an intron splice sites of a retained-intron-containing
TSC2 pre-mRNA that accumulates in the nucleus. Thus, in
embodiments, TSC2 protein is increased using the methods of the
invention to treat a condition caused by TSC2 deficiency.
[0705] In other embodiments, the methods of the invention are used
to increase TSC2 production to treat a condition in a subject in
need thereof. In embodiments, the subject has condition in which
TSC2 is not necessarily deficient relative to wild-type, but where
an increase in TSC2 mitigates the condition nonetheless. In
embodiments, the condition is a caused by a TSC2
haploinsufficiency. In embodiments, the condition is an autosomal
dominant disorder. In embodiments, the condition is an autosomal
recessive disorder.
Tuberous Sclerosis Complex
[0706] Tuberous sclerosis complex is a disease characterized by
tumor growth in multiple organ systems (Au et al., J. Child Neurol.
2004, 19, 699-709). Tumors are usually benign but are occasionally
malignant. Approximately 90% of tuberous sclerosis complex cases
display cortical tuber; facial angiofibroma and renal
angiomyolipoma occur in more than 80% of cases. In addition,
approximately 80% of cases display subependymal nodule,
approximately 50% of cases display cardiac rhabdomyoma, and 51% to
approximately 88% of cases display ungual/subungual fibroma. Tumors
of the central nervous system (CNS) are the leading cause of
morbidity and mortality, followed by renal disease (Au et al., J.
Child Neurol. 2004, 19, 699-709).
[0707] Tuberous sclerosis complex is a genetic disorder with an
autosomal dominant inheritance pattern and a high mutation rate (Au
et al., J. Child Neurol. 2004, 19, 699-709). Linkage of tuberous
sclerosis complex to chromosomal regions 9q34.3 and 16p13.3 led to
the identification of the TSC1 and TSC2 genes, respectively. More
than 69% of tuberous sclerosis complex cases result from
haploinsufficency of TSC2, and disease in approximately two thirds
of patients results from de novo mutation or deletion (Au et al.,
J. Child Neurol. 2004, 19, 699-709). Severe disease is thought to
require a "second hit" reduction of the other allele. The
prevalence of tuberous sclerosis complex is as high as one in 5,800
live births, or approximately 300 births per year in the United
States, with 200 cases per year resulting from mutation of TSC2.
The disease is described, e.g., by OMIM #613254 (Online Mendelian
Inheritance in Man, Johns Hopkins University, 1966-2015),
incorporated by reference herein.
[0708] The TSC2 gene codes for a protein named tuberin. The TSC2
gene contains 41 exons, two of which are alternatively spliced, and
spans approximately 40 kb on chromosome 16 (Au et al., J. Child
Neurol. 2004, 19, 699-709). Tuberin is a 200 kDa protein that forms
a complex with the TSC1 gene product hamartin. The tuberin-hamartin
complex plays a role in the regulation of cell growth, translation,
cell size, and cell adhesion and migration through a variety of
signal cascades. For example, the tuberin-hamartin complex
functions in the phosphatidyl-inositol-3-kinase and protein B
pathway (PI3K/PKB) that regulates translation. Specifically, the
tuberin-hamartin complex suppresses cell growth and translation by
suppressing mTOR kinase activity, resulting in suppression of the
p70 ribosomal protein S6 kinase (S6K) 1 and activation of
eukaryotic translation initiation factor 4E binding protein 1
(4E-BP1). Moreover, the GAP domain of tuberin hydrolyzes guanosine
triphosphate (GTP) bound to the small G protein Rheb, a Ras homolog
enriched in brain, thus preventing mTOR activation.
[0709] Additionally, tuberin and hamartin regulate cell growth and
cell adhesion through the MAP kinase (MAPK) and
E-cadherin-beta-catenin pathways, respectively. Initially
discovered through identification of loss of heterozygosity for
portions of chromosome 16p13, numerous mutations have been
described for TSC2 (Au et al., J. Child Neurol. 2004, 19, 699-709).
Mutations include frameshift and protein truncations, nonsense
mutations, mutations that affect splicing, in-frame mutations,
deletions, insertions, duplications, and large deletions and
rearrangement. Mutations of TSC2 that yield truncated protein
products fail to form the tuberin-hamartin complex, thus resulting
in loss of cell growth regulation. The role of the tuberin-hamartin
complex as a key regulator of multiple signaling pathways is
consistent with the wide spectrum of clinical findings among
patients with tuberous sclerosis complex that involve multiple
organ systems, including neurological and neurobehavioral
abnormalities such as seizures, intellectual disability, and
developmental delay (Au et al., J. Child Neurol. 2004, 19,
699-709).
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[0710] In embodiments, the methods of the present invention exploit
the presence of retained-intron-containing pre-mRNA (RIC pre-mRNA)
transcribed from the TSC2 gene and encoding tuberin protein, in the
cell nucleus. Splicing of the identified TSC2 RIC pre-mRNA species
to produce mature, fully-spliced, TSC2 mRNA, is induced using ASOs
that stimulate splicing out of the retained introns. The resulting
mature TSC2 mRNA can be exported to the cytoplasm and translated,
thereby increasing the amount of tuberin protein in the patient's
cells and alleviating symptoms of tuberous sclerosis complex. This
method, described further below, is known as Targeted Augmentation
of Nuclear Gene Output (TANGO).
TSC2 Nuclear Transcripts
[0711] As described herein in the Examples, the TSC2 gene was
analyzed for intron-retention events and retention of introns 4,
25, 26, 31, and 32 was observed. RNA sequencing (RNAseq),
visualized in the UCSC genome browser, showed TSC2 transcripts
expressed in HCN (human cortical neurons) cells and localized in
either the cytoplasmic or nuclear fraction. In both fractions,
reads were not observed for the majority of the introns. However,
higher read density was detected for introns 4, 25, 26, 31, and 32
in the nuclear fraction compared to the cytoplasmic fraction
indicating that splicing efficiency of introns 4, 25, 26, 31, and
32 is low, resulting in intron retention. The retained-intron
containing pre-mRNA transcripts accumulate primarily in the nucleus
and not translated into the tuberin protein. The read density for
intron 26 in AST is shown in detail in the lower panel indicating
51% intron retention as calculated by bioinformatic analysis. The
percent intron retention (PIR) value for intron 26 was obtained by
averaging four values (87, 83, 20, and 12), each determined in
renal epithelial cells using one of four different algorithms. The
read density for intron 31 in AST is shown in detail in the lower
panel indicating 43% intron retention. The percent intron retention
(PIR) value for intron 31 was obtained by averaging four values
(78, 71, 16, and 8), each determined in renal epithelial cells
using one of four different algorithms. Introns 4 and 32 were not
mapped. Analysis of the ENCODE data (described by, e.g., Tilgner,
et al., 2012, "Deep sequencing of subcellular RNA fractions shows
splicing to be predominantly co-transcriptional in the human genome
but inefficient for lncRNAs," Genome Research 22(9):1616-25) to
identify intron retention events did not identify intron 25, 26, or
31 as retained, and did not map introns 4 and 32.
[0712] Table 3 provides a non-limiting list of target sequences of
a TSC2 RIC pre-mRNA transcript by sequence ID, and ASOs by sequence
ID, useful for increasing production of tuberin protein by
targeting a region of a TSC2 RIC pre-mRNA. In embodiments, other
ASOs useful for these purposes are identified, using, e.g., methods
described herein.
TABLE-US-00002 TABLE 3 List of targets and ASOs targeting the TSC2
gene Target Gene Pre-mRNA Retained Sequence SEQ ID NO: SEQ ID NO:
ASOs Intron SEQ ID NO: TSC2 TSC2:NM_001318829 SEQ ID NOs: 448- 24
5539 SEQ ID NO: SEQ ID NO: 441 669 440 SEQ ID NOs: 670- 3 5536 866
SEQ ID NOs: 867- 29 5540 1099 TSC2:NM_000548 SEQ ID NOs: 32 5544
SEQ ID NO: 442 1100-1303 SEQ ID NOs: 25 5542 1304-1513 SEQ ID NOs:
26 5538 1514-1740 SEQ ID NOs: 4 5536 1741-1937 SEQ ID NOs: 31 5543
1938-2101 TSC2:NM_001077183 SEQ ID NOs: 25 5537 SEQ ID NO: 443
2102-2323 SEQ ID NOs: 4 5536 2324-2520 SEQ ID NOs: 30 5540
2521-2753 TSC2:NM_001114382 SEQ ID NOs: 25 5542 SEQ ID NO: 444
2754-2963 SEQ ID NOs: 26 5538 2964-3190 SEQ ID NOs: 4 5536
3191-3387 SEQ ID NOs: 31 5540 3388-3620 TSC2:NM_001318831 SEQ ID
NOs: 27 5543 SEQ ID NO: 445 3621-3784 SEQ ID NOs: 28 5544 3785-3988
SEQ ID NOs: 22 5537 3989-4210 TSC2:NM_001318832 SEQ ID NOs: 25 5537
SEQ ID NO: 446 4211-4432 SEQ ID NOs: 4 5536 4433-4629 SEQ ID NOs:
30 5540 4630-4862 TSC2:NM_001318827 SEQ ID NOs: 24 5539 SEQ ID NO:
447 4863-5084 SEQ ID NOs: 3 5541 5085-5302 SEQ ID NOs: 29 5540
5303-5302
[0713] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a TSC2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a TSC2
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 440. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 440 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a TSC2 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript
comprising a retained intron at 3, 24, 29 or a combination thereof,
wherein the intron numbering correspond to the mRNA sequence at
NM_001318829. In some embodiments, the ASO targets a TSC2 RIC
pre-mRNA transcript comprising a retained intron at 4, 25, 26, 31,
32 or a combination thereof, wherein the intron numbering
correspond to the mRNA sequence at NM_000548. In some embodiments,
the ASO targets a TSC2 RIC pre-mRNA transcript comprising a
retained intron at 4, 25, 30 or a combination thereof, wherein the
intron numbering correspond to the mRNA sequence at NM_001077183.
In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript
comprising a retained intron at 4, 25, 26, 31, or a combination
thereof, wherein the intron numbering correspond to the mRNA
sequence at NM_001114382. In some embodiments, the ASO targets a
TSC2 RIC pre-mRNA transcript comprising a retained intron at 22,
27, 28 or a combination thereof, wherein the intron numbering
correspond to the mRNA sequence at NM_001318831. In some
embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript
comprising a retained intron at 4, 25, 30 or a combination thereof,
wherein the intron numbering correspond to the mRNA sequence at
NM_001318832. In some embodiments, the ASO targets a TSC2 RIC
pre-mRNA transcript comprising a retained intron at 3, 24, 29 or a
combination thereof, wherein the intron numbering correspond to the
mRNA sequence at NM_001318827.
[0714] In some embodiments, the ASO targets a TSC2 RIC pre-mRNA
sequence according to SEQ ID NO: 441. In some embodiments, the ASO
targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 441
comprising a retained intron 24, a retained intron 3, a retained
intron 29, or a combination thereof. In some embodiments, the ASO
targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 442.
In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence
according to SEQ ID NO: 442 comprising a retained intron 32, a
retained intron 25, a retained intron 26, a retained intron 4, a
retained intron 31 or a combination thereof. In some embodiments,
the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID
NO: 443. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA
sequence according to SEQ ID NO: 443 comprising a retained intron
25, a retained intron 4, a retained intron 30 or a combination
thereof. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA
sequence according to SEQ ID NO: 444. In some embodiments, the ASO
targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 444
comprising a retained intron 25, a retained intron 26, a retained
intron 4, a retained intron 31 or a combination thereof. In some
embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according
to SEQ ID NO: 445. In some embodiments, the ASO targets a TSC2 RIC
pre-mRNA sequence according to SEQ ID NO: 445 comprising a retained
intron 27, a retained intron 28, a retained intron 22 or a
combination thereof. In some embodiments, the ASO targets a TSC2
RIC pre-mRNA sequence according to SEQ ID NO: 446. In some
embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according
to SEQ ID NO: 446 comprising a retained intron 25, a retained
intron 4, a retained intron 30 or a combination thereof. In some
embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according
to SEQ ID NO: 447. In some embodiments, the ASO targets a TSC2 RIC
pre-mRNA sequence according to SEQ ID NO: 447 comprising a retained
intron 24, a retained intron 3, a retained intron 29 or a
combination thereof. In some embodiments, the ASOs disclosed herein
target SEQ ID NOs: 5536-5544. In some embodiments, the ASO has a
sequence according to any one of SEQ ID NOs: 448-5535.
[0715] In some embodiments, the ASO targets exon 24 or exon 25 of a
TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the
intron numbering correspond to the mRNA sequence at NM_001318829.
In some embodiments, the ASO targets an exon 24 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 24. In some embodiments, the ASO targets an
exon 24 sequence about 2 to about 75 or about 4 to about 74
nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 24. In some embodiments,
the ASO targets an exon 25 sequence downstream (or 3') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
24. In some embodiments, the ASO targets an exon 25 sequence about
2 to about 150 or about 2 to about 147 nucleotides downstream (or
3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 24.
[0716] In some embodiments, the ASO targets intron 24 in a TSC2 RIC
pre-mRNA comprising a retained intron 24, wherein the intron
numbering correspond to the mRNA sequence at NM_001318829. In some
embodiments, the ASO targets an intron 24 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 24. In some embodiments, the ASO targets an intron
24 sequence about 6 to about 497 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 24. In some embodiments, the ASO targets an intron
24 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 24. In some embodiments,
the ASO targets an intron 24 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 24.
[0717] In some embodiments, the ASO targets exon 3 or exon 4 of a
TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the
intron numbering correspond to the mRNA sequence at NM_001318829.
In some embodiments, the ASO targets an exon 3 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 3. In some embodiments, the ASO targets an exon
3 sequence about 4 to about 94 nucleotides upstream (or 5') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an exon 4 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an exon 4 sequence about 2 to about 127 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3.
[0718] In some embodiments, the ASO targets intron 3 in a TSC2 RIC
pre-mRNA comprising a retained intron 3, wherein the intron
numbering correspond to the mRNA sequence at NM_001318829. In some
embodiments, the ASO targets an intron 3 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an intron 3
sequence about 6 to about 435 nucleotides downstream (or 3') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an intron 3 sequence
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an intron 3 sequence about 16 to about 432 nucleotides
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3.
[0719] In some embodiments, the ASO targets exon 29 or exon 30 of a
TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the
intron numbering correspond to the mRNA sequence at NM_001318829.
In some embodiments, the ASO targets an exon 29 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 29. In some embodiments, the ASO targets an
exon 29 sequence about 4 to about 184 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an exon 30
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 29. In some embodiments,
the ASO targets an exon 30 sequence about 2 to about 102
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 29.
[0720] In some embodiments, the ASO targets intron 29 in a TSC2 RIC
pre-mRNA comprising a retained intron 29, wherein the intron
numbering correspond to the mRNA sequence at NM_001318829. In some
embodiments, the ASO targets an intron 29 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence about 6 to about 492 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 29. In some embodiments,
the ASO targets an intron 29 sequence about 16 to about 500 or
about 36 to about 500 nucleotides upstream (or 5') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
29.
[0721] In some embodiments, the ASO targets exon 32 or exon 33 of a
TSC2 RIC pre-mRNA comprising a retained intron 32, wherein the
intron numbering correspond to the mRNA sequence at NM_000548. In
some embodiments, the ASO targets an exon 32 sequence upstream (or
5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 32. In some embodiments, the ASO targets an exon 32
sequence about 4 to about 49 nucleotides upstream (or 5') from the
5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 32. In some embodiments, the ASO targets an exon 33 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 32. In some embodiments, the ASO
targets an exon 33 sequence about 2 to about 102 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 32.
[0722] In some embodiments, the ASO targets intron 32 in a TSC2 RIC
pre-mRNA comprising a retained intron 32, wherein the intron
numbering correspond to the mRNA sequence at NM_000548. In some
embodiments, the ASO targets an intron 32 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 32. In some embodiments, the ASO targets an intron
32 sequence about 6 to about 495 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 32. In some embodiments, the ASO targets an intron
32 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 32. In some embodiments,
the ASO targets an intron 32 sequence about 16 to about 500 or
about 36 to about 500 nucleotides upstream (or 5') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
32.
[0723] In some embodiments, the ASO targets exon 25 or exon 26 of a
TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the
intron numbering correspond to the mRNA sequence at NM_000548. In
some embodiments, the ASO targets an exon 25 sequence upstream (or
5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an exon 25
sequence about 4 to about 74 nucleotides upstream (or 5') from the
5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 25. In some embodiments, the ASO targets an exon 26 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 25. In some embodiments, the ASO
targets an exon 26 sequence about 2 to about 112 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 25.
[0724] In some embodiments, the ASO targets intron 25 in a TSC2 RIC
pre-mRNA comprising a retained intron 25, wherein the intron
numbering correspond to the mRNA sequence at NM_000548. In some
embodiments, the ASO targets an intron 25 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence about 6 to about 497 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25. In some embodiments,
the ASO targets an intron 25 sequence about 16 to about 498
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25.
[0725] In some embodiments, the ASO targets exon 26 or exon 27 of a
TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the
intron numbering correspond to the mRNA sequence at NM_000548. In
some embodiments, the ASO targets an exon 26 sequence upstream (or
5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 26. In some embodiments, the ASO targets an exon 26
sequence about 4 to about 111 nucleotides upstream (or 5') from the
5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 26. In some embodiments, the ASO targets an exon 27 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 26. In some embodiments, the ASO
targets an exon 27 sequence about 2 to about 147 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 26.
[0726] In some embodiments, the ASO targets intron 26 in a TSC2 RIC
pre-mRNA comprising a retained intron 26, wherein the intron
numbering correspond to the mRNA sequence at NM_000548. In some
embodiments, the ASO targets an intron 26 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 26. In some embodiments, the ASO targets an intron
26 sequence about 6 to about 500 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 26. In some embodiments, the ASO targets an intron
26 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 26. In some embodiments,
the ASO targets an intron 26 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 26.
[0727] In some embodiments, the ASO targets exon 4 or exon 5 of a
TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_000548. In
some embodiments, the ASO targets an exon 4 sequence upstream (or
5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an exon 4
sequence about 4 to about 94 nucleotides upstream (or 5') from the
5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 127 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0728] In some embodiments, the ASO targets intron 4 in a TSC2 RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_000548. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 6 to about 435 nucleotides downstream (or 3') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 432 nucleotides
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0729] In some embodiments, the ASO targets exon 31 or exon 32 of a
TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the
intron numbering correspond to the mRNA sequence at NM_000548. In
some embodiments, the ASO targets an exon 31 sequence upstream (or
5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 31. In some embodiments, the ASO targets an exon 31
sequence about 4 to about 184 nucleotides upstream (or 5') from the
5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 31. In some embodiments, the ASO targets an exon 32 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 31. In some embodiments, the ASO
targets an exon 32 sequence about 2 to about 52 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 31.
[0730] In some embodiments, the ASO targets intron 31 in a TSC2 RIC
pre-mRNA comprising a retained intron 31, wherein the intron
numbering correspond to the mRNA sequence at NM_000548. In some
embodiments, the ASO targets an intron 31 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 31. In some embodiments, the ASO targets an intron
31 sequence about 6 to about 331 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 31. In some embodiments, the ASO targets an intron
31 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 31. In some embodiments,
the ASO targets an intron 31 sequence about 16 to about 333
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 31.
[0731] In some embodiments, the ASO targets exon 25 or exon 26 of a
TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the
intron numbering correspond to the mRNA sequence at NM_001077183.
In some embodiments, the ASO targets an exon 25 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 25. In some embodiments, the ASO targets an
exon 25 sequence about 4 to about 74 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an exon 26
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25. In some embodiments,
the ASO targets an exon 26 sequence about 2 to about 142
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 25.
[0732] In some embodiments, the ASO targets intron 25 in a TSC2 RIC
pre-mRNA comprising a retained intron 25, wherein the intron
numbering correspond to the mRNA sequence at NM_001077183. In some
embodiments, the ASO targets an intron 25 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence about 6 to about 497 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25. In some embodiments,
the ASO targets an intron 25 sequence about 16 to about 499
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25.
[0733] In some embodiments, the ASO targets exon 4 or exon 5 of a
TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_001077183.
In some embodiments, the ASO targets an exon 4 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 94 nucleotides upstream (or 5') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 127 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0734] In some embodiments, the ASO targets intron 4 in a TSC2 RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_001077183. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 6 to about 435 nucleotides downstream (or 3') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 432 nucleotides
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0735] In some embodiments, the ASO targets exon 30 or exon 31 of a
TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the
intron numbering correspond to the mRNA sequence at NM_001077183.
In some embodiments, the ASO targets an exon 30 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 30. In some embodiments, the ASO targets an
exon 30 sequence about 4 to about 184 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an exon 31
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 30. In some embodiments,
the ASO targets an exon 31 sequence about 2 to about 102
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 30.
[0736] In some embodiments, the ASO targets intron 30 in a TSC2 RIC
pre-mRNA comprising a retained intron 30, wherein the intron
numbering correspond to the mRNA sequence at NM_001077183. In some
embodiments, the ASO targets an intron 30 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence about 6 to about 492 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 30. In some embodiments,
the ASO targets an intron 30 sequence about 16 to about 500 or
about 36 to about 500 nucleotides upstream (or 5') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
30.
[0737] In some embodiments, the ASO targets exon 25 or exon 26 of a
TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the
intron numbering correspond to the mRNA sequence at NM_001114382.
In some embodiments, the ASO targets an exon 25 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 25. In some embodiments, the ASO targets an
exon 25 sequence about 4 to about 74 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an exon 26
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25. In some embodiments,
the ASO targets an exon 26 sequence about 2 to about 112
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 25.
[0738] In some embodiments, the ASO targets intron 25 in a TSC2 RIC
pre-mRNA comprising a retained intron 25, wherein the intron
numbering correspond to the mRNA sequence at NM_001114382. In some
embodiments, the ASO targets an intron 25 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence about 6 to about 497 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25. In some embodiments,
the ASO targets an intron 25 sequence about 16 to about 498
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25.
[0739] In some embodiments, the ASO targets exon 26 or exon 27 of a
TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the
intron numbering correspond to the mRNA sequence at NM_001114382.
In some embodiments, the ASO targets an exon 26 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 26. In some embodiments, the ASO targets an
exon 26 sequence about 4 to about 111 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 26. In some embodiments, the ASO targets an exon 27
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 26. In some embodiments,
the ASO targets an exon 27 sequence about 2 to about 147
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 26.
[0740] In some embodiments, the ASO targets intron 26 in a TSC2 RIC
pre-mRNA comprising a retained intron 26, wherein the intron
numbering correspond to the mRNA sequence at NM_001114382. In some
embodiments, the ASO targets an intron 26 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 26. In some embodiments, the ASO targets an intron
26 sequence about 6 to about 500 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 26. In some embodiments, the ASO targets an intron
26 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 26. In some embodiments,
the ASO targets an intron 26 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 26.
[0741] In some embodiments, the ASO targets exon 4 or exon 5 of a
TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_001114382.
In some embodiments, the ASO targets an exon 4 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 94 nucleotides upstream (or 5') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 127 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0742] In some embodiments, the ASO targets intron 4 in a TSC2 RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_001114382. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 6 to about 435 nucleotides downstream (or 3') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 432 nucleotides
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0743] In some embodiments, the ASO targets exon 31 or exon 32 of a
TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the
intron numbering correspond to the mRNA sequence at NM_001114382.
In some embodiments, the ASO targets an exon 31 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 31. In some embodiments, the ASO targets an
exon 31 sequence about 4 to about 184 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 31. In some embodiments, the ASO targets an exon 32
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 31. In some embodiments,
the ASO targets an exon 32 sequence about 2 to about 102
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 31.
[0744] In some embodiments, the ASO targets intron 31 in a TSC2 RIC
pre-mRNA comprising a retained intron 31, wherein the intron
numbering correspond to the mRNA sequence at NM_001114382. In some
embodiments, the ASO targets an intron 31 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 31. In some embodiments, the ASO targets an intron
31 sequence about 6 to about 492 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 31. In some embodiments, the ASO targets an intron
31 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 31. In some embodiments,
the ASO targets an intron 31 sequence about 16 to about 500 or
about 36 to about 500 nucleotides upstream (or 5') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
31.
[0745] In some embodiments, the ASO targets exon 27 or exon 28 of a
TSC2 RIC pre-mRNA comprising a retained intron 27, wherein the
intron numbering correspond to the mRNA sequence at NM_001318831.
In some embodiments, the ASO targets an exon 27 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 27. In some embodiments, the ASO targets an
exon 27 sequence about 4 to about 184 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 27. In some embodiments, the ASO targets an exon 28
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 27. In some embodiments,
the ASO targets an exon 28 sequence about 2 to about 52 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 27.
[0746] In some embodiments, the ASO targets intron 27 in a TSC2 RIC
pre-mRNA comprising a retained intron 27, wherein the intron
numbering correspond to the mRNA sequence at NM_001318831. In some
embodiments, the ASO targets an intron 27 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 27. In some embodiments, the ASO targets an intron
27 sequence about 6 to about 331 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 27. In some embodiments, the ASO targets an intron
27 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 27. In some embodiments,
the ASO targets an intron 27 sequence about 16 to about 333
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 27.
[0747] In some embodiments, the ASO targets exon 28 or exon 29 of a
TSC2 RIC pre-mRNA comprising a retained intron 28, wherein the
intron numbering correspond to the mRNA sequence at NM_001318831.
In some embodiments, the ASO targets an exon 28 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 28. In some embodiments, the ASO targets an
exon 28 sequence about 4 to about 49 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 28. In some embodiments, the ASO targets an exon 29
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 28. In some embodiments,
the ASO targets an exon 29 sequence about 2 to about 102
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 28.
[0748] In some embodiments, the ASO targets intron 28 in a TSC2 RIC
pre-mRNA comprising a retained intron 28, wherein the intron
numbering correspond to the mRNA sequence at NM_001318831. In some
embodiments, the ASO targets an intron 28 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 28. In some embodiments, the ASO targets an intron
28 sequence about 6 to about 495 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 28. In some embodiments, the ASO targets an intron
28 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 28. In some embodiments,
the ASO targets an intron 28 sequence about 16 to about 500 or
about 36 to about 500 nucleotides upstream (or 5') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
28.
[0749] In some embodiments, the ASO targets exon 22 or exon 23 of a
TSC2 RIC pre-mRNA comprising a retained intron 22, wherein the
intron numbering correspond to the mRNA sequence at NM_001318831.
In some embodiments, the ASO targets an exon 22 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 22. In some embodiments, the ASO targets an
exon 22 sequence about 4 to about 74 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 22. In some embodiments, the ASO targets an exon 23
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 22. In some embodiments,
the ASO targets an exon 23 sequence about 2 to about 142
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 22.
[0750] In some embodiments, the ASO targets intron 22 in a TSC2 RIC
pre-mRNA comprising a retained intron 22, wherein the intron
numbering correspond to the mRNA sequence at NM_001318831. In some
embodiments, the ASO targets an intron 22 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 22. In some embodiments, the ASO targets an intron
22 sequence about 6 to about 497 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 22. In some embodiments, the ASO targets an intron
22 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 22. In some embodiments,
the ASO targets an intron 22 sequence about 16 to about 499
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 22.
[0751] In some embodiments, the ASO targets exon 25 or exon 26 of a
TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the
intron numbering correspond to the mRNA sequence at NM_001318832.
In some embodiments, the ASO targets an exon 25 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 25. In some embodiments, the ASO targets an
exon 25 sequence about 4 to about 74 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an exon 26
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25. In some embodiments,
the ASO targets an exon 26 sequence about 2 to about 142
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 25.
[0752] In some embodiments, the ASO targets intron 25 in a TSC2 RIC
pre-mRNA comprising a retained intron 25, wherein the intron
numbering correspond to the mRNA sequence at NM_001318832. In some
embodiments, the ASO targets an intron 25 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence about 6 to about 497 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 25. In some embodiments, the ASO targets an intron
25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25. In some embodiments,
the ASO targets an intron 25 sequence about 16 to about 499
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 25.
[0753] In some embodiments, the ASO targets exon 4 or exon 5 of a
TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_001318832.
In some embodiments, the ASO targets an exon 4 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 94 nucleotides upstream (or 5') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 127 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0754] In some embodiments, the ASO targets intron 4 in a TSC2 RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_001318832. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 6 to about 435 nucleotides downstream (or 3') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 432 nucleotides
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 4.
[0755] In some embodiments, the ASO targets exon 30 or exon 31 of a
TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the
intron numbering correspond to the mRNA sequence at NM_001318832.
In some embodiments, the ASO targets an exon 30 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 30. In some embodiments, the ASO targets an
exon 30 sequence about 4 to about 184 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an exon 31
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 30. In some embodiments,
the ASO targets an exon 31 sequence about 2 to about 102
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 30.
[0756] In some embodiments, the ASO targets intron 30 in a TSC2 RIC
pre-mRNA comprising a retained intron 30, wherein the intron
numbering correspond to the mRNA sequence at NM_001318832. In some
embodiments, the ASO targets an intron 30 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence about 6 to about 492 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 30. In some embodiments,
the ASO targets an intron 30 sequence about 16 to about 500 or
about 36 to about 500 nucleotides upstream (or 5') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
30.
[0757] In some embodiments, the ASO targets exon 24 or exon 25 of a
TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the
intron numbering correspond to the mRNA sequence at NM_001318827.
In some embodiments, the ASO targets an exon 24 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 24. In some embodiments, the ASO targets an
exon 24 sequence about 4 to about 74 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 24. In some embodiments, the ASO targets an exon 25
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 24. In some embodiments,
the ASO targets an exon 25 sequence about 2 to about 147
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 24.
[0758] In some embodiments, the ASO targets intron 24 in a TSC2 RIC
pre-mRNA comprising a retained intron 24, wherein the intron
numbering correspond to the mRNA sequence at NM_001318827. In some
embodiments, the ASO targets an intron 24 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 24. In some embodiments, the ASO targets an intron
24 sequence about 6 to about 497 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 24. In some embodiments, the ASO targets an intron
24 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 24. In some embodiments,
the ASO targets an intron 24 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 24.
[0759] In some embodiments, the ASO targets exon 3 or exon 4 of a
TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the
intron numbering correspond to the mRNA sequence at NM_001318827.
In some embodiments, the ASO targets an exon 3 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 3. In some embodiments, the ASO targets an exon
3 sequence about 4 to about 69 nucleotides upstream (or 5') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an exon 4 sequence
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an exon 4 sequence about 2 to about 127 nucleotides
downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3.
[0760] In some embodiments, the ASO targets intron 3 in a TSC2 RIC
pre-mRNA comprising a retained intron 3, wherein the intron
numbering correspond to the mRNA sequence at NM_001318827. In some
embodiments, the ASO targets an intron 3 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an intron 3
sequence about 6 to about 499 nucleotides downstream (or 3') from
the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an intron 3 sequence
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an intron 3 sequence about 16 to about 497 nucleotides
upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA
comprising the retained intron 3.
[0761] In some embodiments, the ASO targets exon 29 or exon 30 of a
TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the
intron numbering correspond to the mRNA sequence at NM_001318827.
In some embodiments, the ASO targets an exon 29 sequence upstream
(or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising
the retained intron 29. In some embodiments, the ASO targets an
exon 29 sequence about 4 to about 184 nucleotides upstream (or 5')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an exon 30
sequence downstream (or 3') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 29. In some embodiments,
the ASO targets an exon 30 sequence about 2 to about 102
nucleotides downstream (or 3') from the 3' splice site of a TSC2
RIC pre-mRNA comprising the retained intron 29.
[0762] In some embodiments, the ASO targets intron 29 in a TSC2 RIC
pre-mRNA comprising a retained intron 29, wherein the intron
numbering correspond to the mRNA sequence at NM_001318827. In some
embodiments, the ASO targets an intron 29 sequence downstream (or
3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence about 6 to about 492 nucleotides downstream (or 3')
from the 5' splice site of a TSC2 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC
pre-mRNA comprising the retained intron 29. In some embodiments,
the ASO targets an intron 29 sequence about 16 to about 500 or
about 36 to about 500 nucleotides upstream (or 5') from the 3'
splice site of a TSC2 RIC pre-mRNA comprising the retained intron
29.
[0763] The degree of intron retention can be represented using the
metric percent intron retention (PIR), the percentage of
transcripts in which a given intron is retained. In brief, PIR can
be calculated as the percentage of the average number of reads
mapping to the exon-intron junctions, over the sum of the average
of the exon-intron junction reads plus the exon-exon junction
reads.
Tuberin Protein Expression
[0764] More than 69% of tuberous sclerosis complex cases result
from haploinsufficiency of TSC2, and disease in approximately two
thirds of patients results from de novo mutation or deletion (Au,
K., et al., J. Child Neurol., 2004, 19: 699-709)
[0765] In embodiments, the methods described herein are used to
increase the production of a functional tuberin protein. As used
herein, the term "functional" refers to the amount of activity or
function of a tuberin protein that is necessary to eliminate any
one or more symptoms of tuberous sclerosis complex. In embodiments,
the methods are used to increase the production of a partially
functional tuberin protein. As used herein, the term "partially
functional" refers to any amount of activity or function of the
tuberin protein that is less than the amount of activity or
function that is necessary to eliminate or prevent any one or more
symptoms of a disease or condition. In some embodiments, a
partially functional protein or RNA will have at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 75%, at least 80%, 85%, at least 90%, or at
least 95% less activity relative to the fully functional protein or
RNA.
[0766] In embodiments, the method is a method of increasing the
expression of the tuberin protein by cells of a subject having a
RIC pre-mRNA encoding the tuberin protein, wherein the subject has
tuberous sclerosis complex caused by a deficient amount of activity
of tuberin protein, and wherein the deficient amount of the tuberin
protein is caused by haploinsufficiency of the tuberin protein. In
such an embodiment, the subject has a first allele encoding a
functional tuberin protein, and a second allele from which the
tuberin protein is not produced. In another such embodiment, the
subject has a first allele encoding a functional tuberin protein,
and a second allele encoding a nonfunctional tuberin protein. In
another such embodiment, the subject has a first allele encoding a
functional tuberin protein, and a second allele encoding a
partially functional tuberin protein. In any of these embodiments,
the antisense oligomer binds to a targeted portion of the RIC
pre-mRNA transcribed from the first allele (encoding functional
tuberin protein), thereby inducing constitutive splicing of the
retained intron from the RIC pre-mRNA, and causing an increase in
the level of mature mRNA encoding functional tuberin protein, and
an increase in the expression of the tuberin protein in the cells
of the subject.
[0767] In embodiments, the subject has a first allele encoding a
functional tuberin protein, and a second allele encoding a
partially functional tuberin protein, and the antisense oligomer
binds to a targeted portion of the RIC pre-mRNA transcribed from
the first allele (encoding functional tuberin protein) or a
targeted portion of the RIC pre-mRNA transcribed from the second
allele (encoding partially functional tuberin protein), thereby
inducing constitutive splicing of the retained intron from the RIC
pre-mRNA, and causing an increase in the level of mature mRNA
encoding the tuberin protein, and an increase in the expression of
functional or partially functional tuberin protein in the cells of
the subject.
[0768] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of tuberin
protein in cells of a subject having a RIC pre-mRNA encoding
tuberin protein, wherein the subject has a deficiency, e.g.,
tuberous sclerosis complex, in the amount or function of a tuberin
protein.
[0769] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease or condition is targeted
by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the
disease is targeted by the ASOs. For example, a disease or
condition that is the result of a mutation or deficiency of a first
protein in a particular pathway may be ameliorated by targeting a
RIC pre-mRNA that encodes a second protein, thereby increasing
production of the second protein. In some embodiments, the function
of the second protein is able to compensate for the mutation or
deficiency of the first protein (which is causative of the disease
or condition).
[0770] In embodiments, the subject has:
[0771] a. a first mutant allele from which [0772] i) the tuberin
protein is produced at a reduced level compared to production from
a wild-type allele, [0773] ii) the tuberin protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0774] iii) the tuberin protein or functional RNA is
not produced; and b. a second mutant allele from which [0775] i)
the tuberin protein is produced at a reduced level compared to
production from a wild-type allele, [0776] ii) the tuberin protein
is produced in a form having reduced function compared to an
equivalent wild-type protein, or [0777] iii) the tuberin protein is
not produced, and
[0778] wherein the RIC pre-mRNA is transcribed from the first
allele and/or the second allele. In these embodiments, the ASO
binds to a targeted portion of the RIC pre-mRNA transcribed from
the first allele or the second allele, thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mRNA encoding tuberin
protein and an increase in the expression of the target protein or
functional RNA in the cells of the subject. In these embodiments,
the target protein or functional RNA having an increase in
expression level resulting from the constitutive splicing of the
retained intron from the RIC pre-mRNA is either in a form having
reduced function compared to the equivalent wild-type protein
(partially-functional), or having full function compared to the
equivalent wild-type protein (fully-functional).
[0779] In embodiments, the level of mRNA encoding tuberin protein
is increased 1.1 to 10-fold, when compared to the amount of mRNA
encoding TSC2 that is produced in a control cell, e.g., one that is
not treated with the antisense oligomer or one that is treated with
an antisense oligomer that does not bind to the targeted portion of
the TSC2 RIC pre-mRNA.
[0780] In embodiments, the condition caused by a deficient amount
or activity of tuberin protein is not a condition caused by
alternative or aberrant splicing of the retained intron to which
the ASO is targeted. In embodiments, the condition caused by a
deficient amount or activity of the tuberin protein is not a
condition caused by alternative or aberrant splicing of any
retained intron in a RIC pre-mRNA encoding the tuberin protein. In
embodiments, alternative or aberrant splicing may occur in a
pre-mRNA transcribed from the gene, however the compositions and
methods of the invention do not prevent or correct this alternative
or aberrant splicing.
[0781] In embodiments, a subject treated using the methods of the
invention expresses a partially functional tuberin protein from one
allele, wherein the partially functional tuberin protein is caused
by a frameshift mutation, a nonsense mutation, a missense mutation,
or a partial gene deletion. In embodiments, a subject treated using
the methods of the invention expresses a nonfunctional tuberin
protein from one allele, wherein the nonfunctional tuberin protein
is caused by a frameshift mutation, a nonsense mutation, a missense
mutation, a partial gene deletion, in one allele. In embodiments, a
subject treated using the methods of the invention has a TSC2 whole
gene deletion, in one allele.
[0782] In embodiments, the subject has a TSC2 missense mutation
selected from R611Q/W and R905W. In embodiments, the subject has
TSC2 deletion mutation of at least 6 amino acids in exon 38. In
embodiments, the subject has TSC2 substitution mutation of arginine
611 for tryptophan or glutamine. In embodiments, a subject having
any TSC2 mutation known in the art and described in the literature,
e.g., by (Au, K., et al., J. Child Neurol., 2004, 19: 699-709),
referenced above, is treated using the methods and compositions of
the present invention.
Use of TANGO for Increasing Tuberin Protein Expression
[0783] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) is used in the methods of the invention
to increase expression of a tuberin protein. In these embodiments,
a retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding
tuberin protein is present in the nucleus of a cell. Cells having a
TSC2 RIC pre-mRNA that comprises a retained intron, an exon
flanking the 5' splice site, and an exon flanking the 3' splice
site, encoding the tuberin protein, are contacted with antisense
oligomers (ASOs) that are complementary to a targeted portion of
the RIC pre-mRNA. Hybridization of the ASOs to the targeted portion
of the RIC pre-mRNA results in enhanced splicing at the splice site
(5' splice site or 3' splice site) of the retained intron and
subsequently increases target protein production.
[0784] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[0785] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a RIC pre-mRNA encoding the tuberin protein. In
embodiments, the most abundant retained intron in a RIC pre-mRNA
encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and
26, or 31 and 32. In embodiments, the most abundant retained intron
in a RIC pre-mRNA encoding the tuberin protein is intron 26. In
embodiments, the most abundant retained intron in a RIC pre-mRNA
encoding the tuberin protein is intron 31. In some embodiments, the
most abundant retained intron in a RIC pre-mRNA encoding the
tuberin protein is intron 26, and the second most abundant retained
intron in a RIC pre-mRNA encoding the tuberin protein is intron
31.
[0786] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 65%, about 5% to about 60%, about 5%
to about 55%, about 5% to about 50%, about 5% to about 45%, about
5% to about 40%, about 5% to about 35%, about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 10% to about 100%, about 10% to about 95%, about 10% to
about 90%, about 10% to about 85%, about 10% to about 80%, about
10% to about 75%, about 10% to about 70%, about 10% to about 65%,
about 10% to about 60%, about 10% to about 65%, about 10% to about
60%, about 10% to about 55%, about 10% to about 50%, about 10% to
about 45%, about 10% to about 40%, about 10% to about 35%, about
10% to about 30%, about 10% to about 25%, about 10% to about 20%,
about 15% to about 100%, about 15% to about 95%, about 15% to about
90%, about 15% to about 85%, about 15% to about 80%, about 15% to
about 75%, about 15% to about 70%, about 15% to about 65%, about
15% to about 60%, about 15% to about 65%, about 15% to about 60%,
about 15% to about 55%, about 15% to about 50%, about 15% to about
45%, about 15% to about 40%, about 15% to about 35%, about 15% to
about 30%, about 15% to about 25%, about 20% to about 100%, about
20% to about 95%, about 20% to about 90%, about 20% to about 85%,
about 20% to about 80%, about 20% to about 75%, about 20% to about
70%, about 20% to about 65%, about 20% to about 60%, about 20% to
about 65%, about 20% to about 60%, about 20% to about 55%, about
20% to about 50%, about 20% to about 45%, about 20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about
100%, about 25% to about 95%, about 25% to about 90%, about 25% to
about 85%, about 25% to about 80%, about 25% to about 75%, about
25% to about 70%, about 25% to about 65%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 60%, about 25% to about
55%, about 25% to about 50%, about 25% to about 45%, about 25% to
about 40%, or about 25% to about 35%, retention.
[0787] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g. in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features (e.g.
in the case of an antisense oligomer, the presence of additional,
unrecited nucleobases).
[0788] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g. nucleobase sequence) as well as those which do not materially
affect the character or function of the claimed invention. As used
herein, the term "consisting" is used to indicate the presence of
the recited feature (e.g. nucleobase sequence) alone (so that in
the case of an antisense oligomer consisting of a specified
nucleobase sequence, the presence of additional, unrecited
nucleobases is excluded).
[0789] In embodiments, the targeted region is in a retained intron
that is the second most abundant intron in a RIC pre-mRNA encoding
the tuberin protein. For example, the second most abundant retained
intron may be targeted rather than the most abundant retained
intron due to the uniqueness of the nucleotide sequence of the
second most abundant retained intron, ease of ASO design to target
a particular nucleotide sequence, and/or amount of increase in
protein production resulting from targeting the intron with an ASO.
In embodiments, the retained intron is the second most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the second most
abundant intron in the population of RIC pre-mRNAs encoding the
target protein induces splicing out of two or more retained introns
in the population, including the retained intron to which the
antisense oligomer is targeted or binds. In embodiments,
fully-spliced (mature) RNA encoding the target protein is thereby
produced. In embodiments, the second most retained intron in a RIC
pre-mRNA encoding the tuberin protein is intron 4, 25, 26, 31, 32,
25 and 26, or 31 and 32. In some embodiments, the second most
abundant retained intron in a RIC pre-mRNA encoding the tuberin
protein is intron 31.
[0790] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[0791] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[0792] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for the
TSC2 gene in the published reference genome deposited in the NCBI
repository of biological and scientific information. As used
herein, the "wild-type sequence" refers to the canonical sequence
for the TSC2 gene found at NCBI Gene ID 7249. Also used herein, a
nucleotide position denoted with an "e" indicates the nucleotide is
present in the sequence of an exon (e.g., the exon flanking the 5'
splice site or the exon flanking the 3' splice site).
[0793] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding tuberin protein,
resulting in increased expression of TSC2. As used herein,
"contacting" or administering to cells refers to any method of
providing an ASO in immediate proximity with the cells such that
the ASO and the cells interact. A cell that is contacted with an
ASO will take up or transport the ASO into the cell. The method
involves contacting a condition or disease-associated or condition
or disease-relevant cell with any of the ASOs described herein. In
some embodiments, the ASO may be further modified or attached
(e.g., covalently attached) to another molecule to target the ASO
to a cell type, enhance contact between the ASO and the condition
or disease-associated or condition or disease-relevant cell, or
enhance uptake of the ASO.
[0794] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[0795] In embodiments, contacting a cell that expresses a TSC2 RIC
pre-mRNA with an ASO that is complementary to a targeted portion of
the TSC2 RIC pre-mRNA transcript results in a measurable increase
in the amount of the tuberin protein (e.g., a target protein)
encoded by the pre-mRNA. Methods of measuring or detecting
production of a protein will be evident to one of skill in the art
and include any known method, for example, Western blotting, flow
cytometry, immunofluorescence microscopy, and ELISA.
[0796] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a TSC2 RIC pre-mRNA
transcript results in an increase in the amount of tuberin protein
produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200,
250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of
the protein produced by a cell in the absence of the ASO/absence of
treatment. In embodiments, the total amount of tuberin protein
produced by the cell to which the antisense oligomer was contacted
is increased about 1.1 to about 10-fold, about 1.5 to about
10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about
4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the amount of target protein produced by
an control compound. A control compound can be, for example, an
oligonucleotide that is not complementary to the targeted portion
of the RIC pre-mRNA.
[0797] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a TSC2 RIC pre-mRNA
transcript results in an increase in the amount of mRNA encoding
TSC2, including the mature mRNA encoding the target protein. In
some embodiments, the amount of mRNA encoding tuberin protein, or
the mature mRNA encoding the tuberin protein, is increased by at
least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350,
400, 450, 500, or 1000%, compared to the amount of the protein
produced by a cell in the absence of the ASO/absence of treatment.
In embodiments, the total amount of the mRNA encoding tuberin
protein, or the mature mRNA encoding tuberin protein produced in
the cell to which the antisense oligomer was contacted is increased
about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to
about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold,
about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to
about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold,
about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold compared to the
amount of mature RNA produced in an untreated cell, e.g., an
untreated cell or a cell treated with a control compound. A control
compound can be, for example, an oligonucleotide that is not
complementary to the targeted portion of the TSC2 RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[0798] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of tuberin
protein in cells, for example, in a subject having Tuberous
Sclerosis Complex caused by a deficiency in the amount or activity
of tuberin protein, by increasing the level of mRNA encoding
tuberin protein, or the mature mRNA encoding tuberin protein. In
particular, the methods and compositions as described herein induce
the constitutive splicing of a retained intron from a TSC2 RIC
pre-mRNA transcript encoding tuberin protein, thereby increasing
the level of mRNA encoding tuberin protein, or the mature mRNA
encoding tuberin protein and increasing the expression of tuberin
protein.
[0799] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[0800] In embodiments, constitutive splicing correctly removes a
retained intron from a TSC2 RIC pre-mRNA, wherein the TSC2 RIC
pre-mRNA is transcribed from a wild-type gene or allele, or a
polymorphic gene or allele, that encodes a fully-functional target
protein or functional RNA, and wherein the gene or allele does not
have a mutation that causes alternative splicing or aberrant
splicing of the retained intron.
[0801] In some embodiments, constitutive splicing of a retained
intron from a TSC2 RIC pre-mRNA encoding tuberin protein correctly
removes a retained intron from a TSC2 RIC pre-mRNA encoding tuberin
protein, wherein the TSC2 RIC pre-mRNA is transcribed from a gene
or allele from which the target gene or functional RNA is produced
at a reduced level compared to production from a wild-type allele,
and wherein the gene or allele does not have a mutation that causes
alternative splicing or aberrant splicing of the retained intron.
In these embodiments, the correct removal of the constitutively
spliced retained intron results in production of target protein or
functional RNA that is functional when compared to an equivalent
wild-type protein or functional RNA.
[0802] In other embodiments, constitutive splicing correctly
removes a retained intron from a TSC2 RIC pre-mRNA, wherein the
TSC2 RIC pre-mRNA is transcribed from a gene or allele that encodes
a target protein or functional RNA produced in a form having
reduced function compared to an equivalent wild-type protein or
functional RNA, and wherein the gene or allele does not have a
mutation that causes alternative splicing or aberrant splicing of
the retained intron. In these embodiments, the correct removal of
the constitutively spliced retained intron results in production of
partially functional target protein, or functional RNA that is
partially functional when compared to an equivalent wild-type
protein or functional RNA.
[0803] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[0804] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding tuberin protein or the amount of tuberin protein by
modulating alternative splicing or aberrant splicing of a pre-mRNA
transcribed from the TSC2 gene. Modulation of alternative splicing
or aberrant splicing can be measured using any known method for
analyzing the sequence and length of RNA species, e.g., by RT-PCR
and using methods described elsewhere herein and in the literature.
In embodiments, modulation of alternative or aberrant splicing is
determined based on an increase or decrease in the amount of the
spliced species of interest of at least 10% or 1.1-fold. In
embodiments, modulation is determined based on an increase or
decrease at a level that is at least 10% to 100% or 1.1 to 10-fold,
as described herein regarding determining an increase in mRNA
encoding tuberin protein in the methods of the invention.
[0805] In embodiments, the method is a method wherein the TSC2 RIC
pre-mRNA was produced by partial splicing of a wild-type TSC2
pre-mRNA. In embodiments, the method is a method wherein the TSC2
RIC pre-mRNA was produced by partial splicing of a full-length
wild-type TSC2 pre-mRNA. In embodiments, the TSC2 RIC pre-mRNA was
produced by partial splicing of a full-length TSC2 pre-mRNA. In
these embodiments, a full-length TSC2 pre-mRNA may have a
polymorphism in a splice site of the retained intron that does not
impair correct splicing of the retained intron as compared to
splicing of the retained intron having the wild-type splice site
sequence.
[0806] In embodiments, the mRNA encoding tuberin protein is a
full-length mature mRNA, or a wild-type mature mRNA. In these
embodiments, a full-length mature mRNA may have a polymorphism that
does not affect the activity of the target protein or the
functional RNA encoded by the mature mRNA, as compared to the
activity of tuberin protein encoded by the wild-type mature
mRNA.
Antisense Oligomers
[0807] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of a TSC2 RIC pre-mRNA. As used herein, the
terms "ASO" and "antisense oligomer" are used interchangeably and
refer to an oligomer such as a polynucleotide, comprising
nucleobases that hybridizes to a target nucleic acid (e.g., a TSC2
RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base
pairing (G-U). The ASO may have exact sequence complementary to the
target sequence or near complementarity (e.g., sufficient
complementarity to bind the target sequence and enhancing splicing
at a splice site). ASOs are designed so that they bind (hybridize)
to a target nucleic acid (e.g., a targeted portion of a pre-mRNA
transcript) and remain hybridized under physiological conditions.
Typically, if they hybridize to a site other than the intended
(targeted) nucleic acid sequence, they hybridize to a limited
number of sequences that are not a target nucleic acid (to a few
sites other than a target nucleic acid). Design of an ASO can take
into consideration the occurrence of the nucleic acid sequence of
the targeted portion of the pre-mRNA transcript or a sufficiently
similar nucleic acid sequence in other locations in the genome or
cellular pre-mRNA or transcriptome, such that the likelihood the
ASO will bind other sites and cause "off-target" effects is
limited. Any antisense oligomers known in the art, for example in
PCT Application No. PCT/US2014/054151, published as WO 2015/035091,
titled "Reducing Nonsense-Mediated mRNA Decay," can be used to
practice the methods described herein.
[0808] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[0809] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining non-complementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0810] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[0811] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and
Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by
reference in their entirety.
[0812] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine, 5,
6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[0813] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081
(1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et
al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer
Drug Design 6:539 (1991); Zon et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphorothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[0814] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the invention, including, but not limited
to, any of the ASOs set forth herein in Table 3, comprises an ASO
having phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[0815] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention, including, but not limited to, any of the ASOs set
forth herein in Table 3, comprises about 5-100% Rp, at least about
5% Rp, at least about 10% Rp, at least about 15% Rp, at least about
20% Rp, at least about 25% Rp, at least about 30% Rp, at least
about 35% Rp, at least about 40% Rp, at least about 45% Rp, at
least about 50% Rp, at least about 55% Rp, at least about 60% Rp,
at least about 65% Rp, at least about 70% Rp, at least about 75%
Rp, at least about 80% Rp, at least about 85% Rp, at least about
90% Rp, or at least about 95% Rp, with the remainder Sp, or about
100% Rp. In embodiments, an ASO used in the methods of the
invention, including, but not limited to, any of the ASOs set forth
herein in Table 3, comprises about 10% to about 100% Rp, about 15%
to about 100% Rp, about 20% to about 100% Rp, about 25% to about
100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp,
about 40% to about 100% Rp, about 45% to about 100% Rp, about 50%
to about 100% Rp, about 55% to about 100% Rp, about 60% to about
100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp,
about 75% to about 100% Rp, about 80% to about 100% Rp, about 85%
to about 100% Rp, about 90% to about 100% Rp, or about 95% to about
100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp,
about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45%
to about 55% Rp, with the remainder Sp.
[0816] In embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 3, comprises about 5-100% Sp, at least about 5% Sp, at least
about 10% Sp, at least about 15% Sp, at least about 20% Sp, at
least about 25% Sp, at least about 30% Sp, at least about 35% Sp,
at least about 40% Sp, at least about 45% Sp, at least about 50%
Sp, at least about 55% Sp, at least about 60% Sp, at least about
65% Sp, at least about 70% Sp, at least about 75% Sp, at least
about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at
least about 95% Sp, with the remainder Rp, or about 100% Sp. In
embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 3, comprises about 10% to about 100% Sp, about 15% to about
100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp,
about 30% to about 100% Sp, about 35% to about 100% Sp, about 40%
to about 100% Sp, about 45% to about 100% Sp, about 50% to about
100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp,
about 65% to about 100% Sp, about 70% to about 100% Sp, about 75%
to about 100% Sp, about 80% to about 100% Sp, about 85% to about
100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp,
about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to
about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55%
Sp, with the remainder Rp.
[0817] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[0818] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[0819] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[0820] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[0821] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[0822] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[0823] In other embodiments, the ASOs are complementary to (and
bind to) a targeted portion of a TSC2 RIC pre-mRNA that is
downstream (in the 3' direction) of the 5' splice site of the
retained intron in a TSC2 RIC pre-mRNA (e.g., the direction
designated by positive numbers relative to the 5' splice site)
(FIG. 1). In some embodiments, the ASOs are complementary to a
targeted portion of the TSC2 RIC pre-mRNA that is within the region
+6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100
relative to the 5' splice site of the retained intron. In some
embodiments, the ASO is not complementary to nucleotides +1 to +5
relative to the 5' splice site (the first five nucleotides located
downstream of the 5' splice site). In some embodiments, the ASOs
may be complementary to a targeted portion of a TSC2 RIC pre-mRNA
that is within the region between nucleotides +6 and +50 relative
to the 5' splice site of the retained intron. In some aspects, the
ASOs are complementary to a targeted portion that is within the
region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to
+40, +6 to +30, or +6 to +20 relative to 5' splice site of the
retained intron.
[0824] In some embodiments, the ASOs are complementary to a
targeted region of a TSC2 RIC pre-mRNA that is upstream (5'
relative) of the 3' splice site of the retained intron in a TSC2
RIC pre-mRNA (e.g., in the direction designated by negative
numbers) (FIG. 1). In some embodiments, the ASOs are complementary
to a targeted portion of the TSC2 RIC pre-mRNA that is within the
region -16 to -500, -16 to -400, -16 to -300, -6 to -200, -16 to
-100, relative to the 3' splice site of the retained intron. In
some embodiments, the ASO is not complementary to nucleotides -1 to
-15 relative to the 3' splice site (the first 15 nucleotides
located upstream of the 3' splice site). In some embodiments, the
ASOs are complementary to a targeted portion of the TSC2 RIC
pre-mRNA that is within the region -16 to -50 relative to the 3'
splice site of the retained intron. In some aspects, the ASOs are
complementary to a targeted portion that is within the region -16
to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16 to -40,
or -16 to -30 relative to 3' splice site of the retained
intron.
[0825] In embodiments, the targeted portion of the TSC2 RIC
pre-mRNA is within the region +100 relative to the 5' splice site
of the retained intron to -100 relative to the 3' splice site of
the retained intron.
[0826] In some embodiments, the ASOs are complementary to a
targeted portion of a TSC2 RIC pre-mRNA that is within the exon
flanking the 5' splice site (upstream) of the retained intron (FIG.
1). In some embodiments, the ASOs are complementary to a targeted
portion of the TSC2 RIC pre-mRNA that is within the region +2e to
-4e in the exon flanking the 5' splice site of the retained intron.
In some embodiments, the ASOs are not complementary to nucleotides
-1e to -3e relative to the 5' splice site of the retained intron.
In some embodiments, the ASOs are complementary to a targeted
portion of the TSC2 RIC pre-mRNA that is within the region -4e to
-100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to
-50e, -4 to -40e, -4e to -30e, or -4e to -20e relative to the 5'
splice site of the retained intron.
[0827] In some embodiments, the ASOs are complementary to a
targeted portion of a TSC2 RIC pre-mRNA that is within the exon
flanking the 3' splice site (downstream) of the retained intron
(FIG. 1). In some embodiments, the ASOs are complementary to a
targeted portion to the TSC2 RIC pre-mRNA that is within the region
+2e to -4e in the exon flanking the 3' splice site of the retained
intron. In some embodiments, the ASOs are not complementary to
nucleotide +1e relative to the 3' splice site of the retained
intron. In some embodiments, the ASOs are complementary to a
targeted portion of the TSC2 RIC pre-mRNA that is within the region
+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e,
+2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative to
the 3' splice site of the retained intron. The ASOs may be of any
length suitable for specific binding and effective enhancement of
splicing. In some embodiments, the ASOs consist of 8 to 50
nucleobases. For example, the ASO may be 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some
embodiments, the ASOs consist of more than 50 nucleobases. In some
embodiments, the ASO is from 8 to 50 nucleobases, 8 to 40
nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25
nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50
nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30
nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15
nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35
nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20
nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40
nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25
nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50
nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30
nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15
nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35
nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20
nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35
nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20
nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35
nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20
nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35
nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50
nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to
30 nucleobases in length. In some embodiments, the ASOs are 30
nucleotides in length. In some embodiments, the ASOs are 29
nucleotides in length. In some embodiments, the ASOs are 28
nucleotides in length. In some embodiments, the ASOs are 27
nucleotides in length. In some embodiments, the ASOs are 26
nucleotides in length. In some embodiments, the ASOs are 25
nucleotides in length. In some embodiments, the ASOs are 24
nucleotides in length. In some embodiments, the ASOs are 23
nucleotides in length. In some embodiments, the ASOs are 22
nucleotides in length. In some embodiments, the ASOs are 21
nucleotides in length. In some embodiments, the ASOs are 20
nucleotides in length. In some embodiments, the ASOs are 19
nucleotides in length. In some embodiments, the ASOs are 18
nucleotides in length. In some embodiments, the ASOs are 17
nucleotides in length. In some embodiments, the ASOs are 16
nucleotides in length. In some embodiments, the ASOs are 15
nucleotides in length. In some embodiments, the ASOs are 14
nucleotides in length. In some embodiments, the ASOs are 13
nucleotides in length. In some embodiments, the ASOs are 12
nucleotides in length. In some embodiments, the ASOs are 11
nucleotides in length. In some embodiments, the ASOs are 10
nucleotides in length.
[0828] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[0829] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[0830] In some embodiments, the nucleic acid to be targeted by an
ASO is a TSC2 RIC pre-mRNA expressed in a cell, such as a
eukaryotic cell. In some embodiments, the term "cell" may refer to
a population of cells. In some embodiments, the cell is in a
subject. In some embodiments, the cell is isolated from a subject.
In some embodiments, the cell is ex vivo. In some embodiments, the
cell is a condition or disease-relevant cell or a cell line. In
some embodiments, the cell is in vitro (e.g., in cell culture).
Pharmaceutical Compositions
[0831] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent
or carrier.
[0832] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose. The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphor sulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[0833] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[0834] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancers are a surfactant, fatty
acid, bile salt, chelating agent, or non-chelating
non-surfactant.
[0835] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[0836] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[0837] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[0838] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[0839] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[0840] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. Multiple tissues and organs are affected by
tuberous sclerosis complex, with the brain, kidney, skin, lung and
heart being the most significantly affected tissues. The ASOs of
the present invention may be administered to patients topically to
the skin or by pulmonary delivery to the lung. The ASOs of the
present invention may be administered to patients parenterally, for
example, by intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous injection. In embodiments,
delivery is to the brain, kidney, skin, lung or heart. In
embodiments, a fetus is treated in utero, e.g., by administering
the ASO composition to the fetus directly or indirectly (e.g., via
the mother).
Methods of Identifying Additional ASOs that Enhance Splicing
[0841] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of a TSC2 RIC pre-mRNA, specifically at the target intron. ASOs
that specifically hybridize to different nucleotides within the
target region of the pre-mRNA may be screened to identify
(determine) ASOs that improve the rate and/or extent of splicing of
the target intron. In some embodiments, the ASO may block or
interfere with the binding site(s) of a splicing
repressor(s)/silencer. Any method known in the art may be used to
identify (determine) an ASO that when hybridized to the target
region of the intron results in the desired effect (e.g., enhanced
splicing, protein or functional RNA production). These methods also
can be used for identifying ASOs that enhance splicing of the
retained intron by binding to a targeted region in an exon flanking
the retained intron, or in a non-retained intron. An example of a
method that may be used is provided below.
[0842] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site.
[0843] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[0844] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[0845] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[0846] As described for the ASO walk above, the ASO micro-walk is
performed by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[0847] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by topical application to the skin,
pulmonary delivery to the lung, intrathecal injection,
intracerebroventricular injection, intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. Following administration, the cells, tissues, and/or
organs of the model animals may be assessed to determine the effect
of the ASO treatment by for example evaluating splicing
(efficiency, rate, extent) and protein production by methods known
in the art and described herein. The animal models may also be any
phenotypic or behavioral indication of the disease or disease
severity.
Compositions and Methods for Treatment of Polycystic Kidney
Disease
[0848] Individual introns in primary transcripts of protein-coding
genes having more than one intron are spliced from the primary
transcript with different efficiencies. In most cases only the
fully spliced mRNA is exported through nuclear pores for subsequent
translation in the cytoplasm. Unspliced and partially spliced
transcripts are detectable in the nucleus. It is generally thought
that nuclear accumulation of transcripts that are not fully spliced
is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[0849] Substantial levels of partially-spliced transcripts of the
PKD2 gene, which encodes the PC-2 protein that is deficient in the
debilitating genetic disease, Polycystic Kidney Disease, have been
discovered in the nucleus of human cells. These PKD2 pre-mRNA
species comprise at least one retained intron. The present
invention provides compositions and methods for upregulating
splicing of one or more retained PKD2 introns that are
rate-limiting for the nuclear stages of gene expression to increase
steady-state production of fully-spliced, mature mRNA, and thus,
translated PC-2 protein levels. These compositions and methods
utilize antisense oligomers (ASOs) that promote constitutive
splicing at an intron splice site of a retained-intron-containing
PKD2 pre-mRNA that accumulates in the nucleus. Thus, in
embodiments, PC-2 protein is increased using the methods of the
invention to treat a disease caused by PC-2 deficiency.
[0850] In other embodiments, the methods of the invention are used
to increase PC-2 production to treat a condition in a subject in
need thereof. In embodiments, the subject has condition in which
PC-2 is not necessarily deficient relative to wild-type, but where
an increase in PC-2 mitigates the condition nonetheless. In
embodiments, the condition is a caused by a PC-2
haploinsufficiency.
Polycystic Kidney Disease
[0851] Polycystic Kidney Disease (PKD) is an autosomal dominant
multisystem disorder characterized by the evolution of renal cysts
and a variety of extrarenal manifestations (Torres and Harris,
2009). The main clinical and pathological findings are renal
disease due to the development and enlargement of renal cysts,
which results in renal manifestations such an increase in kidney
volume that correlates directly with the increase in the cyst
volume. Other PKD manifestations include hypertension; endothelial
vasodilation; constrictive nitric oxide synthase activity;
polycystic liver disease; vascular manifestations including
intracranial aneurysms, thoracic aortic dissections and coronary
artery aneurysms; and progressive renal failure that leads to
end-stage renal disease (ESRD) by age 70. While PKD can be
diagnosed in utero or at birth through the use of fetal
ultrasonography, PKD is classically diagnosed later in life through
the detection of renal cysts as determined by renal ultrasound. The
worldwide PKD prevalence is estimated to be between 1:400 and
1:1000, with a male/female sex ratio of -1.2 (Torres and Harris,
2009).
[0852] Mutations in either the PKD1 or PKD2 gene have been reported
to cause PKD. Mutations in PKD1 typically manifest earlier in life
than mutations in PKD2 (age at ESRD 54.3 vs. 74.0 years for PKD1
and PKD2, respectively) and typically result in a more severe
disease state due to the appearance of cysts at a younger age
(Torres and Harris, 2009). Due to this difference in
pathophysiology, the late onset form of PKD generally arises from
mutations in the PKD2 gene. PKD2 encodes the PC-2 protein, a 968
amino acid protein containing a short N-terminal cytoplasmic region
with a ciliary motif, 6 transmembrane domains and a short
C-terminal portion. The human genomic sequence of the PKD2 gene is
set forth at NCBI Gene ID 5311, and the protein at
UniProtKB/Swiss-Prot: Q13563-1, described by, e.g., Mochizuki T, et
al., 1996, "PKD2, a gene for polycystic kidney disease that encodes
an integral membrane protein," Science 272:1339-1342, incorporated
by reference herein. The PKD2 mRNA sequence is set forth at NCBI
Reference Sequence: NM_000297.3.2, described by Yang Y, et al.,
2015, "Oligomerization of the polycystin-2 C-terminal tail and
effects on its Ca2+ binding properties," J. Bio. Chem. 290 (16),
10544-10554, both incorporated by reference herein. Mutations in
PKD2 cause late onset autosomal dominant PKD (ADPKD), the most
prevalent of the inherited renal cystic diseases.
[0853] The PKD2 gene consists of 15 exons and is located on
chromosome 4p22.1. PKD2 mutations in PKD are spread across the
entire protein, with 95 truncating mutations of PKD2 reported in
the ADPKD Mutation Database (maintained by the PKD Foundation, 8330
Ward Parkway, Suite 510, Kansas City, Mo. 64114). Because a
homozygous deficiency in PKD2 is predicted to be incompatible with
live birth, haploinsufficiency is the most likely mechanism of
ADPKD disease manifestation (Torres and Harris, 2009). Mutations
such as nonsense and insertions/deletions are associated with the
classic ADPKD2 phenotype display functional haploinsufficiency. A
PKD missense mutation that results in expression of the PC-2-D511V
protein was predicted to be indistinguishable from wild-type PC-2
in terms of stability (Reynolds, et al., 1999, J. Am. Soc. Nephrol.
10: 2342-2351). The PC-2-D511V variant, despite its stability, was
shown to be dysfunctional due to a predicted disruption in its
ability to act as an ion channel. Thus, even stable variants can
cause the phenotype if the nascent activity is disrupted. The
disease is described, e.g., by OMIM #613095 (Online Mendelian
Inheritance in Man, Johns Hopkins University, 1966-2015),
incorporated by reference herein.
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[0854] In embodiments, the methods disclosed herein exploit the
presence of retained-intron-containing pre-mRNA (RIC pre-mRNA)
transcribed from the PKD2 gene and encoding PC-2 protein, in the
cell nucleus. Splicing of the identified PKD2 RIC pre-mRNA species
to produce mature, fully-spliced, PKD2 mRNA, is induced using ASOs
that stimulate splicing out of the retained introns. The resulting
mature PKD2 mRNA can be exported to the cytoplasm and translated,
thereby increasing the amount of PC-2 protein in the patient's
cells and alleviating symptoms of Polycystic Kidney Disease. This
method, described further below, is known as Targeted Augmentation
of Nuclear Gene Output (TANGO).
PKD2 Nuclear Transcripts
[0855] As described herein in the Examples, the PKD2 gene was
analyzed for intron-retention events and retention of intron 5 was
observed. RNA sequencing (RNAseq), visualized in the UCSC genome
browser, showed PKD2 transcripts expressed in renal epithelial
cells and localized in either the cytoplasmic or nuclear fraction.
In both fractions, reads were not observed for the majority of the
introns. However, higher read density was detected for intron 5 in
the nuclear fraction compared to the cytoplasmic fraction
indicating that splicing efficiency of intron 5 is low, resulting
in intron retention. The retained-intron containing pre-mRNA
transcripts accumulate primarily in the nucleus and are not
translated into the PC-2 protein. The read density for intron 5
indicated 18% intron retention (FIG. 29). The percent intron
retention (PIR) value for intron 5 was obtained by averaging four
values (23, 13, 22, and 14), each determined in renal epithelial
cells using one of four different algorithms. Analysis of the
ENCODE data (described by, e.g., Tilgner, et al., 2012, "Deep
sequencing of subcellular RNA fractions shows splicing to be
predominantly co-transcriptional in the human genome but
inefficient for lncRNAs," Genome Research 22(9):1616-25) to
identify intron retention events did not identify intron 5 as
retained.
[0856] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PKD2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a PKD2
genomic sequence comprising retained intron 5. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 5545. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 5545 comprising a retained intron 5. In some embodiments,
the ASOs disclosed herein target a PKD2 RIC pre-mRNA sequence. In
some embodiments, the ASO targets a PKD2 RIC pre-mRNA sequence
comprising a retained intron 5. In some embodiments, the ASO
targets a PKD2 RIC pre-mRNA sequence according to SEQ ID NO: 5546.
In some embodiments, the ASO targets a PKD2 RIC pre-mRNA sequence
according to SEQ ID NO: 5546 comprising a retained intron 5. In
some embodiments, the ASOs disclosed herein target SEQ ID NO: 5825.
In some embodiments, the ASO has a sequence according to any one of
SEQ ID NOs: 5547-5824.
[0857] In some embodiments, the ASO targets exon 5 of a PKD2 RIC
pre-mRNA comprising a retained intron 5. In some embodiments, the
ASO targets an exon 5 sequence upstream (or 5') from the 5' splice
site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some
embodiments, the ASO targets an exon sequence about 4 to about 204
nucleotides upstream (or 5') from the 5' splice site of a PKD2 RIC
pre-mRNA comprising a retained intron 5. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
5547-5587.
[0858] In some embodiments, the ASO targets intron 5 in a PKD2 RIC
pre-mRNA comprising a retained intron 5. In some embodiments, the
ASO targets an intron 5 sequence downstream (or 3') from the 5'
splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5.
In some embodiments, the ASO targets an intron 5 sequence about 6
to about 497 nucleotides downstream (or 3') from the 5' splice site
of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some
embodiments, the ASO has a sequence according to any one of SEQ ID
NOs: 5588-5684.
[0859] In some embodiments, the ASO targets an intron 5 sequence
upstream (or 5') from the 3' splice site of a PKD2 RIC pre-mRNA
comprising a retained intron 5. In some embodiments, the ASO
targets an intron 5 sequence about 16 to about 496 nucleotides
upstream (or 5') from the 3' splice site of a PKD2 RIC pre-mRNA a
comprising retained intron 5. In some embodiments, the ASO has a
sequence according to any one of SEQ ID NOs: 5685-5781.
[0860] In some embodiments, the ASO targets exon 6 in a PKD2 RIC
pre-mRNA comprising a retained intron 5. In some embodiments, the
ASO targets an exon 6 sequence downstream (or 3') from the 3'
splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5.
In some embodiments, the ASO targets an exon 6 sequence about 2 to
about 212 nucleotides downstream (or 3') from the 3' splice site of
a PKD2 RIC pre-mRNA comprising a retained intron 5. In some
embodiments, the ASO has a sequence according to any one of SEQ ID
NOs: 5782-5824.
[0861] In embodiments, the targeted portion of the PKD2 RIC
pre-mRNA is in intron 5. The PKD2 intron numbering used herein
corresponds to the mRNA sequence at NM_000297.3. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained intron 5 and subsequently increases
PC-2 protein production. It is understood that the intron numbering
may change in reference to a different PKD2 mRNA isoform sequence.
One of skill in the art can determine the corresponding intron
number in any PKD2 isoform based on an intron sequence provided
herein or using the intron number provided in reference to the mRNA
sequence at NM_000297.3. One of skill in the art also can determine
the sequences of flanking exons in any PKD2 isoform for targeting
using the methods of the invention, based on an intron sequence
provided herein or using the intron number provided in reference to
the mRNA sequence at NM_000297.3. In embodiments, the compositions
and methods of the present invention are used to increase the
expression of any known PKD2 isoform, e.g., as described in the
NCBI Gene ID database at Gene ID 5311 (NCBI repository of
biological and scientific information, operated by National Center
for Biotechnology Information, National Library of Medicine, 8600
Rockville Pike, Bethesda, Md. USA 20894), incorporated by reference
herein.
PC-2 Protein Expression
[0862] As described above, PC-2 mutations in ADPKD are spread
across the entire protein, with 95 PC-2 truncating mutations having
been reported in the ADPKD Mutation Database (PKD Foundation).
[0863] In embodiments, the methods described herein are used to
increase the production of a functional PC-2 protein. As used
herein, the term "functional" refers to the amount of activity or
function of a PC-2 protein that is necessary to eliminate any one
or more symptoms of a treated condition, e.g., Polycystic Kidney
Disease. In embodiments, the methods are used to increase the
production of a partially functional PC-2 protein. As used herein,
the term "partially functional" refers to any amount of activity or
function of the PC-2 protein that is less than the amount of
activity or function that is necessary to eliminate or prevent any
one or more symptoms of a disease or condition. In some
embodiments, a partially functional protein or RNA will have at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 75%, at least 80%, 85%, at
least 90%, or at least 95% less activity relative to the fully
functional protein or RNA.
[0864] In embodiments, the method is a method of increasing the
expression of the PC-2 protein by cells of a subject having a RIC
pre-mRNA encoding the PC-2 protein, wherein the subject has
Polycystic Kidney Disease caused by a deficient amount of activity
of PC-2 protein, and wherein the deficient amount of the PC-2
protein is caused by haploinsufficiency of the PC-2 protein. In
such an embodiment, the subject has a first allele encoding a
functional PC-2 protein, and a second allele from which the PC-2
protein is not produced. In another such embodiment, the subject
has a first allele encoding a functional PC-2 protein, and a second
allele encoding a nonfunctional PC-2 protein. In another such
embodiment, the subject has a first allele encoding a functional
PC-2 protein, and a second allele encoding a partially functional
PC-2 protein. In any of these embodiments, the antisense oligomer
binds to a targeted portion of the RIC pre-mRNA transcribed from
the first allele (encoding functional PC-2 protein), thereby
inducing constitutive splicing of the retained intron from the RIC
pre-mRNA, and causing an increase in the level of mature mRNA
encoding functional PC-2 protein, and an increase in the expression
of the PC-2 protein in the cells of the subject.
[0865] In embodiments, the subject has a first allele encoding a
functional PC-2 protein, and a second allele encoding a partially
functional PC-2 protein, and the antisense oligomer binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele (encoding functional PC-2 protein) or a targeted portion of
the RIC pre-mRNA transcribed from the second allele (encoding
partially functional PC-2 protein), thereby inducing constitutive
splicing of the retained intron from the RIC pre-mRNA, and causing
an increase in the level of mature mRNA encoding the PC-2 protein,
and an increase in the expression of functional or partially
functional PC-2 protein in the cells of the subject.
[0866] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of PC-2
protein in cells of a subject having a RIC pre-mRNA encoding PC-2
protein, wherein the subject has a deficiency, e.g., Polycystic
Kidney Disease, in the amount or function of a PC-2 protein.
[0867] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease or condition is targeted
by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the
disease is targeted by the ASOs. For example, a disease that is the
result of a mutation or deficiency of a first protein in a
particular pathway may be ameliorated by targeting a RIC pre-mRNA
that encodes a second protein, thereby increasing production of the
second protein. In some embodiments, the function of the second
protein is able to compensate for the mutation or deficiency of the
first protein.
[0868] In embodiments, the subject has:
[0869] (a) a first mutant allele from which [0870] i) the PC-2
protein is produced at a reduced level compared to production from
a wild-type allele, [0871] ii) the PC-2 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0872] iii) the PC-2 protein or functional RNA is not
produced; and
[0873] (b) a second mutant allele from which [0874] i) the PC-2
protein is produced at a reduced level compared to production from
a wild-type allele, [0875] ii) the PC-2 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0876] iii) the PC-2 protein is not produced, and
wherein the RIC pre-mRNA is transcribed from the first allele
and/or the second allele. In these embodiments, the ASO binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele or the second allele, thereby inducing constitutive splicing
of the retained intron from the RIC pre-mRNA, and causing an
increase in the level of mRNA encoding PC-2 protein and an increase
in the expression of the target protein or functional RNA in the
cells of the subject. In these embodiments, the target protein or
functional RNA having an increase in expression level resulting
from the constitutive splicing of the retained intron from the RIC
pre-mRNA is either in a form having reduced function compared to
the equivalent wild-type protein (partially-functional), or having
full function compared to the equivalent wild-type protein
(fully-functional).
[0877] In embodiments, the level of mRNA encoding PC-2 protein is
increased 1.1 to 10-fold, when compared to the amount of mRNA
encoding PC-2 that is produced in a control cell, e.g., one that is
not treated with the antisense oligomer or one that is treated with
an antisense oligomer that does not bind to the targeted portion of
the PKD2 RIC pre-mRNA.
[0878] In embodiments, the condition caused by a deficient amount
or activity of PC-2 protein is not a condition caused by
alternative or aberrant splicing of the retained intron to which
the ASO is targeted. In embodiments, the condition caused by a
deficient amount or activity of the PC-2 protein is not a condition
caused by alternative or aberrant splicing of any retained intron
in a RIC pre-mRNA encoding the PC-2 protein. In embodiments,
alternative or aberrant splicing may occur in a pre-mRNA
transcribed from the gene, however the compositions and methods of
the invention do not prevent or correct this alternative or
aberrant splicing in the pre-mRNA.
[0879] In embodiments, a subject treated using the methods of the
invention expresses a partially functional PC-2 protein from one
allele, wherein the partially functional PC-2 protein is caused by
a frameshift mutation, a nonsense mutation, a missense mutation, or
a partial gene deletion. In embodiments, a subject treated using
the methods of the invention expresses a nonfunctional PC-2 protein
from one allele, wherein the nonfunctional PC-2 protein is caused
by a frameshift mutation, a nonsense mutation, a missense mutation,
a partial gene deletion, in one allele. In embodiments, a subject
treated using the methods of the invention has a PKD2 whole gene
deletion, in one allele.
[0880] In embodiments, the subject has a PC-2 missense mutation
selected from M1K, P24L, R28P, A35D, R60N, S80L, Q107D, R119H,
G121A, G135V, A147V, A190T, V262M, W292C, R306Q, L314V, R322W,
R322Q, R325P, R325Q, C331S, S332A, Y324C, S349P, A356P, A384P,
G390S, W414G, G418V, T419A, R420G, A421S, R440S, T448K, I452V,
F482C, Y487H, D511V, V516L, L517R, V519M, A552P, I556V, N578D,
N580K, M5831, A615T, F629S, C632R, R638C, L656W, L7151, I758V,
R798C, M800L, S804N, R807Q, R848Q, D886G, R893G, V9091, D919N,
T931M, R945H or S949F. In embodiments, the subject has a PC-2
deletion mutation selected from EX1_EX13del,
IVS2_3'(ABCG2)del80kb*, IVS2_3'(ABCG2)del98 kb,
IVS4+1452_IVS5-965del5722, S378del, F605del, IVS9_3'del28 kb,
2182_2183delAG, L736_N737del2 or R878del. In embodiments, the
subject has PC-2 duplication mutation Ex3dup*. In embodiments, a
subject having any PC-2 mutation known in the art and described in
the literature, e.g., by Chang, et al., 2005, Ren Fail 27: 95-100,
is treated using the methods and compositions of the present
invention.
Use of TANGO for Increasing PC-2 Protein Expression
[0881] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) is used in the methods of the invention
to increase expression of a PC-2 protein. In these embodiments, a
retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding PC-2
protein is present in the nucleus of a cell. Cells having a PKD2
RIC pre-mRNA that comprises a retained intron, an exon flanking the
5' splice site, and an exon flanking the 3' splice site, encoding
the PC-2 protein, are contacted with antisense oligomers (ASOs)
that are complementary to a targeted portion of the RIC pre-mRNA.
Hybridization of the ASOs to the targeted portion of the RIC
pre-mRNA results in enhanced splicing at the splice site (5' splice
site or 3' splice site) of the retained intron and subsequently
increases target protein production.
[0882] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[0883] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a RIC pre-mRNA encoding the PC-2 protein. In embodiments,
the most retained intron in a RIC pre-mRNA encoding the PC-2
protein is intron 5.
[0884] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 55%, about 5% to about 50%, about 5%
to about 45%, about 5% to about 40%, about 5% to about 35%, about
5% to about 30%, about 5% to about 25%, about 5% to about 20%,
about 5% to about 15%, about 10% to about 100%, about 10% to about
95%, about 10% to about 90%, about 10% to about 85%, about 10% to
about 80%, about 10% to about 75%, about 10% to about 70%, about
10% to about 65%, about 10% to about 60%, about 10% to about 55%,
about 10% to about 50%, about 10% to about 45%, about 10% to about
40%, about 10% to about 35%, about 10% to about 30%, about 10% to
about 25%, about 10% to about 20%, about 15% to about 100%, about
15% to about 95%, about 15% to about 90%, about 15% to about 85%,
about 15% to about 80%, about 15% to about 75%, about 15% to about
70%, about 15% to about 65%, about 15% to about 60%, about 15% to
about 55%, about 15% to about 50%, about 15% to about 45%, about
15% to about 40%, about 15% to about 35%, about 15% to about 30%,
about 15% to about 25%, about 20% to about 100%, about 20% to about
95%, about 20% to about 90%, about 20% to about 85%, about 20% to
about 80%, about 20% to about 75%, about 20% to about 70%, about
20% to about 65%, about 20% to about 60%, about 20% to about 55%,
about 20% to about 50%, about 20% to about 45%, about 20% to about
40%, about 20% to about 35%, about 20% to about 30%, about 25% to
about 100%, about 25% to about 95%, about 25% to about 90%, about
25% to about 85%, about 25% to about 80%, about 25% to about 75%,
about 25% to about 70%, about 25% to about 65%, about 25% to about
60%, about 25% to about 55%, about 25% to about 50%, about 25% to
about 45%, about 25% to about 40%, or about 25% to about 35%,
retention.
[0885] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g., in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features
(e.g., in the case of an antisense oligomer, the presence of
additional, unrecited nucleobases).
[0886] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g., nucleobase sequence) as well as those which do not
materially affect the character or function of the claimed
invention.
[0887] As used herein, the term "consisting" is used to indicate
the presence of the recited feature (e.g., nucleobase sequence)
alone (so that in the case of an antisense oligomer consisting of a
specified nucleobase sequence, the presence of additional,
unrecited nucleobases is excluded).
[0888] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[0889] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[0890] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for the
PKD2 gene in the published reference genome deposited in the NCBI
repository of biological and scientific information. As used
herein, the "wild-type sequence" refers to the canonical sequence
for the PKD2 gene found at NCBI Gene ID 5311. Also used herein, a
nucleotide position denoted with an "e" indicates the nucleotide is
present in the sequence of an exon (e.g., the exon flanking the 5'
splice site or the exon flanking the 3' splice site).
[0891] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding PC-2 protein,
resulting in increased expression of PC-2. As used herein,
"contacting" or administering to cells refers to any method of
providing an ASO in immediate proximity with the cells such that
the ASO and the cells interact. A cell that is contacted with an
ASO will take up or transport the ASO into the cell. The method
involves contacting a condition or disease-associated or condition
or disease-relevant cell with any of the ASOs described herein. In
some embodiments, the ASO may be further modified or attached
(e.g., covalently attached) to another molecule to target the ASO
to a cell type, enhance contact between the ASO and the condition
or disease-associated or condition or disease-relevant cell, or
enhance uptake of the ASO.
[0892] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[0893] In embodiments, contacting a cell that expresses a PKD2 RIC
pre-mRNA with an ASO that is complementary to a targeted portion of
the PKD2 RIC pre-mRNA transcript results in a measurable increase
in the amount of the PC-2 protein (e.g., a target protein) encoded
by the pre-mRNA. Methods of measuring or detecting production of a
protein will be evident to one of skill in the art and include any
known method, for example, Western blotting, flow cytometry,
immunofluorescence microscopy, and ELISA.
[0894] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a PKD2 RIC pre-mRNA
transcript results in an increase in the amount of PC-2 protein
produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200,
250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of
the protein produced by a cell in the absence of the ASO/absence of
treatment. In embodiments, the total amount of PC-2 protein
produced by the cell to which the antisense oligomer was contacted
is increased about 1.1 to about 10-fold, about 1.5 to about
10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about
4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about
6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about
1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about
6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2
to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold,
about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about
7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at
least about 2.5-fold, at least about 3-fold, at least about
3.5-fold, at least about 4-fold, at least about 5-fold, or at least
about 10-fold, compared to the amount of target protein produced by
a control compound. A control compound can be, for example, an
oligonucleotide that is not complementary to the targeted portion
of the RIC pre-mRNA.
[0895] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a PKD2 RIC pre-mRNA
transcript results in an increase in the amount of mRNA encoding
PC-2, including the mature mRNA encoding the target protein. In
some embodiments, the amount of mRNA encoding PC-2 protein, or the
mature mRNA encoding the PC-2 protein, is increased by at least 10,
20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450,
500, or 1000%, compared to the amount of the protein produced by a
cell in the absence of the ASO/absence of treatment. In
embodiments, the total amount of the mRNA encoding PC-2 protein, or
the mature mRNA encoding PC-2 protein produced in the cell to which
the antisense oligomer was contacted is increased about 1.1 to
about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold compared to the
amount of mature RNA produced in an untreated cell, e.g., an
untreated cell or a cell treated with a control compound. A control
compound can be, for example, an oligonucleotide that is not
complementary to the targeted portion of the PKD2 RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[0896] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of PC-2
protein in cells, for example, in a subject having Polycystic
Kidney Disease caused by a deficiency in the amount or activity of
PC-2 protein, by increasing the level of mRNA encoding PC-2
protein, or the mature mRNA encoding PC-2 protein. In particular,
the methods and compositions as described herein induce the
constitutive splicing of a retained intron from a PKD2 RIC pre-mRNA
transcript encoding PC-2 protein, thereby increasing the level of
mRNA encoding PC-2 protein, or the mature mRNA encoding PC-2
protein and increasing the expression of PC-2 protein.
[0897] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[0898] In embodiments, constitutive splicing correctly removes a
retained intron from a PKD2 RIC pre-mRNA, wherein the PKD2 RIC
pre-mRNA is transcribed from a wild-type gene or allele, or a
polymorphic gene or allele, that encodes a fully-functional target
protein or functional RNA, and wherein the gene or allele does not
have a mutation that causes alternative splicing or aberrant
splicing of the retained intron.
[0899] In some embodiments, constitutive splicing of a retained
intron from a PKD2 RIC pre-mRNA encoding PC-2 protein correctly
removes a retained intron from a PKD2 RIC pre-mRNA encoding PC-2
protein, wherein the PKD2 RIC pre-mRNA is transcribed from a gene
or allele from which the target gene or functional RNA is produced
at a reduced level compared to production from a wild-type allele,
and wherein the gene or allele does not have a mutation that causes
alternative splicing or aberrant splicing of the retained intron.
In these embodiments, the correct removal of the constitutively
spliced retained intron results in production of target protein or
functional RNA that is functional when compared to an equivalent
wild-type protein or functional RNA.
[0900] In other embodiments, constitutive splicing correctly
removes a retained intron from a PKD2 RIC pre-mRNA, wherein the
PKD2 RIC pre-mRNA is transcribed from a gene or allele that encodes
a target protein or functional RNA produced in a form having
reduced function compared to an equivalent wild-type protein or
functional RNA, and wherein the gene or allele does not have a
mutation that causes alternative splicing or aberrant splicing of
the retained intron. In these embodiments, the correct removal of
the constitutively spliced retained intron results in production of
partially functional target protein, or functional RNA that is
partially functional when compared to an equivalent wild-type
protein or functional RNA.
[0901] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[0902] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding PC-2 protein or the amount of PC-2 protein by
modulating alternative splicing or aberrant splicing of a pre-mRNA
transcribed from the PKD2 gene. Modulation of alternative splicing
or aberrant splicing can be measured using any known method for
analyzing the sequence and length of RNA species, e.g., by RT-PCR
and using methods described elsewhere herein and in the literature.
In embodiments, modulation of alternative or aberrant splicing is
determined based on an increase or decrease in the amount of the
spliced species of interest of at least 10% or 1.1-fold. In
embodiments, modulation is determined based on an increase or
decrease at a level that is at least 10% to 100% or 1.1 to 10-fold,
as described herein regarding determining an increase in mRNA
encoding PC-2 protein in the methods of the invention.
[0903] In embodiments, the methods described herein is a method
wherein the PKD2 RIC pre-mRNA was produced by partial splicing of a
wild-type PKD2 pre-mRNA. In embodiments, the method is a method
wherein the PKD2 RIC pre-mRNA was produced by partial splicing of a
full-length wild-type PKD2 pre-mRNA. In embodiments, the PKD2 RIC
pre-mRNA was produced by partial splicing of a full-length PKD2
pre-mRNA. In these embodiments, a full-length PKD2 pre-mRNA may
have a polymorphism in a splice site of the retained intron that
does not impair correct splicing of the retained intron as compared
to splicing of the retained intron having the wild-type splice site
sequence.
[0904] In embodiments, the mRNA encoding PC-2 protein is a
full-length mature mRNA, or a wild-type mature mRNA. In these
embodiments, a full-length mature mRNA may have a polymorphism that
does not affect the activity of the target protein or the
functional RNA encoded by the mature mRNA, as compared to the
activity of PC-2 protein encoded by the wild-type mature mRNA.
Antisense Oligomers
[0905] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of a PKD2 RIC pre-mRNA. As used herein, the
terms "ASO" and "antisense oligomer" are used interchangeably and
refer to an oligomer such as a polynucleotide, comprising
nucleobases that hybridize to a target nucleic acid (e.g., a PKD2
RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base
pairing (G-U). The ASO may have exact sequence complementary to the
target sequence or near complementarity (e.g., sufficient
complementarity to bind the target sequence and enhancing splicing
at a splice site). ASOs are designed so that they bind (hybridize)
to a target nucleic acid (e.g., a targeted portion of a pre-mRNA
transcript) and remain hybridized under physiological conditions.
Typically, if they hybridize to a site other than the intended
(targeted) nucleic acid sequence, they hybridize to a limited
number of sequences that are not a target nucleic acid (to a few
sites other than a target nucleic acid). Design of an ASO can take
into consideration the occurrence of the nucleic acid sequence of
the targeted portion of the pre-mRNA transcript or a sufficiently
similar nucleic acid sequence in other locations in the genome or
cellular pre-mRNA or transcriptome, such that the likelihood the
ASO will bind other sites and cause "off-target" effects is
limited. Any antisense oligomers known in the art, for example in
PCT Application No. PCT/US2014/054151, published as WO 2015/035091,
titled "Reducing Nonsense-Mediated mRNA Decay," can be used to
practice the methods described herein.
[0906] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[0907] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul, et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0908] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[0909] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and
Stein, Mol. Cancer Ther. 2002, 347-355, herein incorporated by
reference in their entirety.
[0910] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine,
5,6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[0911] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche, et al., Nucleic Acids Res. 14:9081
(1986); Stec, et al., J. Am. Chem. Soc. 106:6077 (1984), Stein, et
al., Nucleic Acids Res. 16:3209 (1988), Zon, et al., Anti Cancer
Drug Design 6:539 (1991); Zon, et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec, et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphorothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[0912] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the disclosure, including, but not limited
to, any of the ASOs set forth herein in Table 5, comprises an ASO
having phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[0913] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention, including, but not limited to, any of the ASOs set
forth herein in Table 5, comprises about 5-100% Rp, at least about
5% Rp, at least about 10% Rp, at least about 15% Rp, at least about
20% Rp, at least about 25% Rp, at least about 30% Rp, at least
about 35% Rp, at least about 40% Rp, at least about 45% Rp, at
least about 50% Rp, at least about 55% Rp, at least about 60% Rp,
at least about 65% Rp, at least about 70% Rp, at least about 75%
Rp, at least about 80% Rp, at least about 85% Rp, at least about
90% Rp, or at least about 95% Rp, with the remainder Sp, or about
100% Rp. In embodiments, an ASO used in the methods of the
invention, including, but not limited to, any of the ASOs set forth
herein in Table 5, comprises about 10% to about 100% Rp, about 15%
to about 100% Rp, about 20% to about 100% Rp, about 25% to about
100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp,
about 40% to about 100% Rp, about 45% to about 100% Rp, about 50%
to about 100% Rp, about 55% to about 100% Rp, about 60% to about
100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp,
about 75% to about 100% Rp, about 80% to about 100% Rp, about 85%
to about 100% Rp, about 90% to about 100% Rp, or about 95% to about
100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp,
about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45%
to about 55% Rp, with the remainder Sp.
[0914] In embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 5, comprises about 5-100% Sp, at least about 5% Sp, at least
about 10% Sp, at least about 15% Sp, at least about 20% Sp, at
least about 25% Sp, at least about 30% Sp, at least about 35% Sp,
at least about 40% Sp, at least about 45% Sp, at least about 50%
Sp, at least about 55% Sp, at least about 60% Sp, at least about
65% Sp, at least about 70% Sp, at least about 75% Sp, at least
about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at
least about 95% Sp, with the remainder Rp, or about 100% Sp. In
embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 5, comprises about 10% to about 100% Sp, about 15% to about
100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp,
about 30% to about 100% Sp, about 35% to about 100% Sp, about 40%
to about 100% Sp, about 45% to about 100% Sp, about 50% to about
100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp,
about 65% to about 100% Sp, about 70% to about 100% Sp, about 75%
to about 100% Sp, about 80% to about 100% Sp, about 85% to about
100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp,
about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to
about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55%
Sp, with the remainder Rp.
[0915] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[0916] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[0917] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[0918] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary, et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary, et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[0919] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[0920] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[0921] In some embodiments, the ASOs are complementary to (and bind
to) a targeted portion of a PKD2 RIC pre-mRNA that is downstream
(in the 3' direction) of the 5' splice site of the retained intron
in a PKD2 RIC pre-mRNA (e.g., the direction designated by positive
numbers relative to the 5' splice site) (FIG. 1). In some
embodiments, the ASOs are complementary to a targeted portion of
the PKD2 RIC pre-mRNA that is within the region of about +6 to
about +500 relative to the 5' splice site of the retained intron.
In some embodiments, the ASO is not complementary to nucleotides +1
to +5 relative to the 5' splice site (the first five nucleotides
located downstream of the 5' splice site). In some embodiments, the
ASOs may be complementary to a targeted portion of a PKD2 RIC
pre-mRNA that is within the region between nucleotides +6 and +497
relative to the 5' splice site of the retained intron. In some
aspects, the ASOs are complementary to a targeted portion that is
within the region about +6 to about +500, about +6 to about +490,
about +6 to about +480, about +6 to about +470, about +6 to about
+460, about +6 to about +450, about +6 to about +440, about +6 to
about +430, about +6 to about +420, about +6 to about +410, about
+6 to about +400, about +6 to about +390, about +6 to about +380,
about +6 to about +370, about +6 to about +360, about +6 to about
+350, about +6 to about +340, about +6 to about +330, about +6 to
about +320, about +6 to about +310, about +6 to about +300, about
+6 to about +290, about +6 to about +280, about +6 to about +270,
about +6 to about +260, about +6 to about +250, about +6 to about
+240, about +6 to about +230, about +6 to about +220, about +6 to
about +210, about +6 to about +200, about +6 to about +190, about
+6 to about +180, about +6 to about +170, about +6 to about +160,
about +6 to about +150, about +6 to about +140, about +6 to about
+130, about +6 to about +120, about +6 to about +110, about +6 to
about +100, about +6 to about +90, about +6 to about +80, about +6
to about +70, about +6 to about +60, about +6 to about +50, about
+6 to about +40, about +6 to about +30, or about +6 to about +20
relative to 5' splice site of the retained intron.
[0922] In some embodiments, the ASOs are complementary to (and bind
to) a targeted portion of a PKD2 RIC pre-mRNA that is upstream (in
the 5' direction) of the 5' splice site of the retained intron in a
PKD2 RIC pre-mRNA (e.g., the direction designated by negative
numbers relative to the 5' splice site) (FIG. 1). In some
embodiments, the ASOs are complementary to a targeted portion of
the PKD2 RIC pre-mRNA that is within the region of about -4e to
about -210e relative to the 5' splice site of the retained intron.
In some embodiments, the ASO is not complementary to nucleotides
-1e to -3e relative to the 5' splice site (the first three
nucleotides located upstream of the 5' splice site). In some
embodiments, the ASOs may be complementary to a targeted portion of
a PKD2 RIC pre-mRNA that is within the region between nucleotides
-4e and about -204e relative to the 5' splice site of the retained
intron. In some aspects, the ASOs are complementary to a targeted
portion that is within the region about -4e to about -210e, about
-4e to about -200e, about -4e to about -190e, about -4e to about
-180e, about -4e to about -170e, about -4e to about -160e, about
-4e to about -150e, about -4e to about -140e, about -4e to about
-130e, about -4e to about -120e, about -4e to about -110e, about
-4e to about -100e, about -4e to about -90e, about -4e to about
-80e, about -4e to about -70e, about -4e to about -60e, about -4e
to about -50e, about -4e to about -40e, about -4e to about -30e, or
about -4e to about -20e relative to 5' splice site of the retained
intron.
[0923] In some embodiments, the ASOs are complementary to a
targeted region of a PKD2 RIC pre-mRNA that is upstream (in the 5'
direction) of the 3' splice site of the retained intron in a PKD2
RIC pre-mRNA (e.g., in the direction designated by negative
numbers) (FIG. 1). In some embodiments, the ASOs are complementary
to a targeted portion of the PKD2 RIC pre-mRNA that is within the
region of about -16 to about -500 relative to the 3' splice site of
the retained intron. In some embodiments, the ASO is not
complementary to nucleotides -1 to -15 relative to the 3' splice
site (the first 15 nucleotides located upstream of the 3' splice
site). In some embodiments, the ASOs are complementary to a
targeted portion of the PKD2 RIC pre-mRNA that is within the region
-16 to -496 relative to the 3' splice site of the retained intron.
In some aspects, the ASOs are complementary to a targeted portion
that is within the region about -16 to about -500, about -16 to
about -490, about -16 to about -480, about -16 to about -470, about
-16 to about -460, about -16 to about -450, about -16 to about
-440, about -16 to about -430, about -16 to about -420, about -16
to about -410, about -16 to about -400, about -16 to about -390,
about -16 to about -380, about -16 to about -370, about -16 to
about -360, about -16 to about -350, about -16 to about -340, about
-16 to about -330, about -16 to about -320, about -16 to about
-310, about -16 to about -300, about -16 to about -290, about -16
to about -280, about -16 to about -270, about -16 to about -260,
about -16 to about -250, about -16 to about -240, about -16 to
about -230, about -16 to about -220, about -16 to about -210, about
-16 to about -200, about -16 to about -190, about -16 to about
-180, about -16 to about -170, about -16 to about -160, about -16
to about -150, about -16 to about -140, about -16 to about -130,
about -16 to about -120, about -16 to about -110, about -16 to
about -100, about -16 to about -90, about -16 to about -80, about
-16 to about -70, about -16 to about -60, about -16 to about -50,
about -16 to about -40, or about -16 to about -30 relative to 3'
splice site of the retained intron.
[0924] In some embodiments, the ASOs are complementary to a
targeted region of a PKD2 RIC pre-mRNA that is downstream (in the
3' direction) of the 3' splice site of the retained intron in a
PKD2 RIC pre-mRNA (e.g., in the direction designated by positive
numbers) (FIG. 1). In some embodiments, the ASOs are complementary
to a targeted portion of the PKD2 RIC pre-mRNA that is within the
region of about +2e to about +220e relative to the 3' splice site
of the retained intron. In some embodiments, the ASO is not
complementary to nucleotides +1e relative to the 3' splice site
(the first nucleotide located downstream of the 3' splice site). In
some embodiments, the ASOs may be complementary to a targeted
portion of a PKD2 RIC pre-mRNA that is within the region between
nucleotides +2e and +212e relative to the 3' splice site of the
retained intron. In some aspects, the ASOs are complementary to a
targeted portion that is within the region about +2e to about
+220e, about +2e to about +210e, about +2e to about +200e, about
+2e to about +190e, about +2e to about +180e, about +2e to about
+170e, about +2e to about +160e, about +2e to about +150e, about
+2e to about +140e, about +2e to about +130e, about +2e to about
+120e, about +2e to about +110e, about +2e to about +100e, about
+2e to about +90e, about +2e to about +80e, about +2e to about
+70e, about +2e to about +60e, about +2e to about +50e, about +2e
to about +40e, about +2e to about +30e, about +2e to about +20e, or
about +2e to about +10e relative to 3' splice site of the retained
intron.
[0925] In embodiments, the targeted portion of the PKD2 RIC
pre-mRNA is within the region +100 relative to the 5' splice site
of the retained intron to -100 relative to the 3' splice site of
the retained intron.
[0926] The ASOs may be of any length suitable for specific binding
and effective enhancement of splicing. In some embodiments, the
ASOs consist of 8 to 50 nucleobases. For example, the ASO may be 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases
in length. In some embodiments, the ASOs consist of more than 50
nucleobases. In some embodiments, the ASO is from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40
nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25
nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40
nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25
nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40
nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25
nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40
nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25
nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35
nucleobases, or 25 to 30 nucleobases in length. In some
embodiments, the ASOs are 18 nucleotides in length. In some
embodiments, the ASOs are 15 nucleotides in length. In some
embodiments, the ASOs are 25 nucleotides in length.
[0927] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[0928] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[0929] In some embodiments, the nucleic acid to be targeted by an
ASO is a PKD2 RIC pre-mRNA expressed in a cell, such as a
eukaryotic cell. In some embodiments, the term "cell" may refer to
a population of cells. In some embodiments, the cell is in a
subject. In some embodiments, the cell is isolated from a subject.
In some embodiments, the cell is ex vivo. In some embodiments, the
cell is a condition or disease-relevant cell or a cell line. In
some embodiments, the cell is in vitro (e.g., in cell culture).
Pharmaceutical Compositions
[0930] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent,
or carrier.
[0931] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose). The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[0932] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[0933] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancer is a surfactant, fatty acid,
bile salt, chelating agent, or non-chelating nonsurfactant.
[0934] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[0935] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[0936] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[0937] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[0938] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[0939] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. Multiple tissues and organs are affected by
Polycystic Kidney Disease, with the kidney being the most
significantly affected tissue. The ASOs of the present invention
may be administered to patients parenterally, for example, by
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection.
[0940] Subjects are evaluated for response to treatment using any
appropriate markers. In embodiments, subjects with kidney disease
are evaluated for response to treatment by measuring specific
markers for kidney disease, including creatinine, creatinine
clearance, blood pressure, 24-hour urine volume, 24-hour urine
protein, vWAg and platelet aggregation by arachidonic acid.
Methods of Identifying Additional ASOs that Enhance Splicing
[0941] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of a PKD2 RIC pre-mRNA, specifically at the target intron. ASOs
that specifically hybridize to different nucleotides within the
target region of the pre-mRNA may be screened to identify
(determine) ASOs that improve the rate and/or extent of splicing of
the target intron. In some embodiments, the ASO may block or
interfere with the binding site(s) of a splicing
repressor(s)/silencer. Any method known in the art may be used to
identify (determine) an ASO that when hybridized to the target
region of the intron results in the desired effect (e.g., enhanced
splicing, protein or functional RNA production). These methods also
can be used for identifying ASOs that enhance splicing of the
retained intron by binding to a targeted region in an exon flanking
the retained intron, or in a non-retained intron. An example of a
method that may be used is provided below.
[0942] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site. In some embodiments,
the ASOs can be tiled from about 210 nucleotides upstream of the 5'
splice site, to about 500 nucleotides downstream of the 5' splice
site. In some embodiments, the ASOs can be tiled from about 500
nucleotides upstream of the 3' splice site, to about 220
nucleotides downstream of the 3' splice site.
[0943] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[0944] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[0945] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[0946] As described for the ASO walk above, the ASO micro-walk is
performed by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[0947] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. Following administration, the cells, tissues, and/or
organs of the model animals may be assessed to determine the effect
of the ASO treatment by for example evaluating splicing
(efficiency, rate, extent) and protein production by methods known
in the art and described herein. The animal models may also be any
phenotypic or behavioral indication of the disease or disease
severity.
Compositions and Methods for Treatment of Retinitis Pigmentosa 18
and Retinitis Pigmentosa 13
[0948] Individual introns in primary transcripts of protein-coding
genes having more than one intron are spliced from the primary
transcript with different efficiencies. In most cases only the
fully spliced mRNA is exported through nuclear pores for subsequent
translation in the cytoplasm. Unspliced and partially spliced
transcripts are detectable in the nucleus. It is generally thought
that nuclear accumulation of transcripts that are not fully spliced
is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[0949] Substantial levels of partially-spliced transcripts of the
PRPF3 and PRPF8 genes, which encode the PRPF3 protein, deficient in
RP18, and the PRPF8 protein, deficient in RP13, respectively, have
been discovered in the nucleus of human cells. These PRPF3 and
PRPF8 pre-mRNA species comprise at least one retained intron. The
present invention provides compositions and methods for
upregulating splicing of one or more retained PRPF3 or PRPF8
introns that are rate-limiting for the nuclear stages of gene
expression to increase steady-state production of fully-spliced,
mature mRNA, and thus, translated PRPF3 or PRPF8 protein levels,
respectively. These compositions and methods utilize antisense
oligomers (ASOs) that promote constitutive splicing at an intron
splice site of a retained-intron-containing PRPF3 or PRPF8 pre-mRNA
that accumulates in the nucleus. Thus, in embodiments, PRPF3 or
PRPF8 protein is increased using the methods of the invention to
treat a condition caused by PRPF3 or PRPF8 protein deficiency. In
other embodiments, the methods of the invention are used to
increase PRPF3 or PRPF8 production to treat a condition in a
subject in need thereof. In embodiments, the subject has condition
in which PRPF3 or PRPF8 is not necessarily deficient relative to
wild-type, but where an increase in PRPF3 or PRPF8 mitigates the
condition nonetheless. In embodiments, the condition is a caused by
a PRPF3 or PRPF8 haploinsufficiency.
Retinitis Pigmentosa
[0950] Retinitis pigmentosa (RP) describes a debilitating group of
eye disorders, including RP18, caused by a deficiency in the PRPF3
protein, and RP13, caused by a deficiency in the PRPF8 protein.
Subjects having RP experience loss of night vision in the early
phase of the disorder. Over time, loss of vision begins to occur,
which eventually leads to tunnel vision. This loss in vision can be
attributed to a dysfunction in the rod photoreceptor system. As the
disorder progresses, the afflicted patient may lose a significant
portion of their cone photoreceptors before experiencing loss of
visual acuity (Berger et al., 2010).
[0951] The overall prevalence of RP diseases is estimated to be
about 1:3500. The mutation of over 40 genes has been correlated to
incidence of RP disease through three patterns of inheritance:
autosomal dominant, autosomal recessive, and X-linked. The genes
that have been implicated in the progression of RP diseases can be
grouped into five main categories: i) phototransduction, ii)
retinal metabolism, iii) tissue development and maintenance, iv)
cellular structure, and v) splicing. The genes that comprise the
splicing category (PRPF3, PRPF8, PRP31 and PAP1) are responsible
for the assembly of the spliceosome protein complex that is
responsible for removing intronic sequences from pre-mRNA. Studies
have suggested that the progression of at least some cases of RP is
due to haploinsuffiency, meaning that the presence of a single
dysfunctional allele results in diminished expression of the
corresponding protein (Berger et al., 2010).
PRPF3
[0952] The PRPF3 gene, which spans 16 exons and is located at
1q21.1, encodes the pre-mRNA processing factor 3 (PRPF3) protein.
The PRPF3 canonical mRNA sequence is set forth at NCBI Reference
Sequence: NM_004698. PRPF3 is a 77 kD, 682 amino acid protein that
is involved in spliceosome assembly. Dysfunction of PRPF3 has been
implicated in the progression of RP 18. It has been speculated that
a retina-specific splicing element may interact with PRPF3 and
generate the rod photoreceptor-specific phenotype.
[0953] Missense mutations P493S and T494M in PRPF3 have both been
shown to display the RP18 phenotype, linking dysfunction of PRPF3
with the RP 18 phenotype (Berger et al., 2010). RP18 and the PRPF3
gene are described by, e.g., OMIM #601414 (Online Mendelian
Inheritance in Man, Johns Hopkins University, 1966-2015),
incorporated by reference herein.
PRPF8
[0954] The PRPF8 gene, which spans 42 exons and is located at
17p13.3, codes for the pre-mRNA processing factor 8 (PRPF8)
protein. The PRPF8 canonical mRNA sequence is set forth at NCBI
Reference Sequence: NM_006445. PRPF8 is a 220 kD, 2,334 amino acid
protein that is involved in spliceosome assembly. Dysfunction of
PRPF8 has been implicated in the progression of RP13. PRPF8 mutants
H2309P, H2309R, R2310K, P2301T, F2304L, R2310G and F2314L were
found to result in the clinical phenotype of RP13, linking
dysfunction of PRPF8 with RP13 (Berger et al., 2010). RP13 and the
RP8 gene are described by, e.g., OMIM #600059 (Online Mendelian
Inheritance in Man, Johns Hopkins University, 1966-2015),
incorporated by reference herein.
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[0955] In embodiments, the methods of the present invention exploit
the presence of retained-intron-containing pre-mRNA (RIC pre-mRNA)
transcribed from the PRPF3 or the PRPF8 gene in the cell nucleus.
Splicing of the identified RIC pre-mRNA species to produce mature,
fully-spliced, mRNA, is induced using ASOs that stimulate splicing
out of the retained introns. The resulting mature mRNA can be
exported to the cytoplasm and translated, thereby increasing the
amount of PRPF3 protein or PRPF8 protein in the patient's cells and
alleviating symptoms of RP18 or RP13, respectively. This method,
described further below, is known as Targeted Augmentation of
Nuclear Gene Output (TANGO).
PRPF3 Nuclear Transcripts
[0956] As described herein in the Examples, the PRPF3 gene was
analyzed for intron-retention events and retention of introns,
e.g., introns 12 and 13, was observed. RNA sequencing (RNAseq),
visualized in the UCSC genome browser, showed PRPF3 transcripts
expressed in ARPE-19 cells and localized in either the cytoplasmic
or nuclear fraction. In both fractions, reads were not observed for
the majority of the introns. However, higher read density was
detected for introns 12 and 13 in the nuclear fraction compared to
the cytoplasmic fraction indicating that splicing efficiency of
introns 12 and 13 is low, resulting in intron retention. The
retained-intron containing pre-mRNA transcripts accumulate
primarily in the nucleus and are not translated into the PRPF3
protein. The read density for introns 12 and 13, indicated 26%, and
33% intron retention, respectively. The percent intron retention
(PIR) value for intron 12 was obtained by averaging four values
(37, 35, 16, and 15). The PIR value for intron 13 was obtained
similarly, by averaging the four values 34, 33, 34, and 31. Each
value was determined in ARPE-19 (retinal pigmented epithelial)
cells using one of four different algorithms.
[0957] In some embodiments, the PRPF3 intron number corresponds to
the mRNA sequence at NM_004698. In some embodiments, the targeted
portion of the PRPF3 RIC pre-mRNA is in intron 12 and/or 13. In
embodiments, hybridization of an ASO to the targeted portion of a
PRPF3 RIC pre-mRNA results in enhanced splicing at the splice site
(5' splice site or 3' splice site) of at least one of retained
PRPF3 introns 12 or 13 and subsequently increases PRPF3 protein
production. It is understood that the intron numbering may change
in reference to a different PRPF3 isoform sequence. One of skill in
the art can determine the corresponding intron number in any PRPF3
isoform based on an intron sequence provided herein or using the
intron number provided in reference to the mRNA sequence at
NM_004698. One of skill in the art also can determine the sequences
of flanking exons in any PRPF3 isoform for targeting using the
methods of the invention, based on an intron sequence provided
herein or using the intron number provided in reference to the mRNA
sequence at NM_004698. In embodiments, the compositions and methods
of the present invention are used to increase the expression of any
known PRPF3 isoform, e.g., as described in the NCBI Gene ID
database at Gene ID 9129 (NCBI repository of biological and
scientific information, operated by National Center for
Biotechnology Information, National Library of Medicine, 8600
Rockville Pike, Bethesda, Md. USA 20894), incorporated by reference
herein.
[0958] In some embodiments, the PRPF8 intron numbering corresponds
to the mRNA sequence at NM_006445. In embodiments, the targeted
portion of the PRPF8 RIC pre-mRNA is in intron 31. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of at least one of retained PRPF8 intron 31 and
subsequently increases PRPF8 protein production. It is understood
that the intron numbering may change in reference to a different
PRPF8 isoform sequence. One of skill in the art can determine the
corresponding intron number in any PRPF8 isoform based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_006445. One of skill in the
art also can determine the sequences of flanking exons in any PRPF8
isoform for targeting using the methods of the invention, based on
an intron sequence provided herein or using the intron number
provided in reference to the mRNA sequence at NM_006445. In
embodiments, the compositions and methods of the present invention
are used to increase the expression of any known PRPF8 isoform,
e.g., as described in the NCBI Gene ID database at Gene ID 10594
(NCBI repository of biological and scientific information),
incorporated by reference herein.
PRPF3
[0959] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PRPF3 genomic sequence (a PRPF3 RIC
pre-mRNA). In some embodiments, the PRPF3 genomic sequence is SEQ
ID NO: 5826. In some embodiments, the PRPF3 RIC pre-mRNA is SEQ ID
NO: 5828.
PRPF3: Retained Intron 12
[0960] In some embodiments, the PRPF3 RIC pre-mRNA transcript
comprises retained intron 12. In some embodiments, when the PRPF3
RIC pre-mRNA transcript comprises retained intron 12, the ASOs
disclosed herein target SEQ ID NO: 6272. In some embodiments, when
the PRPF3 RIC pre-mRNA transcript comprises retained intron 12, the
ASO has a sequence according to any one of SEQ ID NOs: 5830-6064.
In some embodiments, the ASOs target a PRPF3 RIC pre-mRNA
sequence.
[0961] In some embodiments, the ASO targets exon 12 of PRPF3 RIC
pre-mRNA comprising a retained intron 12. In some embodiments, the
ASO targets an exon 12 sequence upstream (or 5') from the 5' splice
site of a PRPF3 RIC pre-mRNA comprising a retained intron 12. In
some embodiments, the ASO targets an exon sequence about 4 to about
94 nucleotides upstream (or 5') from the 5' splice site of a PRPF3
RIC pre-mRNA comprising a retained intron 12. In some embodiments,
the ASO has a sequence according to any one of SEQ ID NOs:
5830-5848.
[0962] In some embodiments, the ASO targets intron 12 in a PRPF3
RIC pre-mRNA comprising a retained intron 12. In some embodiments,
the ASO targets an intron 12 sequence downstream (or 3') from the
5' splice site of a PRPF3 RIC pre-mRNA comprising a retained intron
12. In some embodiments, the ASO targets an intron 12 sequence
about 6 to about 498 nucleotides downstream (or 3') from the 5'
splice site of a PRPF3 RIC pre-mRNA comprising a retained intron
12. In some embodiments, the ASO has a sequence according to any
one of SEQ ID NOs: 5849-5946.
[0963] In some embodiments, the ASO targets an intron 12 sequence
upstream (or 5') from the 3' splice site of a PRPF3 RIC pre-mRNA
comprising a retained intron 12. In some embodiments, the ASO
targets an intron 12 sequence about 16 to about 496 nucleotides
upstream (or 5') from the 3' splice site of a PRPF3 RIC pre-mRNA a
comprising retained intron 12. In some embodiments, the ASO has a
sequence according to any one of SEQ ID NOs: 5947-6043.
[0964] In some embodiments, the ASO targets exon 13 in a PRPF3 RIC
pre-mRNA comprising a retained intron 12. In some embodiments, the
ASO targets an exon 13 sequence downstream (or 3') from the 3'
splice site of a PRPF3 RIC pre-mRNA comprising a retained intron
12. In some embodiments, the ASO targets an exon 13 sequence about
2 to about 102 nucleotides downstream (or 3') from the 3' splice
site of a PRPF3 RIC pre-mRNA comprising a retained intron 12. In
some embodiments, the ASO has a sequence according to any one of
SEQ ID NOs: 6044-6064.
PRPF3: Retained Intron 13
[0965] In some embodiments, the PRPF3 RIC pre-mRNA transcript
comprises retained intron 13. In some embodiments, when the PRPF3
RIC pre-mRNA transcript comprises retained intron 13, the ASOs
disclosed herein target SEQ ID NO: 6271. In some embodiments, when
the PRPF3 RIC pre-mRNA transcript comprises retained intron 13, the
ASO has a sequence according to any one of SEQ ID NOs: 6065-6148.
In some embodiments, the ASOs target a PRPF3 RIC pre-mRNA
sequence.
[0966] In some embodiments, the ASO targets exon 13 of PRPF3 RIC
pre-mRNA comprising a retained intron 13. In some embodiments, the
ASO targets an exon 13 sequence upstream (or 5') from the 5' splice
site of a PRPF3 RIC pre-mRNA comprising a retained intron 13. In
some embodiments, the ASO targets an exon sequence about 4 to about
99 nucleotides upstream (or 5') from the 5' splice site of a PRPF3
RIC pre-mRNA comprising a retained intron 13. In some embodiments,
the ASO has a sequence according to any one of SEQ ID NOs:
6065-6084.
[0967] In some embodiments, the ASO targets intron 13 in a PRPF3
RIC pre-mRNA comprising a retained intron 13. In some embodiments,
the ASO targets an intron 13 sequence downstream (or 3') from the
5' splice site of a PRPF3 RIC pre-mRNA comprising a retained intron
13. In some embodiments, the ASO targets an intron 13 sequence
about 6 to about 146 nucleotides downstream (or 3') from the 5'
splice site of a PRPF3 RIC pre-mRNA comprising a retained intron
13. In some embodiments, the ASO has a sequence according to any
one of SEQ ID NOs: 6085-6111.
[0968] In some embodiments, the ASO targets an intron 13 sequence
upstream (or 5') from the 3' splice site of a PRPF3 RIC pre-mRNA
comprising a retained intron 13. In some embodiments, the ASO
targets an intron 13 sequence about 16 to about 146 nucleotides
upstream (or 5') from the 3' splice site of a PRPF3 RIC pre-mRNA a
comprising retained intron 13. In some embodiments, the ASO has a
sequence according to any one of SEQ ID NOs: 6112-6135.
[0969] In some embodiments, the ASO targets exon 14 in a PRPF3 RIC
pre-mRNA comprising a retained intron 13. In some embodiments, the
ASO targets an exon 14 sequence downstream (or 3') from the 3'
splice site of a PRPF3 RIC pre-mRNA comprising a retained intron
13. In some embodiments, the ASO targets an exon 14 sequence about
2 to about 67 nucleotides downstream (or 3') from the 3' splice
site of a PRPF3 RIC pre-mRNA comprising a retained intron 13. In
some embodiments, the ASO has a sequence according to any one of
SEQ ID NOs: 6136-6148.
PRPF8
[0970] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PRPF8 genomic sequence (a PRPF8 RIC
pre-mRNA). In some embodiments, the PRPF8 genomic sequence is SEQ
ID NO. 5827. In some embodiments, the PRPF8 RIC pre-mRNA is SEQ ID
NO. 5829.
PRPF8: Retained Intron 31
[0971] In some embodiments, the PRPF8 RIC pre-mRNA transcript
comprises retained intron 31. In some embodiments, when the PRPF8
RIC pre-mRNA transcript comprises retained intron 31, the ASOs
disclosed herein target SEQ ID NO: 6273. In some embodiments, when
the PRPF8 RIC pre-mRNA transcript comprises retained intron 31, the
ASO has a sequence according to any one of SEQ ID NOs: 6149-6270.
In some embodiments, the ASOs target a PRPF8 RIC pre-mRNA
sequence.
[0972] In some embodiments, the ASO targets exon 31 of PRPF8 RIC
pre-mRNA comprising a retained intron 31. In some embodiments, the
ASO targets an exon 31 sequence upstream (or 5') from the 5' splice
site of a PRPF8 RIC pre-mRNA comprising a retained intron 31. In
some embodiments, the ASO targets an exon sequence about 4 to about
144 nucleotides upstream (or 5') from the 5' splice site of a PRPF8
RIC pre-mRNA comprising a retained intron 31. In some embodiments,
the ASO has a sequence according to any one of SEQ ID NOs:
6149-6175.
[0973] In some embodiments, the ASO targets intron 31 in a PRPF8
RIC pre-mRNA comprising a retained intron 31. In some embodiments,
the ASO targets an intron 31 sequence downstream (or 3') from the
5' splice site of a PRPF8 RIC pre-mRNA comprising a retained intron
31. In some embodiments, the ASO targets an intron 31 sequence
about 6 to about 156 nucleotides downstream (or 3') from the 5'
splice site of a PRPF8 RIC pre-mRNA comprising a retained intron
31. In some embodiments, the ASO has a sequence according to any
one of SEQ ID NOs: 6176-6206.
[0974] In some embodiments, the ASO targets an intron 31 sequence
upstream (or 5') from the 3' splice site of a PRPF8 RIC pre-mRNA
comprising a retained intron 31. In some embodiments, the ASO
targets an intron 31 sequence about 16 to about 156 nucleotides
upstream (or 5') from the 3' splice site of a PRPF8 RIC pre-mRNA a
comprising retained intron 31. In some embodiments, the ASO has a
sequence according to any one of SEQ ID NOs: 6207-6235.
[0975] In some embodiments, the ASO targets exon 32 in a PRPF8 RIC
pre-mRNA comprising a retained intron 31. In some embodiments, the
ASO targets an exon 32 sequence downstream (or 3') from the 3'
splice site of a PRPF8 RIC pre-mRNA comprising a retained intron
31. In some embodiments, the ASO targets an exon 32 sequence about
2 to about 172 nucleotides downstream (or 3') from the 3' splice
site of a PRPF8 RIC pre-mRNA comprising a retained intron 31. In
some embodiments, the ASO has a sequence according to any one of
SEQ ID NOs: 6236-6270.
Protein Expression
[0976] As described above, PRPF3 and PRPF8 mutations in RP diseases
are spread across the entire protein. In some embodiments, the
methods described herein are used to increase the production of a
functional PRPF3 protein. In other embodiments, the methods
described herein are used to increase the production of a
functional PRPF8 protein. In other embodiments, the methods
described herein are used to increase the production of a
functional PRPF3 protein or PRPF8 protein. As used herein, the term
"functional" refers to the amount of activity or function of a
PRPF3 or PRPF8 protein that is necessary to eliminate any one or
more symptoms of an RP disease. In some embodiments, the methods
are used to increase the production of a partially functional PRPF3
protein. In other embodiments, the methods are used to increase the
production of a partially functional PRPF8 protein. In other
embodiments, the methods are used to increase the production of a
partially functional PRPF3 or PRPF8 protein. As used herein, the
term "partially functional" refers to any amount of activity or
function of the PRPF3 or PRPF8 protein that is less than the amount
of activity or function that is necessary to eliminate or prevent
any one or more symptoms of a disease. In some embodiments, a
partially functional protein or RNA will have at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 75%, at least 80%, 85%, at least 90%, or at
least 95% less activity relative to the fully functional protein or
RNA.
[0977] In embodiments, the method is a method of increasing the
expression of the PRPF3 protein by cells of a subject having a RIC
pre-mRNA encoding the PRPF3 protein, wherein the subject has an RP
disease caused by a deficient amount of activity of PRPF3 protein,
and wherein the deficient amount of the PRPF3 protein is caused by
haploinsufficiency of the PRPF3 protein. In such an embodiment, the
subject has a first allele encoding a functional PRPF3 protein, and
a second allele from which the PRPF3 protein is not produced. In
another such embodiment, the subject has a first allele encoding a
functional PRPF3 protein, and a second allele encoding a
nonfunctional PRPF3 protein. In another such embodiment, the
subject has a first allele encoding a functional PRPF3 protein, and
a second allele encoding a partially functional PRPF3 protein. In
any of these embodiments, the antisense oligomer binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele (encoding functional PRPF3 protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding
functional PRPF3 protein, and an increase in the expression of the
PRPF3 protein in the cells of the subject.
[0978] In embodiments, the method is a method of increasing the
expression of the PRPF8 protein by cells of a subject having a RIC
pre-mRNA encoding the PRPF8 protein, wherein the subject has an RP
disease caused by a deficient amount of activity of PRPF8 protein,
and wherein the deficient amount of the PRPF8 protein is caused by
haploinsufficiency of the PRPF8 protein. In such an embodiment, the
subject has a first allele encoding a functional PRPF8 protein, and
a second allele from which the PRPF8 protein is not produced. In
another such embodiment, the subject has a first allele encoding a
functional PRPF8 protein, and a second allele encoding a
nonfunctional PRPF8 protein. In another such embodiment, the
subject has a first allele encoding a functional PRPF8 protein, and
a second allele encoding a partially functional PRPF8 protein. In
any of these embodiments, the antisense oligomer binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele (encoding functional PRPF8 protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding
functional PRPF8 protein, and an increase in the expression of the
PRPF8 protein in the cells of the subject.
[0979] In some embodiments, the subject has a first allele encoding
a functional PRPF3 protein, and a second allele encoding a
partially functional PRPF3 protein, and the antisense oligomer
binds to a targeted portion of the RIC pre-mRNA transcribed from
the first allele (encoding functional PRPF3 protein) or a targeted
portion of the RIC pre-mRNA transcribed from the second allele
(encoding partially functional PRPF3 protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding the
PRPF3 protein, and an increase in the expression of functional or
partially functional PRPF3 protein in the cells of the subject.
[0980] In other embodiments, the subject has a first allele
encoding a functional PRPF8 protein, and a second allele encoding a
partially functional PRPF8 protein, and the antisense oligomer
binds to a targeted portion of the RIC pre-mRNA transcribed from
the first allele (encoding functional PRPF8 protein) or a targeted
portion of the RIC pre-mRNA transcribed from the second allele
(encoding partially functional PRPF8 protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding the
PRPF8 protein, and an increase in the expression of functional or
partially functional PRPF8 protein in the cells of the subject.
[0981] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of PRPF3
protein in cells of a subject having a RIC pre-mRNA encoding PRPF3
protein, wherein the subject has a deficiency, e.g., RP18, in the
amount or function of a PRPF3 protein.
[0982] In other related embodiments, the method is a method of
using an ASO to increase the expression of a protein or functional
RNA. In embodiments, an ASO is used to increase the expression of
PRPF8 protein in cells of a subject having a RIC pre-mRNA encoding
PRPF8 protein, wherein the subject has a deficiency, e.g., RP13, in
the amount or function of a PRPF8 protein.
[0983] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease is targeted by the ASOs
described herein. In some embodiments, a RIC pre-mRNA transcript
that encodes a protein that is not causative of the disease is
targeted by the ASOs. For example, a disease that is the result of
a mutation or deficiency of a first protein in a particular pathway
may be ameliorated by targeting a RIC pre-mRNA that encodes a
second protein, thereby increasing production of the second
protein. In some embodiments, the function of the second protein is
able to compensate for the mutation or deficiency of the first
protein (which is causative of the disease or condition).
[0984] In embodiments, the subject has:
[0985] a. a first mutant allele from which [0986] i) the PRPF3
protein is produced at a reduced level compared to production from
a wild-type allele, [0987] ii) the PRPF3 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0988] iii) the PRPF3 protein or functional RNA is not
produced; and
[0989] b. a second mutant allele from which [0990] i) the PRPF3
protein is produced at a reduced level compared to production from
a wild-type allele, [0991] ii) the PRPF3 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0992] iii) the PRPF3 protein is not produced, and
wherein the RIC pre-mRNA is transcribed from the first allele
and/or the second allele. In these embodiments, the ASO binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele or the second allele, thereby inducing constitutive splicing
of the retained intron from the RIC pre-mRNA, and causing an
increase in the level of mRNA encoding PRPF3 protein and an
increase in the expression of the target protein or functional RNA
in the cells of the subject. In these embodiments, the target
protein or functional RNA having an increase in expression level
resulting from the constitutive splicing of the retained intron
from the RIC pre-mRNA is either in a form having reduced function
compared to the equivalent wild-type protein
(partially-functional), or having full function compared to the
equivalent wild-type protein (fully-functional).
[0993] In embodiments, the subject has:
[0994] a. a first mutant allele from which [0995] i. the PRPF8
protein is produced at a reduced level compared to production from
a wild-type allele, [0996] ii. the PRPF8 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [0997] iii. the PRPF8 protein or functional RNA is not
produced; and
[0998] b. a second mutant allele from which [0999] i. the PRPF8
protein is produced at a reduced level compared to production from
a wild-type allele, [1000] ii. the PRPF8 protein is produced in a
form having reduced function compared to an equivalent wild-type
protein, or [1001] iii. the PRPF8 protein is not produced, and
wherein the RIC pre-mRNA is transcribed from the first allele
and/or the second allele. In these embodiments, the ASO binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele or the second allele, thereby inducing constitutive splicing
of the retained intron from the RIC pre-mRNA, and causing an
increase in the level of mRNA encoding PRPF8 protein and an
increase in the expression of the target protein or functional RNA
in the cells of the subject. In these embodiments, the target
protein or functional RNA having an increase in expression level
resulting from the constitutive splicing of the retained intron
from the RIC pre-mRNA is either in a form having reduced function
compared to the equivalent wild-type protein
(partially-functional), or having full function compared to the
equivalent wild-type protein (fully-functional).
[1002] In embodiments, the level of mRNA encoding PRPF3 or PRPF8
protein is increased 1.1 to 10-fold, when compared to the amount of
mRNA encoding PRPF3 or PRPF8 that is produced in a control cell,
e.g., one that is not treated with the antisense oligomer or one
that is treated with an antisense oligomer that does not bind to
the targeted portion of the PRPF3 or PRPF8 RIC pre-mRNA.
[1003] In embodiments, the condition caused by a deficient amount
or activity of PRPF3 or PRPF8 protein is not a condition caused by
alternative or aberrant splicing of the retained intron to which
the ASO is targeted. In embodiments, the condition caused by a
deficient amount or activity of the PRPF3 or PRPF8 protein is not a
condition caused by alternative or aberrant splicing of any
retained intron in a RIC pre-mRNA encoding the PRPF3 or PRPF8
protein.
[1004] In embodiments, a subject treated using the methods of the
invention expresses a partially functional PRPF3 or PRPF8 protein
from one allele, wherein the partially functional PRPF3 or PRPF8
protein is caused by a frameshift mutation, a nonsense mutation, a
missense mutation, or a partial gene deletion. In embodiments, a
subject treated using the methods of the invention expresses a
nonfunctional PRPF3 or PRPF8 protein from one allele, wherein the
nonfunctional PRPF3 or PRPF8 protein is caused by a frameshift
mutation, a nonsense mutation, a missense mutation, a partial gene
deletion, in one allele. In embodiments, a subject treated using
the methods of the invention has a PRPF3 or PRPF8 whole gene
deletion, in one allele.
[1005] In some embodiments, the subject has a PRPF3 missense
mutation selected from P493S and T494M. In other embodiments, the
subject has a PRPF8 missense mutation selected from H2309P, H2309R,
R2310K, P2301T, F2304L, R2310G and F2314L. In embodiments, a
subject having any mutation known in the art and described in the
literature, e.g., by McKie et al 2001 referenced above, is treated
using the methods and compositions of the present invention.
Use of TANGO for Increasing Protein Expression
[1006] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) is used in the methods of the invention
to increase expression of a PRPF3 or PRPF8 protein. In these
embodiments, a retained-intron-containing pre-mRNA (RIC pre-mRNA)
encoding PRPF3 or PRPF8 protein is present in the nucleus of a
cell. Cells having a PRPF3 or PRPF8 RIC pre-mRNA that comprises a
retained intron, an exon flanking the 5' splice site, and an exon
flanking the 3' splice site, encoding the PRPF3 or PRPF8 protein,
are contacted with antisense oligomers (ASOs) that are
complementary to a targeted portion of the RIC pre-mRNA.
Hybridization of the ASOs to the targeted portion of the RIC
pre-mRNA results in enhanced splicing at the splice site (5' splice
site or 3' splice site) of the retained intron and subsequently
increases target protein production.
[1007] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[1008] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a RIC pre-mRNA encoding the PRPF3 and/or PRPF8 protein.
In embodiments, the most retained intron in a RIC pre-mRNA encoding
the PRPF3 protein is intron 12. In embodiments, the most retained
intron in a RIC pre-mRNA encoding the PRPF3 protein is intron 13.
In some embodiments, the most retained intron in a RIC pre-mRNA
encoding the PRPF8 protein is intron 31.
[1009] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 65%, about 5% to about 60%, about 5%
to about 55%, about 5% to about 50%, about 5% to about 45%, about
5% to about 40%, about 5% to about 35%, about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 10% to about 100%, about 10% to about 95%, about 10% to
about 90%, about 10% to about 85%, about 10% to about 80%, about
10% to about 75%, about 10% to about 70%, about 10% to about 65%,
about 10% to about 60%, about 10% to about 65%, about 10% to about
60%, about 10% to about 55%, about 10% to about 50%, about 10% to
about 45%, about 10% to about 40%, about 10% to about 35%, about
10% to about 30%, about 10% to about 25%, about 10% to about 20%,
about 15% to about 100%, about 15% to about 95%, about 15% to about
90%, about 15% to about 85%, about 15% to about 80%, about 15% to
about 75%, about 15% to about 70%, about 15% to about 65%, about
15% to about 60%, about 15% to about 65%, about 15% to about 60%,
about 15% to about 55%, about 15% to about 50%, about 15% to about
45%, about 15% to about 40%, about 15% to about 35%, about 15% to
about 30%, about 15% to about 25%, about 20% to about 100%, about
20% to about 95%, about 20% to about 90%, about 20% to about 85%,
about 20% to about 80%, about 20% to about 75%, about 20% to about
70%, about 20% to about 65%, about 20% to about 60%, about 20% to
about 65%, about 20% to about 60%, about 20% to about 55%, about
20% to about 50%, about 20% to about 45%, about 20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about
100%, about 25% to about 95%, about 25% to about 90%, about 25% to
about 85%, about 25% to about 80%, about 25% to about 75%, about
25% to about 70%, about 25% to about 65%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 60%, about 25% to about
55%, about 25% to about 50%, about 25% to about 45%, about 25% to
about 40%, or about 25% to about 35%, retention. ENCODE data
(described by, e.g., Tilgner, et al., 2012, "Deep sequencing of
subcellular RNA fractions shows splicing to be predominantly
co-transcriptional in the human genome but inefficient for
lncRNAs," Genome Research 22(9):1616-25) can be used to aid in
identifying retained introns.
[1010] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g. in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features (e.g.
in the case of an antisense oligomer, the presence of additional,
unrecited nucleobases).
[1011] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g. nucleobase sequence) as well as those which do not materially
affect the character or function of the claimed invention. As used
herein, the term "consisting" is used to indicate the presence of
the recited feature (e.g. nucleobase sequence) alone (so that in
the case of an antisense oligomer consisting of a specified
nucleobase sequence, the presence of additional, unrecited
nucleobases is excluded).
[1012] In embodiments, the targeted region is in a retained intron
that is the second most abundant intron in a population of RIC
pre-mRNA encoding the PRPF3 or PRPF8 protein. For example, the
second most abundant retained intron may be targeted rather than
the most abundant retained intron due to the uniqueness of the
nucleotide sequence of the second most abundant retained intron,
ease of ASO design to target a particular nucleotide sequence,
and/or amount of increase in protein production resulting from
targeting the intron with an ASO. In embodiments, the retained
intron is the second most abundant intron in a population of RIC
pre-mRNAs transcribed from the gene encoding the target protein in
a cell, wherein the population of RIC pre-mRNAs comprises two or
more retained introns. In embodiments, an antisense oligomer
targeted to the second most abundant intron in the population of
RIC pre-mRNAs encoding the target protein induces splicing out of
two or more retained introns in the population, including the
retained intron to which the antisense oligomer is targeted or
binds. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced. In embodiments, the second-most
abundant retained intron in a population of RIC pre-mRNA encoding
the PRPF3 protein is intron 12. In embodiments, the second-most
abundant retained intron in a population of RIC pre-mRNA encoding
the PRPF3 protein is intron 13.
[1013] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[1014] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[1015] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for the
PRPF3 or PRPF8 gene in the published reference genome deposited in
the NCBI repository of biological and scientific information. As
used herein, the "wild-type PRPF3 sequence" refers to the canonical
sequence available at NCBI Gene ID 9129. As used herein, the
"wild-type PRPF8 sequence" refers to the canonical sequence
available at NCBI Gene ID 10594. Also used herein, a nucleotide
position denoted with an "e" indicates the nucleotide is present in
the sequence of an exon (e.g., the exon flanking the 5' splice site
or the exon flanking the 3' splice site).
[1016] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding PRPF3 or PRPF8
protein, resulting in increased expression of the PRPF3 or PRPF8
protein, respectively. As used herein, "contacting" or
administering to cells refers to any method of providing an ASO in
immediate proximity with the cells such that the ASO and the cells
interact. A cell that is contacted with an ASO will take up or
transport the ASO into the cell. The method involves contacting a
condition or disease-associated or condition or disease-relevant
cell with any of the ASOs described herein. In some embodiments,
the ASO may be further modified or attached (e.g., covalently
attached) to another molecule to target the ASO to a cell type,
enhance contact between the ASO and the condition or
disease-associated or condition or disease-relevant cell, or
enhance uptake of the ASO.
[1017] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[1018] In embodiments, contacting a cell that expresses a PRPF3 or
PRPF8 RIC pre-mRNA with an ASO that is complementary to a targeted
portion of the PRPF3 or PRPF8 RIC pre-mRNA transcript results in a
measurable increase in the amount of the PRPF3 or PRPF8 protein
(e.g., a target protein) encoded by the pre-mRNA. Methods of
measuring or detecting production of a protein will be evident to
one of skill in the art and include any known method, for example,
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA.
[1019] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a PRPF3 and/or PRPF8 RIC
pre-mRNA transcript results in an increase in the amount of PRPF3
and/or PRPF8 protein produced by at least 10, 20, 30, 40, 50, 60,
80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared
to the amount of the protein produced by a cell in the absence of
the ASO/absence of treatment. In embodiments, the total amount of
PRPF3 or PRPF8 protein produced by the cell to which the antisense
oligomer was contacted is increased about 1.1 to about 10-fold,
about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to
about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,
about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to
about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold,
about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about
8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3
to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,
about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about
9-fold, at least about 1.1-fold, at least about 1.5-fold, at least
about 2-fold, at least about 2.5-fold, at least about 3-fold, at
least about 3.5-fold, at least about 4-fold, at least about 5-fold,
or at least about 10-fold, compared to the amount of target protein
produced by an control compound. A control compound can be, for
example, an oligonucleotide that is not complementary to the
targeted portion of the RIC pre-mRNA.
[1020] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a PRPF3 or PRPF8 RIC
pre-mRNA transcript results in an increase in the amount of mRNA
encoding PRPF3 or PRPF8, including the mature mRNA encoding the
target protein. In some embodiments, the amount of mRNA encoding
PRPF3 or PRPF8 protein, or the mature mRNA encoding the PRPF3 or
PRPF8 protein, is increased by at least 10, 20, 30, 40, 50, 60, 80,
100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to
the amount of the protein produced by a cell in the absence of the
ASO/absence of treatment. In embodiments, the total amount of the
mRNA encoding PRPF3 or PRPF8 protein, or the mature mRNA encoding
PRPF3 or PRPF8 protein produced in the cell to which the antisense
oligomer was contacted is increased about 1.1 to about 10-fold,
about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to
about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,
about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to
about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold,
about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about
8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3
to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,
about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about
9-fold, at least about 1.1-fold, at least about 1.5-fold, at least
about 2-fold, at least about 2.5-fold, at least about 3-fold, at
least about 3.5-fold, at least about 4-fold, at least about 5-fold,
or at least about 10-fold compared to the amount of mature RNA
produced in an untreated cell, e.g., an untreated cell or a cell
treated with a control compound. A control compound can be, for
example, an oligonucleotide that is not complementary to the
targeted portion of the PRPF3 or PRPF8 RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[1021] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of PRPF3
or PRPF8 protein in cells, for example, in a subject having RP
caused by a deficiency in the amount or activity of PRPF3 or PRPF8
protein, by increasing the level of mRNA encoding PRPF3 or PRPF8
protein, or the mature mRNA encoding PRPF3 or PRPF8 protein. In
particular, the methods and compositions as described herein induce
the constitutive splicing of a retained intron from a PRPF3 or
PRPF8 RIC pre-mRNA transcript encoding PRPF3 or PRPF8 protein,
thereby increasing the level of mRNA encoding PRPF3 or PRPF8
protein, or the mature mRNA encoding PRPF3 or PRPF8 protein and
increasing the expression of PRPF3 or PRPF8 protein.
[1022] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[1023] In embodiments, constitutive splicing correctly removes a
retained intron from a PRPF3 or PRPF8 RIC pre-mRNA, wherein the
PRPF3 or PRPF8 RIC pre-mRNA is transcribed from a wild-type gene or
allele, or a polymorphic gene or allele, that encodes a
fully-functional target protein or functional RNA, and wherein the
gene or allele does not have a mutation that causes alternative
splicing or aberrant splicing of the retained intron.
[1024] In some embodiments, constitutive splicing of a retained
intron from a PRPF3 or PRPF8 RIC pre-mRNA encoding PRPF3 or PRPF8
protein correctly removes a retained intron from a PRPF3 or PRPF8
RIC pre-mRNA encoding PRPF3 or PRPF8 protein, wherein the PRPF3 or
PRPF8 RIC pre-mRNA is transcribed from a gene or allele from which
the target gene or functional RNA is produced at a reduced level
compared to production from a wild-type allele, and wherein the
gene or allele does not have a mutation that causes alternative
splicing or aberrant splicing of the retained intron. In these
embodiments, the correct removal of the constitutively spliced
retained intron results in production of target protein or
functional RNA that is functional when compared to an equivalent
wild-type protein or functional RNA.
[1025] In other embodiments, constitutive splicing correctly
removes a retained intron from a PRPF3 or PRPF8 RIC pre-mRNA,
wherein the PRPF3 or PRPF8 RIC pre-mRNA is transcribed from a gene
or allele that encodes a target protein or functional RNA produced
in a form having reduced function compared to an equivalent
wild-type protein or functional RNA, and wherein the gene or allele
does not have a mutation that causes alternative splicing or
aberrant splicing of the retained intron. In these embodiments, the
correct removal of the constitutively spliced retained intron
results in production of partially functional target protein, or
functional RNA that is partially functional when compared to an
equivalent wild-type protein or functional RNA.
[1026] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[1027] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding PRPF3 or PRPF8 protein or the amount of PRPF3 or
PRPF8 protein by modulating alternative splicing or aberrant
splicing of a pre-mRNA transcribed from the PRPF3 or PRPF8 gene.
Modulation of alternative splicing or aberrant splicing can be
measured using any known method for analyzing the sequence and
length of RNA species, e.g., by RT-PCR and using methods described
elsewhere herein and in the literature. In embodiments, modulation
of alternative or aberrant splicing is determined based on an
increase or decrease in the amount of the spliced species of
interest of at least 10% or 1.1-fold. In embodiments, modulation is
determined based on an increase or decrease at a level that is at
least 10% to 100% or 1.1 to 10-fold, as described herein regarding
determining an increase in mRNA encoding PRPF3 or PRPF8 protein in
the methods of the invention.
[1028] In embodiments, the method is a method wherein the PRPF3 or
PRPF8 RIC pre-mRNA was produced by partial splicing of a wild-type
PRPF3 or PRPF8 pre-mRNA. In embodiments, the method is a method
wherein the PRPF3 or PRPF8 RIC pre-mRNA was produced by partial
splicing of a full-length wild-type PRPF3 or PRPF8 pre-mRNA. In
embodiments, the PRPF3 or PRPF8 RIC pre-mRNA was produced by
partial splicing of a full-length PRPF3 or PRPF8 pre-mRNA. In these
embodiments, a full-length PRPF3 or PRPF8 pre-mRNA may have a
polymorphism in a splice site of the retained intron that does not
impair correct splicing of the retained intron as compared to
splicing of the retained intron having the wild-type splice site
sequence.
[1029] In embodiments, the mRNA encoding PRPF3 or PRPF8 protein is
a full-length mature mRNA, or a wild-type mature mRNA. In these
embodiments, a full-length mature mRNA may have a polymorphism that
does not affect the activity of the target protein or the
functional RNA encoded by the mature mRNA, as compared to the
activity of PRPF3 or PRPF8 protein encoded by the wild-type mature
mRNA.
Antisense Oligomers
[1030] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of a PRPF3 or PRPF8 RIC pre-mRNA. As used
herein, the terms "ASO" and "antisense oligomer" are used
interchangeably and refer to an oligomer such as a polynucleotide,
comprising nucleobases that hybridizes to a target nucleic acid
(e.g., a PRPF3 or PRPF8 RIC pre-mRNA) sequence by Watson-Crick base
pairing or wobble base pairing (G-U). The ASO may have exact
sequence complementary to the target sequence or near
complementarity (e.g., sufficient complementarity to bind the
target sequence and enhancing splicing at a splice site). ASOs are
designed so that they bind (hybridize) to a target nucleic acid
(e.g., a targeted portion of a pre-mRNA transcript) and remain
hybridized under physiological conditions. Typically, if they
hybridize to a site other than the intended (targeted) nucleic acid
sequence, they hybridize to a limited number of sequences that are
not a target nucleic acid (to a few sites other than a target
nucleic acid). Design of an ASO can take into consideration the
occurrence of the nucleic acid sequence of the targeted portion of
the pre-mRNA transcript or a sufficiently similar nucleic acid
sequence in other locations in the genome or cellular pre-mRNA or
transcriptome, such that the likelihood the ASO will bind other
sites and cause "off-target" effects is limited. Any antisense
oligomers known in the art, for example in PCT Application No.
PCT/US2014/054151, published as WO 2015/035091, titled "Reducing
Nonsense-Mediated mRNA Decay," can be used to practice the methods
described herein.
[1031] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[1032] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[1033] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[1034] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and
Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by
reference in their entirety.
[1035] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine,
5,6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[1036] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081
(1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et
al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer
Drug Design 6:539 (1991); Zon et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[1037] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the invention, including, but not limited
to, any of the ASOs set forth herein in Table 6, comprises an ASO
having phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[1038] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention, including, but not limited to, any of the ASOs set
forth herein in Table 1, comprises about 5-100% Rp, at least about
5% Rp, at least about 10% Rp, at least about 15% Rp, at least about
20% Rp, at least about 25% Rp, at least about 30% Rp, at least
about 35% Rp, at least about 40% Rp, at least about 45% Rp, at
least about 50% Rp, at least about 55% Rp, at least about 60% Rp,
at least about 65% Rp, at least about 70% Rp, at least about 75%
Rp, at least about 80% Rp, at least about 85% Rp, at least about
90% Rp, or at least about 95% Rp, with the remainder Sp, or about
100% Rp. In embodiments, an ASO used in the methods of the
invention, including, but not limited to, any of the ASOs set forth
herein in Table 6, comprises about 10% to about 100% Rp, about 15%
to about 100% Rp, about 20% to about 100% Rp, about 25% to about
100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp,
about 40% to about 100% Rp, about 45% to about 100% Rp, about 50%
to about 100% Rp, about 55% to about 100% Rp, about 60% to about
100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp,
about 75% to about 100% Rp, about 80% to about 100% Rp, about 85%
to about 100% Rp, about 90% to about 100% Rp, or about 95% to about
100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp,
about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45%
to about 55% Rp, with the remainder Sp.
[1039] In embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 6, comprises about 5-100% Sp, at least about 5% Sp, at least
about 10% Sp, at least about 15% Sp, at least about 20% Sp, at
least about 25% Sp, at least about 30% Sp, at least about 35% Sp,
at least about 40% Sp, at least about 45% Sp, at least about 50%
Sp, at least about 55% Sp, at least about 60% Sp, at least about
65% Sp, at least about 70% Sp, at least about 75% Sp, at least
about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at
least about 95% Sp, with the remainder Rp, or about 100% Sp. In
embodiments, an ASO used in the methods of the invention,
including, but not limited to, any of the ASOs set forth herein in
Table 6, comprises about 10% to about 100% Sp, about 15% to about
100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp,
about 30% to about 100% Sp, about 35% to about 100% Sp, about 40%
to about 100% Sp, about 45% to about 100% Sp, about 50% to about
100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp,
about 65% to about 100% Sp, about 70% to about 100% Sp, about 75%
to about 100% Sp, about 80% to about 100% Sp, about 85% to about
100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp,
about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to
about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55%
Sp, with the remainder Rp.
[1040] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[1041] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[1042] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[1043] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[1044] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[1045] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[1046] In other embodiments, the ASOs are complementary to (and
bind to) a targeted portion of a PRPF3 or PRPF8 RIC pre-mRNA that
is downstream (in the 3' direction) of the 5' splice site of the
retained intron in a PRPF3 or PRPF8 RIC pre-mRNA (e.g., the
direction designated by positive numbers relative to the 5' splice
site) (FIG. 1). In some embodiments, the ASOs are complementary to
a targeted portion of the PRPF3 or PRPF8 RIC pre-mRNA that is
within the region +6 to +100 relative to the 5' splice site of the
retained intron. In some embodiments, the ASO is not complementary
to nucleotides +1 to +5 relative to the 5' splice site (the first
five nucleotides located downstream of the 5' splice site). In some
embodiments, the ASOs may be complementary to a targeted portion of
a PRPF3 or PRPF8 RIC pre-mRNA that is within the region between
nucleotides +6 and +50 relative to the 5' splice site of the
retained intron. In some aspects, the ASOs are complementary to a
targeted portion that is within the region +6 to +90, +6 to +80, +6
to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20
relative to 5' splice site of the retained intron.
[1047] In some embodiments, the ASOs are complementary to a
targeted region of a PRPF3 or PRPF8 RIC pre-mRNA that is upstream
(5' relative) of the 3' splice site of the retained intron in a
PRPF3 or PRPF8 RIC pre-mRNA (e.g., in the direction designated by
negative numbers) (FIG. 1). In some embodiments, the ASOs are
complementary to a targeted portion of the PRPF3 or PRPF8 RIC
pre-mRNA that is within the region -16 to -100 relative to the 3'
splice site of the retained intron. In some embodiments, the ASO is
not complementary to nucleotides -1 to -15 relative to the 3'
splice site (the first 15 nucleotides located upstream of the 3'
splice site). In some embodiments, the ASOs are complementary to a
targeted portion of the PRPF3 or PRPF8 RIC pre-mRNA that is within
the region -16 to -50 relative to the 3' splice site of the
retained intron. In some aspects, the ASOs are complementary to a
targeted portion that is within the region -16 to -90, -16 to -80,
-16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30
relative to 3' splice site of the retained intron.
[1048] In embodiments, the targeted portion of the PRPF3 or PRPF8
RIC pre-mRNA is within the region +100 relative to the 5' splice
site of the retained intron to -100 relative to the 3' splice site
of the retained intron.
[1049] In some embodiments, the ASOs are complementary to a
targeted portion of a PRPF3 or PRPF8 RIC pre-mRNA that is within
the exon flanking the 5' splice site (upstream) of the retained
intron (FIG. 1). In some embodiments, the ASOs are complementary to
a targeted portion of the PRPF3 or PRPF8 RIC pre-mRNA that is
within the region +2e to -4e in the exon flanking the 5' splice
site of the retained intron. In some embodiments, the ASOs are not
complementary to nucleotides -1e to -3e relative to the 5' splice
site of the retained intron. In some embodiments, the ASOs are
complementary to a targeted portion of the PRPF3 or PRPF8 RIC
pre-mRNA that is within the region -4e to -100e, -4e to -90e, -4e
to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to -40e, -4e to
-30e, or -4e to -20e relative to the 5' splice site of the retained
intron.
[1050] In some embodiments, the ASOs are complementary to a
targeted portion of a PRPF3 or PRPF8 RIC pre-mRNA that is within
the exon flanking the 3' splice site (downstream) of the retained
intron (FIG. 1). In some embodiments, the ASOs are complementary to
a targeted portion to the PRPF3 or PRPF8 RIC pre-mRNA that is
within the region +2e to -4e in the exon flanking the 3' splice
site of the retained intron. In some embodiments, the ASOs are not
complementary to nucleotide +1e relative to the 3' splice site of
the retained intron. In some embodiments, the ASOs are
complementary to a targeted portion of the PRPF3 or PRPF8 RIC
pre-mRNA that is within the region +2e to +100e, +2e to +90e, +2e
to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to
+30e, or +2 to +20e relative to the 3' splice site of the retained
intron. The ASOs may be of any length suitable for specific binding
and effective enhancement of splicing. In some embodiments, the
ASOs consist of 8 to 50 nucleobases. For example, the ASO may be 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases
in length. In some embodiments, the ASOs consist of more than 50
nucleobases. In some embodiments, the ASO is from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40
nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25
nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40
nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25
nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40
nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25
nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40
nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25
nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35
nucleobases, or 25 to 30 nucleobases in length. In some
embodiments, the ASOs are 18 nucleotides in length. In some
embodiments, the ASOs are 15 nucleotides in length. In some
embodiments, the ASOs are 25 nucleotides in length.
[1051] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[1052] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[1053] In some embodiments, the nucleic acid to be targeted by an
ASO is a PRPF3 or PRPF8 RIC pre-mRNA expressed in a cell, such as a
eukaryotic cell. In some embodiments, the term "cell" may refer to
a population of cells. In some embodiments, the cell is in a
subject. In some embodiments, the cell is isolated from a subject.
In some embodiments, the cell is ex vivo. In some embodiments, the
cell is a condition or disease-relevant cell or a cell line. In
some embodiments, the cell is in vitro (e.g., in cell culture).
Pharmaceutical Compositions
[1054] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent
or carrier.
[1055] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose. The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[1056] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[1057] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancer is a surfactant, fatty acid,
bile salt, chelating agent, or non-chelating nonsurfactant.
[1058] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[1059] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[1060] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[1061] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[1062] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[1063] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. The eye is the most significantly affected tissue
in RP13 and RP18. An ASO of the present invention may therefore be
administered to a subject by intravitreal injection, by subretinal
injection, or by topical application to the eye. In embodiments, an
ASO of the present invention administered to a subject via an
implant, e.g., an encapsulated drug implanted in the eye. In
embodiments, RPE cells treated ex vivo with an ASO of the present
invention are implanted in the eye, e.g., in encapsulated form. In
embodiments, therapy is administered in conjunction with existing
treatments for RP, e.g., oral acetazolamide, calcium channel
blockers, lutein or zeaxanthin, oral valproic acid, or an
immunosuppressive agent. In embodiments, ASOs are administered
parenterally. In embodiments, ASOs of the present invention are
administered to a subject orally, by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. In embodiments, a fetus is treated in utero, e.g., by
administering the ASO composition to the fetus directly or
indirectly (e.g., via the mother).
[1064] In embodiments, subjects treated using the methods and
compositions are evaluated for improvement in condition using any
methods known and described in the art.
Methods of Identifying Additional ASOs that Enhance Splicing
[1065] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of a PRPF3 or PRPF8 RIC pre-mRNA, specifically at the target
intron. ASOs that specifically hybridize to different nucleotides
within the target region of the pre-mRNA may be screened to
identify (determine) ASOs that improve the rate and/or extent of
splicing of the target intron. In some embodiments, the ASO may
block or interfere with the binding site(s) of a splicing
repressor(s)/silencer. Any method known in the art may be used to
identify (determine) an ASO that when hybridized to the target
region of the intron results in the desired effect (e.g., enhanced
splicing, protein or functional RNA production). These methods also
can be used for identifying ASOs that enhance splicing of the
retained intron by binding to a targeted region in an exon flanking
the retained intron, or in a non-retained intron. An example of a
method that may be used is provided below.
[1066] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site.
[1067] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1068] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[1069] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[1070] As described for the ASO walk above, the ASO micro-walk is
performed by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1071] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by intravitreal injection, by subretinal
injection, by topical application to the eye, or by an encapsulated
drug implanted in the eye. Following administration, the cells,
tissues, and/or organs of the model animals may be assessed to
determine the effect of the ASO treatment by for example evaluating
splicing (efficiency, rate, extent) and protein production by
methods known in the art and described herein. The animal models
may also be any phenotypic or behavioral indication of the disease
or disease severity.
Compositions and Methods for Treatment of Central Nervous System
Diseases
[1072] Individual introns in primary transcripts of protein-coding
genes having more than one intron are spliced from the primary
transcript with different efficiencies. In most cases only the
fully spliced mRNA is exported through nuclear pores for subsequent
translation in the cytoplasm. Unspliced and partially spliced
transcripts are detectable in the nucleus. It is generally thought
that nuclear accumulation of transcripts that are not fully spliced
is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[1073] Substantial levels of partially-spliced transcripts encoding
the: ATP1A2 protein, deficient in Familial hemiplegic migraine-2,
Familial Basilar migraine, and Alternating hemiplegia of childhood;
CACNA1A protein, deficient in Episodic ataxia type 2 (EA2),
Familial hemiplegic migraine, and Spinocerebellar ataxia type 6;
SETD5 protein, deficient in Mental retardation-23 (MRD23), and 3p25
microdeletion syndrome; SHANK3 protein, deficient in
Phelan-McDermid syndrome (PHMDS), and Schizophrenia-15 (SCZD15);
NF2 protein, deficient in Neurofibromatosis, type 2, Meningioma,
NF2-related, and Schwannomatosis 1; DNMT1 protein, deficient in
Hereditary sensory neuropathy type IE (HSN1E), Autosomal dominant
cerebellar ataxia, deafness, and narcolepsy (ADCADN); TCF4 protein,
deficient in Pitt-hopkins syndrome; RAI1 protein, deficient in
Smith-magenis syndrome; PEX1 protein, deficient in PEROXISOME
BIOGENESIS DISORDER 1A (Zellweger syndrome)(PBD1A), and Heimler
syndrome-1 (HMLR1); ARSA protein, deficient in Metachromatic
Leukodystrophy; EIF2B5 protein/EIF2B1 protein/EIF2B2, deficient in
Leukoencephalopathy with vanishing white matter (VWM); NPC1
protein, deficient in Niemann-Pick disease type C1 and Niemann-Pick
disease type D; ADAR protein, deficient in Aicardi-Goutieres
syndrome-6 (AGS6); MFSD8 protein, deficient in Neuronal ceroid
lipofuscinosis-7; STXBP1 protein, deficient in early infantile
epileptic encephalopathy-4; PRICKLE2 protein, deficient in
progressive myoclonic epilepsy 5; PRRT2 protein, deficient in
familial infantile convulsion with paroxysmal choreoathetosis,
episodic kinesigenic dyskinesia 1, or benign familial infantile
seizuers-2; IDUA protein, deficient in attenuated MPS-1
(Hurler-scheie syndrome); and STX1B protein, deficient in
generalized Epilepsy with febrile seizure plus type 9 have been
discovered in the nucleus of human cells. These ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, and STX1B
pre-mRNA species comprise at least one retained intron. The present
invention provides compositions and methods for upregulating
splicing of one or more retained ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B introns that
are rate-limiting for the nuclear stages of gene expression to
increase steady-state production of fully-spliced, mature mRNA, and
thus, translated ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein levels. These
compositions and methods can utilize antisense oligomers (ASOs)
that promote constitutive splicing at intron splice sites of a
retained-intron-containing ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B pre-mRNA (RIC
pre-mRNA) that accumulates in the nucleus. Thus, in embodiments,
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein can be increased using the methods of the
invention to treat a condition caused by ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
deficiency.
[1074] In some embodiments, disclosed herein are compositions and
methods for upregulating splicing of one or more retained ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
introns that are rate-limiting for the nuclear stages of gene
expression to increase steady-state production of fully-spliced,
mature mRNA, and thus, translated ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein levels. These
compositions and methods can utilize antisense oligomers (ASOs)
that promote constitutive splicing at intron splice sites of a
retained-intron-containing ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B pre-mRNA (RIC pre-mRNA) that
accumulates in the nucleus. Thus, in embodiments, ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein can
be increased using the methods of the invention to treat a
condition caused by ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B deficiency.
[1075] In some embodiments, disclosed herein are compositions and
methods for upregulating splicing of one or more retained ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B introns that
are rate-limiting for the nuclear stages of gene expression to
increase steady-state production of fully-spliced, mature mRNA, and
thus, translated ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B protein levels. These compositions and methods can
utilize antisense oligomers (ASOs) that promote constitutive
splicing at intron splice sites of a retained-intron-containing
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
pre-mRNA (RIC pre-mRNA) that accumulates in the nucleus. Thus, in
embodiments, ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B protein can be increased using the methods of the
invention to treat a condition caused by ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B deficiency.
[1076] In other embodiments, the methods of the invention can be
used to increase ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B production to treat a
condition in a subject in need thereof. In embodiments, the subject
has a condition in which ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B is not necessarily
deficient relative to wild-type, but where an increase in ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B mitigates the condition nonetheless. In embodiments, the
condition can be caused by a ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
haploinsufficiency.
[1077] In other embodiments, the methods of the invention can be
used to increase ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B production to treat a condition in a
subject in need thereof. In embodiments, the subject has a
condition in which ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B is not necessarily deficient relative to
wild-type, but where an increase in ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B mitigates the condition
nonetheless. In embodiments, the condition can be caused by a
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or
STX1B haploinsufficiency.
[1078] In additional embodiments, the methods of the invention can
be used to increase ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B production to treat a condition in a
subject in need thereof. In embodiments, the subject has a
condition in which ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B is not necessarily deficient relative to
wild-type, but where an increase in ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B mitigates the condition
nonetheless. In embodiments, the condition can be caused by a
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
haploinsufficiency.
[1079] In embodiments, the described compositions and methods are
used to treat a subject or patient having a CNS disease or
condition that is caused by a deficiency in the target protein. In
embodiments the described compositions and methods are used to
treat a subject or patient having a CNS disease or condition that
is not caused by a deficiency in the target protein. In
embodiments, the subject or patient having a CNS disease or
condition can benefit from increased production of the target
protein by supplementing normal production of the target protein.
In embodiments, the target protein acts on a secondary target to
ameliorate or treat the CNS disease or condition in the subject. In
embodiments, the secondary target protein is deficient in the
subject. In embodiments, the secondary target protein is not
deficient in the subject.
ATP1A2
[1080] ATP1A2, encodes the .alpha.2 isoform of Na+K+-ATPase (sodium
potassium ATPase). The .alpha.2 isoform belongs to the family of
P-type cation transport ATPases, and to the subfamily of
Na+/K+-ATPases. Na+/K+-ATPase is an integral membrane protein
responsible for establishing and maintaining the electrochemical
gradients of Na and K ions across the plasma membrane. These
gradients are essential for osmoregulation, for sodium-coupled
transport of a variety of organic and inorganic molecules, and for
electrical excitability of nerve and muscle.
[1081] Na+/K+-ATPase transfers Na+ out and K+ into the cell, using
ATP as energy source. Na+/K+ ATPases are present in all human
cells; however, the .alpha.2 isoform is mainly restricted to the
brain and muscle. Na+/K+-ATPases that include the .alpha.-2 subunit
are primarily found in glia cells, particularly in astrocytes,
where they contribute to the glial clearance of high extracellular
K+ to prevent further depolarization. Through its action in glia,
the Na+/K+-ATPase plays a critical role in the normal function of
neurons. Communication between neurons depend neurotransmitters.
Briefly, neurons release neurotransmitters, which bind to receptor
proteins on neighboring neurons. After the neurotransmitters have
had their effect, neurotransmitters detach from their receptors and
are removed from the spaces between neurons by glia. The Na+/K+
ATPase helps regulate this process by stimulating glia to clear
neurotransmitters from the spaces between neurons. Na+/K+-ATPases
also removes excess potassium ions from these spaces. In humans,
mutations in ATP1A2 are associated with genetic diseases, for
example familial hemiplegic migraine-2 (FHM2), familial basilar
migraine and alternating hemiplegia of childhood.
Familial Hemiplegic Migraine-2
[1082] Familial hemiplegic migraine-2 (FHM2) and familial basilar
migraine are caused by heterozygous mutation in ATP1A2. FHM2 is a
monogenic variant of migraine with aura. Migraines can cause
intense, throbbing pain in one area of the head, often accompanied
by nausea, vomiting, and extreme sensitivity to light and sound.
These recurrent headaches can begin in childhood or adolescence and
can be triggered by certain foods, emotional stress, and minor head
trauma. Each headache may last from a few hours to a few days. In
some types of migraine, including familial hemiplegic migraine, a
pattern of neurological symptoms called an aura precedes the
headache. The most common symptoms associated with an aura are
temporary visual changes such as blind spots (scotomas), flashing
lights, zig-zagging lines, and double vision. In people with
familial hemiplegic migraine, auras are also characterized by
temporary numbness or weakness, often affecting one side of the
body (hemiparesis). Additional features of an aura can include
difficulty with speech, confusion, and drowsiness. An aura can
develops gradually over a few minutes and lasts about an hour.
Familial hemiplegic migraine episodes includes fever, seizures,
prolonged weakness, coma, and, rarely, death. Although most people
with familial hemiplegic migraine recover completely between
episodes, neurological symptoms such as memory loss and problems
with attention can last for weeks or months. About 20 percent of
people with this condition develop mild but permanent difficulty
coordinating movements (ataxia), which may worsen with time, and
rapid, involuntary eye movements called nystagmus.
[1083] FHM 2 is caused by mutations present in ATP1A2. The
mutations and deficiencies in ATP1A2 that cause FHM2 are
responsible for approximately 20% of FHM in families. There are
over 50 mutations in ATP1A2 that have been identified in
association with FHM2. Almost all FHM2 mutations are non-synonymous
SNPs, but there are also small deletions and a mutation affecting
the stop codon, causing an extension of the ATP1A2 protein by 27
amino acid residues. Most of the mutations are associated with pure
FHM without additional clinical symptoms. ATP1A2 is expressed
primarily in astrocytes in the adult, where it appears functionally
coupled to various transporters (glutamate transporter and
Na.sup.+/Ca.sup.2+ exchanger), and is essential in the clearance of
released glutamate and potassium from the extracellular space
during neuronal activity. FHM2 mutations that cause loss of
function of ATP1A2 may lead to decreased glutamate clearance and an
increase of potassium in the synaptic cleft during neuronal
activity, which could lead to prolonged recovery time after
neuronal excitation, and may render the brain to be more
susceptible to cortical spreading depression. Cortical spreading
depression is a wave of continual strong neuronal depolarization
that slowly progresses across the cortex, generating a brief
intense spike of activity that is followed by long-lasting neural
suppression. Cortical spreading depression has been shown to
activate the trigeminovascular system which is responsible for the
headache associated with migraines.
[1084] There are two hypothesized mechanisms for the effects of
ATP1A2 mutations. First, the mutations cause an increase in
extracellular potassium, which can result in the impaired clearance
of potassium ions and therefore induce Cortical spreading
depression. Second, since the distribution of ATP1A2 is
co-localized with the Na.sup.+/Ca.sup.2+ exchanger, the mutations
to ATP1A2 would cause intracellular sodium to increase, which
increases intracellular calcium levels through the
Na.sup.+/Ca.sup.2+ exchanger, resulting in glutamate release and a
decrease in glutamate clearance which can also lead to Cortical
spreading depression. Both hypotheses result in making the brain
more susceptible to Cortical spreading depression and therefore
migraines with aura.
[1085] Recently, a number of ATP1A2 mutations were reported to be
associated with FHM and cerebellar problems, specifically motor
problems, childhood convulsions, epilepsy, and mental retardation.
Some ATP1A2 mutations have been shown to be associated with
non-hemiplegic migraine phenotypes, such as basilar migraine and
the common migraine.
Familial Basilar Migraine
[1086] Basilar migraine is a subtype of migraine with aura in which
the aura symptoms originate from the brainstem or reflect the
simultaneous involvement of both hemispheres.
Alternating Hemiplegia of Childhood
[1087] Alternating hemiplegia of childhood is an autosomal dominant
condition. Alternating hemiplegia of childhood can result from new
mutations in the gene and occur in people with no history of the
disorder in their family. The primary feature of this condition is
recurrent episodes of temporary paralysis, often affecting one side
of the body (hemiplegia). During some episodes, the paralysis
alternates from one side to the other or affects both sides of the
body at the same time. The known ATP1A2 gene mutation associated
with this condition replaces a single amino acid in Na+/K+ ATPase:
the amino acid threonine is replaced with the amino acid asparagine
at protein position 378. This genetic change can impair the
protein's ability to transport ions. In addition to paralysis,
affected individuals can have sudden attacks of uncontrollable
muscle activity; these can cause involuntary limb movements
(choreoathetosis), muscle tensing (dystonia), movement of the eyes
(nystagmus), or shortness of breath (dyspnea). Individuals with
alternating hemiplegia of childhood may also experience sudden
redness and warmth (flushing) or unusual paleness (pallor) of the
skin. These attacks can occur during or separately from episodes of
hemiplegia. The episodes of hemiplegia or uncontrolled movements
can be triggered by certain factors, such as stress, extreme
tiredness, cold temperatures, or bathing, although the trigger is
not always known. The number and length of the episodes initially
worsen throughout childhood but then begin to decrease over time.
The uncontrollable muscle movements may disappear entirely, but the
episodes of hemiplegia occur throughout life. Alternating
hemiplegia of childhood also causes mild to severe cognitive
problems. Almost all affected individuals have some level of
developmental delay and intellectual disability. Their cognitive
functioning typically declines over time.
CACNA1A
[1088] The CACNA1A gene encodes the alpha-1 subunit of the calcium
channel CaV2.1. Voltage-dependent Ca(2+) channels not only mediate
the entry of Ca(2+) ions into excitable cells but are also involved
in a variety of Ca(2+)-dependent processes, including muscle
contraction, hormone or neurotransmitter release, and gene
expression. Diriong et al. (1995) noted that calcium channels are
multisubunit complexes and that the channel activity is directed by
a pore-forming alpha-1 subunit, which is often sufficient to
generate voltage-sensitive Ca(2+) channel activity. This subunit
forms the pore through which calcium ions can flow. CaV2.1 channels
play an essential role in neuronal communication. These channels
help control the release of neurotransmitters, and are involved in
neuronal plasticity. Different mutations in the alpha-1 subunit the
CaV2.1 calcium channel are responsible for familial hemiplegic
migraine (FHM), episodic ataxia type 2 (EA2), and spinocerebellar
ataxia type 6 (SCA6). Missense and splice site mutations have been
found in FHM and episodic ataxia type 2 (EA2), respectively,
whereas a CAG repeat in the CACNA1A gene was found expanded in
patients with spinocerebellar ataxia type 6 (SCA6).
Episodic Ataxia Type 2 (EA2)
[1089] Episodic ataxia is a group of related conditions that affect
the nervous system and cause problems with movement. People with
episodic ataxia have recurrent episodes of poor coordination and
balance (ataxia). During these episodes, many affected individuals
also experience dizziness (vertigo), nausea and vomiting, migraine
headaches, blurred or double vision, slurred speech, and ringing in
the ears (tinnitus). Seizures, muscle weakness, and paralysis
affecting one side of the body (hemiplegia) may also occur during
attacks. Additionally, some affected individuals have a muscle
abnormality called myokymia during or between episodes. This
abnormality can cause muscle cramping, stiffness, and continuous,
fine muscle twitching that appears as rippling under the skin.
[1090] Episodes of ataxia and other symptoms can begin anytime from
early childhood to adulthood. They can be triggered by
environmental factors such as emotional stress, caffeine, alcohol,
certain medications, physical activity, and illness. The frequency
of attacks ranges from several per day to one or two per year.
Between episodes, some affected individuals continue to experience
ataxia, which may worsen over time, as well as involuntary eye
movements called nystagmus.
[1091] Researchers have identified at least seven types of episodic
ataxia, designated type 1 through type 7. The types are
distinguished by their pattern of signs and symptoms, age of onset,
length of attacks, and, when known, genetic cause.
[1092] More than 50 mutations in the CACNA1A gene have been found
to cause episodic ataxia type 2 (EA2), the most common form of
episodic ataxia. In addition to problems with coordination and
balance (ataxia), EA2 is associated with involuntary eye movements
called nystagmus. The CACNA1A mutations responsible for EA2 reduce
the production of functional CaV2.1 channels or prevent these
channels from reaching the cell membrane, where they are needed to
transport calcium ions. A decrease in the number of these channels
reduces the total flow of calcium ions into neurons, which disrupts
the release of neurotransmitters in the brain. Although changes in
signaling between neurons underlie the episodes of uncoordinated
movement seen in people with episodic ataxia, it is unclear how
altered calcium ion transport causes the specific features of the
condition.
Familial Hemiplegic Migraine
[1093] At least 20 mutations in the CACNA1A gene have been
identified in people with familial hemiplegic migraine type 1
(FHM1). FHM is characterized by an aura of hemiplegia that is
always associated with at least one other aura symptom (e.g.,
hemianopsia, hemisensory deficit, aphasia). The aura is followed by
a moderate to severe headache. In FHM1, the aura includes temporary
numbness or weakness on one side of the body (hemiparesis). Like
EA2, FHM1 is commonly associated with ataxia and nystagmus. Most of
the mutations that cause FHM1 change single amino acids in the
CaV2.1 channel. The most common mutation, which has been found in
more than a dozen affected families, replaces the amino acid
threonine with the amino acid methionine at protein position
666.
[1094] The CACNA1A mutations responsible for familial hemiplegic
migraine change the structure of the CaV2.1 channel. The altered
channels open more easily than usual, which increases the inward
flow of calcium ions. A greater influx of calcium ions through
CaV2.1 channels increases the cell's release of neurotransmitters.
The resulting changes in signaling between neurons lead to
development of these severe headaches in people with familial
hemiplegic migraine. Approximately 50% of families with FHM,
including all those with permanent cerebellar symptoms, have
missense mutations in CACNA1A [Battistini et al 1999, Ducros et al
1999, Friend et al 1999].
Spinocerebellar Ataxia Type 6
[1095] Spinocerebellar ataxia type 6 is a late-onset, autosomal
dominant disorder. Spinocerebellar ataxia type 6 (SCA6) is another
disorder caused by CACNA1A gene mutations. The major features of
this condition include progressive ataxia, nystagmus, and impaired
speech (dysarthria), most often beginning in a person's forties or
fifties. SCA6 results from an increased number of copies
(expansion) of the CAG trinucleotide repeat in the CACNA1A gene.
Most cases of SCA6 are a result of CAG repeat expansion beyond the
normal range, i.e., more than 19 repeats. In people with this
condition, the CAG segment is repeated from 20 to more than 30
times. An increase in the length of the CAG segment leads to the
production of an abnormally long version of the alpha-1 subunit.
The abnormal subunit is found in the cell membrane as well as in
the cytoplasm, where it clusters together and forms aggregates. The
effect these aggregates have on cell functioning is unknown. The
lack of normal calcium channels impairs the cells' ability to
transport calcium ions. These changes alter the release of
neurotransmitters in the brain and eventually lead to the death of
neurons. Certain neurons called Purkinje cells seem to be
particularly sensitive to a disruption in calcium transport.
Purkinje cells are located in the part of the brain that
coordinates movement (cerebellum). Over time, the loss of Purkinje
cells and other cells of the cerebellum causes the movement
problems characteristic of SCA6.
SETD5
[1096] SETD5 expression in adult brain, followed by spinal cord,
most isolated adult brain regions, and ovary. Much lower expression
was detected in other adult peripheral tissues and in fetal brain
and liver. The SETD5 gene encodes a 1,442-residue protein that is a
putative methyltransferase. Mutations in this gene have been
associated with autosomal dominant mental retardation-23.
Mental Retardation-23 (MRD23)
[1097] Autosomal dominant mental retardation-23 (MRD23) is caused
by heterozygous mutation in the SETD5 gene on chromosome 3p25.
Mental retardation, autosomal dominant 23 (MRD23) is a disorder
characterized by significantly below average general intellectual
functioning associated with impairments in adaptive behavior and
manifested during the developmental period. MRD23 patients manifest
moderate to severe intellectual disability with additional variable
features of brachycephaly, a low hairline, depressed nasal bridge,
prominent high nasal root, tubular nose, upslanting palpebral
fissures, long and smooth philtrum, micrognathia, thin upper lip,
and crowded teeth. Behavioral problems, including
obsessive-compulsive disorder, hand flapping with ritualized
behavior, and autism, are prominent features. The disease is caused
by mutations affecting the gene represented in this entry.
3p25 Microdeletion Syndrome
[1098] De novo loss-of-function mutations in SETD5, encoding a
methyltransferase in a 3p25 microdeletion syndrome critical region,
causes intellectual disability. Characteristic features of the
distal 3p-syndrome include low birth weight, microcephaly,
trigonocephaly, hypotonia, psychomotor and growth retardation,
ptosis, telecanthus, downslanting palpebral fissures, and
micrognathia. Postaxial polydactyly, renal anomalies, cleft palate,
congenital heart defects (especially atrioventricular septal
defects), preauricular pits, sacral dimple, and gastrointestinal
anomalies are variable features. Although intellectual deficits are
almost invariably associated with cytogenetically visible 3p
deletions, rare patients with a 3p26-p25 deletion and normal
intelligence or only mild abnormalities have been described.
SHANK3
[1099] In humans, SHANK3 encodes SH3 and multiple ankyrin repeat
domains 3 (SHANK3), also known as proline-rich synapse-associated
protein 2 (ProSAP2). Northern blot analysis indicated that human
PSAP2 is expressed primarily in brain as 7- and 8-kb transcripts.
The SHANK3 gene spans 60 kb and contains 22 exons. In rats and
humans, PSAP2 is expressed preferentially in cerebral cortex and
cerebellum. This gene is a member of the Shank gene family. Shank
proteins are multidomain scaffold proteins of the postsynaptic
density that connect neurotransmitter receptors, ion channels, and
other membrane proteins to the actin cytoskeleton and
G-protein-coupled signaling pathways. Shank proteins also play a
role in synapse formation and dendritic spine maturation. SHANK3 is
one of the genes disrupted in patients with the 22q13.3 deletion
syndrome, also known as Phelan-McDermid syndrome.
Phelan-McDermid Syndrome (PHMDS)
[1100] Phelan-McDermid syndrome (PHMDS) can be caused by a
heterozygous contiguous gene deletion at chromosome 22q13 or by
mutation in the SHANK3 gene. Phelan-McDermid syndrome is a
developmental disorder with variable features. Common features
include neonatal hypotonia, global developmental delay, normal to
accelerated growth, absent to severely delayed speech, autistic
behavior, and minor dysmorphic features. Other less common features
associated with this syndrome included increased tolerance to pain,
dysplastic toenails, chewing behavior, fleshy hands, dysplastic
ears, pointed chin, dolichocephaly, ptosis, tendency to overheat,
and epicanthic folds. Researchers have showed that Phelan-McDermid
syndrome neurons have reduced SHANK3 expression and major defects
in excitatory, but not inhibitory, synaptic transmission.
Excitatory synaptic transmission in Phelan-McDermid syndrome
neurons could be corrected by restoring SHANK3 expression or by
treating neurons with insulin-like growth factor-1. IGF1 treatment
promoted formation of mature excitatory synapses that lack SHANK3
but contained PSD95 and NMDA receptors with fast deactivation
kinetics.
Schizophrenia-15 (SCZD15)
[1101] Susceptibility to schizophrenia-15 (SCZD15) has been
associated with mutation in the SH3 and multiple ankyrin repeat
domains-3 gene (SHANK3). Schizophrenia-15 is a complex,
multifactorial psychotic disorder or group of disorders
characterized by disturbances in the form and content of thought
(e.g. delusions, hallucinations), in mood (e.g. inappropriate
affect), in sense of self and relationship to the external world
(e.g. loss of ego boundaries, withdrawal), and in behavior (e.g.,
bizarre or apparently purposeless behavior). Although it affects
emotions, it is distinguished from mood disorders in which such
disturbances are primary. Similarly, there may be mild impairment
of cognitive function, and it is distinguished from the dementias
in which disturbed cognitive function is considered primary. Some
patients manifest schizophrenic as well as bipolar disorder
symptoms and are often given the diagnosis of schizoaffective
disorder.
NF2
[1102] The NF2 gene encodes the protein merlin, also known as
schwannomin. Merlin is a membrane-cytoskeleton scaffolding protein,
i.e. linking actin filaments to cell membrane or membrane
glycoproteins. Human merlin is predominantly found in nervous
tissue, but also in several other fetal tissues, and is mainly
located in adherens junctions. NF2 belongs to the tumor suppressor
group of genes. The phosphorylation of serine 518 is known to alter
the functional state of merlin. The signaling pathway of merlin is
proposed to include several salient cell growth controlling
molecules, including eIF3c, CD44, protein kinase A, and p21
activated kinases. Mutations of the NF2 gene cause a human
autosomal dominant disease called neurofibromatosis type 2. It is
characterized by the development of tumors of the nervous system,
most commonly of bilateral vestibular schwannomas (also called
acoustic neuromas).
Neurofibromatosis, Type 2
[1103] Neurofibromatosis type II is caused by mutation in the gene
encoding neurofibromin-2, which is also called merlin, on
chromosome 22q12.2. Neurofibromatosis type II is an inheritable
disorder with an autosomal dominant mode of transmission. Incidence
of the disease is about 1 in 60,000. Through statistics, it is
suspected that one-half of cases are inherited, and one-half are
the result of new, de novo mutations.
[1104] More than 200 mutations in the NF2 gene have been identified
in people with neurofibromatosis type 2. About 90 percent of NF2
mutations result in an abnormally shortened version of the merlin
protein. This short protein cannot perform its normal tumor
suppressor function in cells. Research suggests that the loss of
merlin allows cells, especially Schwann cells, to multiply too
frequently and form noncancerous tumors. The most common tumors in
neurofibromatosis type 2 are vestibular schwannomas, which develop
along the nerve that carries information from the inner ear to the
brain. Other tumors affecting the nervous system also occur in
people with this condition. Somatic mutations in the NF2 gene are
involved in the development of several types of tumors, both
noncancerous (benign) and cancerous (malignant).
[1105] It is known that merlin's deficiency can result in
unmediated progression through the cell cycle due to the lack of
contact-mediated tumor suppression, sufficient to result in the
tumors characteristic of Neurofibromatosis type II. Mutations of NF
II is presumed to result in either a failure to synthesize merlin
or the production of a defective peptide that lacks the normal
tumor-suppressive effect.
Meningioma, NF2-Related
[1106] Somatic mutations in the gene encoding merlin NF2 on
chromosome 22q12 have also been found in tumor tissues of a subset
of patients with meningiomas without other features of
neurofibromatosis-2. Meningioma, is a relatively common neoplasm of
the central nervous system that arises from arachnoidal cells. The
majority are well differentiated vascular tumors which grow slowly
and have a low potential to be invasive, although malignant
subtypes occur. Meningiomas have a predilection to arise from the
parasagittal region, cerebral convexity, sphenoidal ridge,
olfactory groove, and spinal canal. Meningiomas tend to present in
the fourth to sixth decades of life with signs indicative of a
slowly progressive mass lesion. Specific clinical manifestations
depend on the location of the tumor, but may include intracranial
hypertension, cranial neuropathies, ataxia, and other focal
neurologic signs. Most meningiomas are benign; only a very small
percentage of meningiomas become malignant. Many meningiomas are
asymptomatic, producing no symptoms throughout a person's life, and
if discovered, require no treatment other than periodic
observation. Typically, symptomatic meningiomas are treated with
either radiosurgery or conventional surgery. Historical evidence of
meningiomas has been found going back hundreds of years, with some
successful surgeries for their removal beginning in the 1800s.
Schwannomatosis 1
[1107] Individual schwannoma tumors from patients with
schwannomatosis have been found to harbor somatic mutations in the
neurofibromin-2 gene (NF2). Schwannomas are benign tumors of the
peripheral nerve sheath that usually occur singly in otherwise
normal individuals. Multiple schwannomas in the same individual
suggest an underlying tumor predisposition syndrome. The most
common such syndrome is neurofibromatosis II. The hallmark of NF2
is the development of bilateral vestibular nerve schwannomas, but
two-thirds or more of all NF2-affected individuals develop
schwannomas in other locations, and dermal schwannomas (or
neurilemmomas) may precede vestibular tumors in NF2-affected
children.
DNMT1
[1108] The DNMT1 gene encodes the enzyme DNA
(cytosine-5)-methyltransferase 1. This enzyme is involved in DNA
methylation. In particular, the enzyme helps add methyl groups to
cytosines. DNA methylation is important in many cellular functions.
These functions include gene silencing, regulating reactions
involving proteins and lipids, and controlling the processing of
neurotransmitters. DNA (cytosine-5)-methyltransferase 1 is active
in the adult nervous system. Although its specific function is not
well understood, the enzyme may help regulate neuron maturation and
differentiation, the ability of neurons to migrate where needed and
connect with each other, and neuron survival. In vitro functional
expression studies in E. coli and HeLa cells showed that the
mutations affected proper folding of DNMT1 and resulted in
premature degradation, reduced methyltransferase activity, and
impaired heterochromatin binding during the G2 cell cycle phase,
leading to global hypomethylation and site-specific
hypermethylation. These changes indicated epigenetic
dysregulation.
Hereditary Sensory Neuropathy Type IE (HSN1E)
[1109] Hereditary sensory neuropathy type IE (HSN1E) is caused by
heterozygous mutation in the DNMT1 gene on chromosome 19p13.
Hereditary sensory neuropathy type IE is an autosomal dominant
neurodegenerative disorder characterized by adult onset of
progressive peripheral sensory loss associated with progressive
hearing impairment and early-onset dementia. At least three DNMT1
gene mutations have been identified in people with hereditary
sensory and autonomic neuropathy (HSAN IE), a disorder
characterized by a gradual dementia, deafness, and sensory problems
in the feet. HSAN IE is further characterized by impaired function
of sensory neurons, which transmit information about sensations
such as pain, temperature, and touch. Sensations in the feet and
legs are particularly affected in people with HSAN IE. Gradual loss
of sensation in the feet (peripheral neuropathy), which usually
begins in adolescence or early adulthood, can lead to difficulty
walking. Affected individuals may not be aware of injuries to their
feet, which can lead to open sores and infections. If these
complications are severe, amputation of the affected areas may be
required. HSAN IE is also characterized by a loss of the ability to
sweat (sudomotor function), especially on the hands and feet.
Sweating is a function of the autonomic nervous system, which also
controls involuntary body functions such as heart rate, digestion,
and breathing. These other autonomic functions are unaffected in
people with HSAN IE. The severity of the signs and symptoms of HSAN
IE and their age of onset are variable, even within the same
family. The mutations in exon 20, reduce or eliminate the DNA
(cytosine-5)-methyltransferase 1 enzyme's methylation function. As
a result, maintenance of the neurons that make up the nervous
system is impaired. However, it is not known how the mutations
cause the specific signs and symptoms of HSAN IE.
Autosomal Dominant Cerebellar Ataxia, Deafness, and Narcolepsy
(ADCADN)
[1110] Autosomal dominant cerebellar ataxia, deafness, and
narcolepsy (ADCADN) is caused by heterozygous mutation in the DNMT1
gene on chromosome 19p13. ADCADN is an autosomal dominant
neurologic disorder characterized by adult onset of progressive
cerebellar ataxia, narcolepsy/cataplexy, sensorineural deafness,
and dementia. More variable features include optic atrophy, sensory
neuropathy, psychosis, and depression.
[1111] At least three DNMT1 gene mutations have been identified in
people with autosomal dominant cerebellar ataxia, deafness, and
narcolepsy. The mutations associated with this disorder are in exon
2 the DNMT1 gene. Mutations in different locations within the gene
may affect the DNA (cytosine-5)-methyltransferase 1 enzyme
differently, which can lead to particular combinations of signs and
symptoms.
TCF4
[1112] TCF4 encodes transcription factor 4, a basic
helix-loop-helix transcription factor. The encoded protein
recognizes an Ephrussi-box (`E-box`) binding site (`CANNTG`)--a
motif first identified in immunoglobulin enhancers. This gene is
broadly expressed, and may play an important role in nervous system
development. The TCF4 protein is found in the brain, muscles,
lungs, and heart. This protein also appears to be active in various
tissues before birth. The TCF4 protein plays a role cell
differentiation and apoptosis.
Pitt-Hopkins Syndrome
[1113] Pitt-Hopkins syndrome is characterized by mental
retardation, wide mouth and distinctive facial features, and
intermittent hyperventilation followed by apnea. Pitt-Hopkins
syndrome is linked to haploinsufficiency of the TCF4 transcription
factor gene. At least 50 mutations in the TCF4 gene have been found
to cause Pitt-Hopkins syndrome. Some mutations delete a nucleotide
within the TCF4 gene, while other mutations delete the TCF4 gene as
well as a number of genes that surround it. Still other TCF4 gene
mutations replace single nucleotides. The size of the mutation does
not appear to affect the severity of the condition; individuals
with large deletions and those with single nucleotide changes seem
to have similar signs and symptoms. TCF4 gene mutations disrupt the
protein's ability to bind to DNA and control the activity of
certain genes. These gene mutations typically do not affect the
TCF4 protein's ability to bind to other proteins. The TCF4
protein's inability to bind to DNA and control the activity of
certain genes, particularly those genes involved in nervous system
development and function, contributes to the signs and symptoms of
Pitt-Hopkins syndrome. It is also likely that the loss of the
normal proteins that are attached to the nonfunctional TCF4
proteins contribute to the features of this condition.
RAI1
[1114] The RAI1 gene encodes Retinoic acid-induced protein 1.
Retinoic acid-induced protein 1 is an important transcriptional
regulator of the circadian clock components: CLOCK, ARNTL/BMAL1,
ARNTL2/BMAL2, PER1/3, CRY1/2, NR1D1/2 and RORA/C. Retinoic
acid-induced protein 1 positively regulates the transcriptional
activity of CLOCK a core component of the circadian clock. Retinoic
acid-induced protein 1 regulates transcription through chromatin
remodeling by interacting with other proteins in chromatin as well
as proteins in the basic transcriptional machinery. Retinoic
acid-induced protein 1 may be important for embryonic and postnatal
development. Retinoic acid-induced protein 1 may be involved in
neuronal differentiation. Mutations in one copy of this gene lead
to the production of a nonfunctional version of the RAI1 protein or
reduce the amount of this protein that is produced.
Smith-Magenis Syndrome
[1115] Smith-Magenis syndrome (SMS) is caused in most cases (90%)
by a 3.7-Mb interstitial deletion in chromosome 17p11.2. The
disorder can also be caused by mutations in the RAI1 gene, which is
within the Smith-Magenis chromosome region. Smith-Magenis syndrome
is a developmental disorder that affects many parts of the body.
The major features of this condition include mild to moderate
intellectual disability, delayed speech and language skills,
distinctive facial features, sleep disturbances, and behavioral
problems. Most people with Smith-Magenis syndrome have a broad,
square-shaped face with deep-set eyes, full cheeks, and a prominent
lower jaw. The middle of the face and the bridge of the nose often
appear flattened. The mouth tends to turn downward with a full,
outward-curving upper lip. These facial differences can be subtle
in early childhood, but they usually become more distinctive in
later childhood and adulthood. Dental abnormalities are also common
in affected individuals. Disrupted sleep patterns are
characteristic of Smith-Magenis syndrome, typically beginning early
in life. Affected individuals may be very sleepy during the day,
but they have trouble falling asleep and awaken several times each
night. Individuals with Smith-Magenis syndrome have affectionate,
engaging personalities, but most also have behavioral problems.
These include frequent temper tantrums and outbursts, aggression,
anxiety, impulsiveness, and difficulty paying attention.
Self-injury, including biting, hitting, head banging, and skin
picking, is very common. Repetitive self-hugging is a behavioral
trait that may be unique to Smith-Magenis syndrome. Individuals
with this condition also compulsively lick their fingers and flip
pages of books and magazines (a behavior known as "lick and flip").
Other signs and symptoms of Smith-Magenis syndrome include short
stature, abnormal curvature of the spine (scoliosis), reduced
sensitivity to pain and temperature, and a hoarse voice. Some
people with this disorder have ear abnormalities that lead to
hearing loss. Affected individuals may have eye abnormalities that
cause nearsightedness (myopia) and other vision problems. Although
less common, heart and kidney defects also have been reported in
people with Smith-Magenis syndrome. A small percentage of
individuals with Smith-Magenis syndrome have a mutation in the RAIL
gene instead of a chromosomal deletion. Although these individuals
have many of the major features of the condition, they are less
likely than people with a chromosomal deletion to have short
stature, hearing loss, and heart or kidney abnormalities.
PEX1
[1116] The PEX1 gene encodes peroxisomal biogenesis factor 1
(Pex1p), which is part of a group of proteins called peroxins.
Peroxins are essential for the formation and normal functioning of
cell structures called peroxisomes. Peroxisomes are sac-like
compartments that contain enzymes needed to break down many
different substances, including fatty acids and certain toxic
compounds. They are also important for the production of lipids
used in digestion and in the nervous system. Peroxins assist in the
biogenesis of peroxisomes by producing the membrane that separates
the peroxisome from the rest of the cell and by importing enzymes
into the peroxisome. Pex1p enables other peroxins to bring enzymes
into the peroxisome.
Peroxisome Biogenesis Disorder 1a (Zellweger Syndrome)(PBD1A)
[1117] Zellweger syndrome (PBD1A) is caused by homozygous or
compound heterozygous mutation in the PEX1 gene on chromosome
7q21-q22. Zellweger syndrome is an autosomal recessive systemic
disorder characterized clinically by severe neurologic dysfunction,
craniofacial abnormalities, and liver dysfunction, and
biochemically by the absence of peroxisomes. Most severely affected
individuals with classic Zellweger syndrome phenotype die within
the first year of life.
[1118] At least 114 mutations in the PEX1 gene have been identified
in people with Zellweger spectrum disorder. The conditions'
features, which vary in severity, can include weak muscle tone
(hypotonia), developmental delay, and vision and hearing problems.
Mutations in the PEX1 gene are the most common cause of Zellweger
spectrum disorder and are found in nearly 70 percent of affected
individuals. There are two common PEX1 gene mutations found in
people with Zellweger spectrum disorder. One mutation replaces the
amino acid glycine with the amino acid aspartic acid at position
843 in Pex1p (written as Gly843Asp or G843D). This mutation leads
to reduced levels of the protein. Individuals who have the G843D
mutation tend to have signs and symptoms that are at the
less-severe end of the condition spectrum. The other common
mutation, which is known as the 1700fs mutation, leads to the
production of an abnormally short, nonfunctional Pex1p. Individuals
who have the 1700fs mutation often have signs and symptoms that are
at the severe end of the condition spectrum. Mutations in the PEX1
gene that cause Zellweger spectrum disorder reduce or eliminate the
activity of the Pex1p protein. Without enough functional Pex1p,
enzymes are not properly imported into peroxisomes. As a result,
cells contain empty peroxisomes that cannot carry out their usual
functions. The severe end of the condition spectrum is caused by
the absence of functional peroxisomes within cells. The less severe
end of the condition spectrum results from mutations that allow
some peroxisomes to form.
Heimler Syndrome-1 (HMLR1)
[1119] Heimler syndrome-1 (HMLR1) is caused by homozygous or
compound heterozygous mutations in the PEX1 gene on chromosome
7q21. Heimler syndrome-1 (HMLR1), represents the mildest end of the
peroxisomal biogenesis disorder spectrum. Heimler syndrome-1 is a
rare autosomal recessive disorder characterized by sensorineural
hearing loss, enamel hypoplasia of the secondary dentition, and
nail abnormalities.
ARSA
[1120] The ARSA encodes enzyme arylsulfatase A. This enzyme is
localized in lysosomes. Arylsulfatase A aids the processing of
sulfatides. For example, Arylsulfatase A hydrolyzes cerebroside
sulfate to cerebroside and sulfate. Sulfatides are a subgroup of
sphingolipids, a category of fats that are important components of
cell membranes. Sulfatides are abundant in the nervous system's
white matter, consisting of nerve fibers covered by myelin.
Metachromatic Leukodystrophy
[1121] Metachromatic leukodystrophy is caused by mutation in the
arylsulfatase A gene (ARSA) on chromosome 22q13. Metachromatic
leukodystrophy is an inherited disorder characterized by the
accumulation of sulfatides in cells. This accumulation especially
affects cells in the nervous system that produce myelin. Sulfatide
accumulation in myelin-producing cells causes progressive
destruction of white matter (leukodystrophy) throughout the nervous
system, including in the central nervous system and the nerves
connecting the brain and spinal cord to muscles and sensory cells
that detect sensations such as touch, pain, heat, and sound (the
peripheral nervous system). In individuals with metachromatic
leukodystrophy, white matter damage causes progressive
deterioration of intellectual functions and motor skills, such as
the ability to walk. Affected individuals also develop loss of
sensation in the extremities (peripheral neuropathy), incontinence,
seizures, paralysis, an inability to speak, blindness, and hearing
loss. Eventually they lose awareness of their surroundings and
become unresponsive. While neurological problems are the primary
feature of metachromatic leukodystrophy, effects of sulfatide
accumulation on other organs and tissues have been reported, most
often involving the gallbladder.
[1122] The most common form of metachromatic leukodystrophy,
affecting about 50 to 60 percent of all individuals with this
disorder, is called the late infantile form. This form of the
disorder usually appears in the second year of life. Affected
children lose any speech they have developed, become weak, and
develop problems with walking (gait disturbance). As the disorder
worsens, muscle tone generally first decreases, and then increases
to the point of rigidity. Individuals with the late infantile form
of metachromatic leukodystrophy typically do not survive past
childhood. In 20 to 30 percent of individuals with metachromatic
leukodystrophy, onset occurs between the age of 4 and adolescence.
In this juvenile form, the first signs of the disorder may be
behavioral problems and increasing difficulty with schoolwork.
Progression of the disorder is slower than in the late infantile
form, and affected individuals may survive for about 20 years after
diagnosis.
[1123] The adult form of metachromatic leukodystrophy affects
approximately 15 to 20 percent of individuals with the disorder. In
this form, the first symptoms appear during the teenage years or
later. Often behavioral problems such as alcoholism, drug abuse, or
difficulties at school or work are the first symptoms to appear.
The affected individual may experience psychiatric symptoms such as
delusions or hallucinations. People with the adult form of
metachromatic leukodystrophy may survive for 20 to 30 years after
diagnosis. During this time there may be some periods of relative
stability and other periods of more rapid decline.
EIF2b5/EIF2B1/EIF2B2
[1124] The EIF2B5, EIF2B1 and EIF2B2 genes encodes a subunit of
eukaryotic translation initiation factor 2B (eIF2B), a
heteropentameric guanine nucleotide exchange factor necessary for
the proper function of the translation initiation factor eIF2, and
an essential regulator for protein synthesis. eIF2B catalyzes the
exchange of GDP for GTP. Under some conditions, eIF2B increases
protein synthesis under other conditions, eLF2B slows protein
synthesis. Proper regulation of protein synthesis is vital for
ensuring that the correct levels of protein are available for the
cell to cope with changing conditions. Mutations in eIF2B can cause
partial loss of eIF2B function. Impairment of eIF2B function makes
it more difficult for cells to regulate protein synthesis and deal
with changing conditions and stress.
Leukoencephalopathy with Vanishing White Matter (VWM)
[1125] Leukoencephalopathy with vanishing white matter (VWM) can be
caused by homozygous or compound heterozygous mutation in any of
the 5 genes encoding subunits of the translation initiation factor
EIF2B: EIF2B1 on chromosome 12q24, EIF2B2 on chromosome 14q24,
EIF2B3 on chromosome 1p34, EIF2B4 on chromosome 2p23, or EIF2B5 on
chromosome 3q27. Vanishing white matter leukodystrophy is an
autosomal recessive neurologic disorder characterized by variable
neurologic features, including progressive cerebellar ataxia,
spasticity, and cognitive impairment associated with white matter
lesions on brain imaging. The age at onset can range from early
infancy to adulthood. Rapid neurologic deterioration can occur
following minor head trauma. Female mutation carriers may develop
ovarian failure, manifest as primary amenorrhea or as secondary
amenorrhea lasting more than 6 months, associated with elevated
gonadotropin levels at age less than 40 years.
[1126] Neurologic deterioration usually begins in late infancy or
early childhood, but juvenile- and adult-onset cases have been
described. Mild and severe cases have been observed, even within
families. The neurologic signs include progressive cerebellar
ataxia, spasticity, inconstant optic atrophy, and relatively
preserved mental abilities. Disease is chronic-progressive with, in
most individuals, additional episodes of rapid deterioration
following febrile infections or minor head trauma. Head trauma
leads only to motor deterioration, whereas infections with fever
may end in coma. Death occurs after a variable period of several
years to a few decades, usually following an episode of fever and
coma. MRI is diagnostic and shows a diffuse abnormality of the
cerebral white matter beginning in the presymptomatic stage. Both
MRI and magnetic resonance spectroscopy indicate that, with time,
an increasing amount of the abnormal white matter vanishes and is
replaced by cerebrospinal fluid. The mode of inheritance is
autosomal recessive.
NPC1
[1127] NPC1 encodes Niemann-Pick disease, type C1 (NPC1), a large
protein that resides in the limiting membrane of endosomes and
lysosomes and mediates intracellular cholesterol trafficking via
binding of cholesterol to its N-terminal domain. It is predicted to
have a cytoplasmic C-terminus, 13 transmembrane domains, and 3
large loops in the lumen of the endosome--the last loop being at
the N-terminus. This protein transports low-density lipoproteins to
late endosomal/lysosomal compartments where they are hydrolized and
released as free cholesterol.
[1128] Mutations in NPC1 leads to a shortage of NPC1, which
prevents movement of cholesterol and other lipids, leading to their
accumulation in cells. Because these lipids are not in their proper
location in cells, many normal cell functions that require lipids
(such as cell membrane formation) are impaired. The accumulation of
lipids as well as the cell dysfunction eventually leads to cell
death, causing the tissue and organ damage seen in Niemann-Pick
disease types C1.
Niemann-Pick Disease Type C1 and Niemann-Pick Disease Type D
[1129] Niemann-Pick disease type C1 and Niemann-Pick disease type
D, also known as the Nova Scotian type, are caused by mutation in
the NPC1 gene. Niemann-Pick type C (NPC) disease is an autosomal
recessive lipid storage disorder characterized by progressive
neurodegeneration. Approximately 95% of cases are caused by
mutations in the NPC1 gene, referred to as type C1; 5% are caused
by mutations in the NPC2 gene, referred to as type C2. The clinical
manifestations of types C1 and C2 are similar because the
respective genes are both involved in egress of lipids,
particularly cholesterol, from late endosomes or lysosomes.
Niemann-Pick disease types C1 and C2 usually become apparent in
childhood, although signs and symptoms can develop at any time.
People with these types usually develop difficulty coordinating
movements (ataxia), an inability to move the eyes vertically
(vertical supranuclear gaze palsy), poor muscle tone (dystonia),
severe liver disease, and interstitial lung disease. Individuals
with Niemann-Pick disease types C1 and C2 have problems with speech
and swallowing that worsen over time, eventually interfering with
feeding. Affected individuals often experience progressive decline
in intellectual function and about one-third have seizures.
ADAR
[1130] ADAR (adenosine deaminase acting on RNA) encodes
Double-stranded RNA-specific adenosine deaminase. ADAR catalyzes
the deamination of adenosine to inosine in dsRNA substrates,
induces translation within the nucleus, possibly at the surface of
the nucleolus. ADAR catalyzes the hydrolytic deamination of
adenosine to inosine in double-stranded RNA (dsRNA) referred to as
A-to-I RNA editing. This may affect gene expression and function in
a number of ways that include mRNA translation by changing codons
and hence the amino acid sequence of proteins; pre-mRNA splicing by
altering splice site recognition sequences; RNA stability by
changing sequences involved in nuclease recognition; genetic
stability in the case of RNA virus genomes by changing sequences
during viral RNA replication; and RNA structure-dependent
activities such as microRNA production or targeting or protein-RNA
interactions. ADAR can edit both viral and cellular RNAs and can
edit RNAs at multiple sites (hyper-editing) or at specific sites
(site-specific editing). ADAR cellular RNA substrates include:
bladder cancer-associated protein (BLCAP), neurotransmitter
receptors for glutamate (GRIA2) and serotonin (HTR2C) and GABA
receptor (GABRA3). Site-specific RNA editing of transcripts
encoding these proteins results in amino acid substitutions which
consequently alters their functional activities. Exhibits low-level
editing at the GRIA2 Q/R site, but edits efficiently at the R/G
site and HOTSPOT1. Its viral RNA substrates include: hepatitis C
virus (HCV), vesicular stomatitis virus (VSV), measles virus (MV),
hepatitis delta virus (HDV), and human immunodeficiency virus type
1 (HIV-1). ADAR exhibits either a proviral (HDV, MV, VSV and HIV-1)
or an antiviral effect (HCV) and this can be editing-dependent (HDV
and HCV), editing-independent (VSV and MV) or both (HIV-1). ADAR
impairs HCV replication via RNA editing at multiple sites. ADAR
enhances the replication of MV, VSV and HIV-1 through an
editing-independent mechanism via suppression of EIF2AK2/PKR
activation and function. ADAR stimulates both the release and
infectivity of HIV-1 viral particles by an editing-dependent
mechanism where it associates with viral RNAs and edits adenosines
in the 5'UTR and the Rev and Tat coding sequence. ADAR can enhance
viral replication of HDV via A-to-I editing at a site designated as
amber/W, thereby changing an UAG amber stop codon to an UIG
tryptophan (W) codon that permits synthesis of the large delta
antigen (L-HDAg) which has a key role in the assembly of viral
particles. However, high levels of ADAR1 inhibit HDV
replication.
Aicardi-Goutieres Syndrome-6 (AGS6)
[1131] Aicardi-Goutieres syndrome-6 (AGS6) can be caused by
homozygous, compound heterozygous, or heterozygous mutation in the
ADAR gene on chromosome 1q21.3. Aicardi-Goutieres syndrome (AGS)
manifests as an early-onset encephalopathy that usually, but not
always, results in severe intellectual and physical handicap. A
subgroup of infants with AGS present at birth with abnormal
neurologic findings, hepatosplenomegaly, elevated liver enzymes,
and thrombocytopenia, a picture highly suggestive of congenital
infection. Otherwise, most affected infants present at variable
times after the first few weeks of life, frequently after a period
of apparently normal development. Typically, they demonstrate the
subacute onset of a severe encephalopathy characterized by extreme
irritability, intermittent sterile pyrexias, loss of skills, and
slowing of head growth. Over time, as many as 40% develop chilblain
skin lesions on the fingers, toes, and ears. It is becoming
apparent that atypical, sometimes milder, cases of AGS exist, and
thus the true extent of the phenotype associated with mutation of
the AGS-related genes is not yet known. For example, mutation of
ADAR has recently been associated with a clinical presentation of
acute bilateral striatal necrosis.
MFSD8
[1132] MFSD8 gene, encodes a putative lysosomal transporter, on
chromosome 4q28. The MFSD8 protein is found in lysosomes. The MFSD8
protein belongs to a large group of related proteins called the
major facilitator superfamily of secondary active transporter
proteins. Proteins in this family move certain molecules between
structures in cells or in and out of cells. While it is likely that
the MFSD8 protein transports molecules, the specific molecules it
moves are unknown. The MFSD8 protein probably transports substances
across the membranes of lysosomes.
Neuronal Ceroid Lipofuscinosis-7
[1133] Neuronal ceroid lipofuscinosis-7 (CLN7) is caused by
homozygous or compound heterozygous mutation in the MFSD8 gene,
which encodes a putative lysosomal transporter, on chromosome 4q28.
The neuronal ceroid-lipofuscinoses (NCLs) are a group of inherited,
neurodegenerative, lysosomal storage disorders characterized by
progressive intellectual and motor deterioration, seizures, and
early death. Visual loss is a feature of most forms. Clinical
phenotypes have been characterized traditionally according to the
age of onset and order of appearance of clinical features into
infantile, late-infantile, juvenile, adult, and Northern epilepsy
(also known as progressive epilepsy with mental retardation).
STXBP1
[1134] STXBP1 gene, encodes a syntaxin-binding protein. The encoded
protein can play a role in release of neurotransmitters via
regulation of syntaxin, a transmembrane attachment protein
receptor. Mutations in STXBP1 gene is associated with infantile
epileptic encephalopathy-4. STXBP1 can participate in the
regulation of synaptic vesicle docking and fusion, through
interaction with GTP-binding proteins. STXBP1 can interact with
syntaxins 1, 2, and 3 but not syntaxin 4. STXBP1 can play a role in
determining the specificity of intracellular fusion reactions.
[1135] In humans, STXBP1 is expressed as a 4-kb transcript in
various tissues. The highest levels of expression can be observed
in the retina and cerebellum.
Early Infantile Epileptic Encephalopathy-4
[1136] Epileptic encephalopathy, early infantile, 4 (EIEE4) is a
severe form of epilepsy characterized by frequent tonic seizures or
spasms beginning in infancy with a specific EEG finding of
suppression-burst patterns, characterized by high-voltage bursts
alternating with almost flat suppression phases. Affected
individuals can have neonatal or infantile onset of seizures,
profound mental retardation, and MRI evidence of brain
hypomyelination.
[1137] In some cases, in a patient with early infantile epileptic
encephalopathy-4, a heterozygous 1631G-A transition in the STXBP1
gene, resulting in a gly544-to-asp (G544D) substitution can be
identified. In some cases, a patient with early infantile epileptic
encephalopathy-4 can develop seizures by age 10 days with a
suppression-burst pattern on EEG. In some cases, a patient with
early infantile epileptic encephalopathy-4 can have profound mental
retardation and spastic paraplegia at about age 37 years.
[1138] In some cases, in a patient with early infantile epileptic
encephalopathy-4, a heterozygous 539G-A transition in the STXBP1
gene, resulting in a cys180-to-tyr (C180Y) substitution can be
identified. In some cases, a patient with early infantile epileptic
encephalopathy-4 can have infantile onset of tonic and myoclonic
seizures with suppression-burst pattern and hypsarrhythmia, delayed
brain myelination, and spastic quadriplegia. In embodiments,
mutations in the STXBP1 gene can impair structural stability, lower
thermostability, and decrease binding to several functional
synaptic proteins.
[1139] In some cases, in a patient with early infantile epileptic
encephalopathy-4, a heterozygous 1328T-G transversion in the STXBP1
gene, resulting in a met443-to-arg (M443R) substitution can be
identified. In some cases, a patient with early infantile epileptic
encephalopathy-4 can develop tonic seizures at age 6 weeks and can
later have profoundly delayed development. In some cases, a patient
with early infantile epileptic encephalopathy-4 can have delayed
brain myelination.
[1140] In some cases, in a patient with early infantile epileptic
encephalopathy-4, a heterozygous 251T-A transversion in the STXBP1
gene, resulting in a val84-to-asp (V84D) substitution can be
identified. In some cases, a patient with early infantile epileptic
encephalopathy-4 can develop tonic seizures at age 2 months with
suppression-burst pattern and hypsarrhythmia, and can later show
profound mental retardation.
[1141] In some cases, in a patient with Early infantile epileptic
encephalopathy-4, a de novo heterozygous 1162C-T transition in exon
14 of the STXBP1 gene, resulting in an arg388-to-ter (R388X)
substitution, predicted to truncate the domain-3 region, which
together with domain-1 provides a binding surface for syntaxin-1
can be identified. In some cases, a patient with early infantile
epileptic encephalopathy-4 can have severe mental retardation, with
hypotonia, abnormal gait, tremor, and/or seizures.
[1142] In some cases, in a patient with early infantile epileptic
encephalopathy-4, a de novo heterozygous G-to-A transition in
intron 3 of the STXBP1 gene, resulting in the creation of a stop
codon downstream of exon 3 can be identified. In some cases, a
patient with early infantile epileptic encephalopathy-4 can have
severe mental retardation, with hypotonia, abnormal gait, tremor,
and/or seizures.
[1143] In some cases, in a patient with Early infantile epileptic
encephalopathy-4, a de novo heterozygous 847G-A transition in the
STXBP1 gene, resulting in a glu283-to-lys (E283K) substitution at a
highly conserved residue can be identified.
PRICKLE2
[1144] The PRICKLE2 gene encodes a postsynaptic protein involved in
neuronal architecture and function, prickle planar cell polarity
protein 2. Mutations in this gene are associated with progressive
myoclonic epilepsy type 5.
Progressive Myoclonic Epilepsy 5
[1145] Epilepsy, progressive myoclonic 5 (EPM5) is a
neurodegenerative disorder characterized by myoclonic seizures and
variable neurologic symptoms including cognitive decline and
persistent movement abnormalities.
[1146] In some cases, a heterozygosity for a complex mutation in
the PRICKLE2 gene, a 443G-A transition, resulting in an
arg148-to-his (R148H) substitution, and a 457G-A transition,
resulting in a val153-to-ile (V153I) substitution can be identified
in a progressive myoclonic epilepsy patient. In some cases, a
heterozygous 1813G-T transversion in the PRICKLE2 gene, resulting
in a val605-to-phe (V605F) substitution can be identified in a
progressive myoclonic epilepsy patient.
PRRT2
[1147] The PRRT2 gene provides instructions for making the
proline-rich transmembrane protein 2 (PRRT2). The function of this
protein is thought to be involved in signaling in the brain.
Studies show that PRRT2 protein interacts with SNAP25, which is
involved in signaling between nerve cells in the brain. SNAP25
helps control the release of neurotransmitters.
Familial Infantile Convulsion with Paroxysmal Choreoathetosis
[1148] Infantile Convulsions and paroxysmal ChoreoAthetosis (ICCA)
syndrome is a neurological condition characterized by the
occurrence of seizures during the first year of life (Benign
familial infantile epilepsy) and choreoathetotic dyskinetic attacks
during childhood or adolescence. Benign familial infantile epilepsy
can begin at 3 to 12 months of age with a family history of the
same type of seizures. Seizures are afebrile, partial or sometimes
generalized, and normally disappear after the first year of life.
During childhood or adolescence, affected individuals present with
paroxysmal kinesigenic dyskinesia with frequent and recurrent
episodic choreoathetotic or dystonic movements that last less than
1 minute. The attacks can be triggered by the initiation of
voluntary movements or startle. The association with other
paroxysmal disorders such as migraine, with or without aura,
hemiplegic migraine, episodic ataxia and tics has also been
described. The genetic loci of ICCA syndrome have been described on
chromosomes 16p11.2-q12.1, 16q13-q22.1 and 3q29-29. Mutations in
the Proline-rich transmembrane protein 2 (PRRT2) gene, located on
16p11.2, have recently been found in families affected by ICCA
syndrome. This gene encodes a membrane protein that interacts with
the presynaptic protein SNAP-25 but the mechanism leading to the
disease remains unknown. The diagnosis is mainly clinical, based on
the appearance of infantile convulsions with benign evolution
followed by kinesigenic dyskinesia attacks later on. Genetic
testing can confirm the diagnosis. Differential diagnosis can
include other paroxysmal dystonias such as paroxysmal
exertion-induced dyskinesia and paroxysmal non-kinesigenic
dyskinesia triggered by drugs or food intake (such as caffeine and
alcohol). ICCA syndrome can present as sporadic or familial; in the
latter case, it is transmitted as an autosomal dominant trait that
can be variably expressed within the same family. Antiepileptic
drugs, mainly phenytoin or carbamazepine, can be effective in
controlling seizures and dyskinesia during the active phase of the
disorder.
[1149] In some cases, in patients familial infantile convulsions
with paroxysmal choreoathetosis, a heterozygous 950G-A transition
in the PRRT2 gene, resulting in a ser317-to-asn (S317N)
substitution in a highly conserved residue can be identified.
[1150] In some cases, in patients with familial infantile
convulsions with paroxysmal choreoathetosis, a heterozygous C-to-T
transition in the PRRT2 gene, resulting in an arg240-to-ter (R240X)
substitution can be identified. In some cases, in patients with
familial infantile convulsions with paroxysmal choreoathetosis, a
heterozygous 1-bp insertion (516insT) in the PRRT2 gene, resulting
in premature termination at residue 173 can be identified. In some
cases, in patients with familial infantile convulsions with
paroxysmal choreoathetosis, a heterozygous 562C-T transition in the
PRRT2 gene, resulting in a gln188-to-ter (Q188X) substitution can
be identified.
Episodic Kinesigenic Dyskinesia 1
[1151] Episodic kinesigenic dyskinesia 1 can be referred to as
familial paroxysmal kinesigenic dyskinesia. Familial paroxysmal
kinesigenic dyskinesia is a disorder characterized by episodes of
abnormal movement that range from mild to severe. In the condition
name, paroxysmal indicates that the abnormal movements come and go
over time, kinesigenic indicates that episodes are triggered by
movement, and dyskinesia refers to involuntary movement of the
body.
[1152] People with familial paroxysmal kinesigenic dyskinesia
experience episodes of irregular jerking or shaking movements that
are induced by sudden motion, such as standing up quickly or being
startled. An episode may involve slow, prolonged muscle
contractions (dystonia); small, fast, "dance-like" motions
(chorea); writhing movements of the limbs (athetosis); or, rarely,
flailing movements of the limbs (ballismus). Familial paroxysmal
kinesigenic dyskinesia may affect one or both sides of the body.
The type of abnormal movement varies among affected individuals,
even among members of the same family. In many people with familial
paroxysmal kinesigenic dyskinesia, a pattern of symptoms called an
aura immediately precedes the episode. The aura is often described
as a crawling or tingling sensation in the affected body part.
Individuals with this condition do not lose consciousness during an
episode and do not experience any symptoms between episodes.
[1153] Individuals with familial paroxysmal kinesigenic dyskinesia
usually begin to show signs and symptoms of the disorder during
childhood or adolescence. Episodes typically last less than five
minutes, and the frequency of episodes ranges from one per month to
100 per day. In most affected individuals, episodes occur less
often with age.
[1154] In some people with familial paroxysmal kinesigenic
dyskinesia the disorder begins in infancy with recurring seizures
called benign infantile convulsions. These seizures usually develop
in the first year of life and stop by age 3. When benign infantile
convulsions are associated with familial paroxysmal kinesigenic
dyskinesia, the condition is known as infantile convulsions and
choreoathetosis (ICCA). In families with ICCA, some individuals
develop only benign infantile convulsions, some have only familial
paroxysmal kinesigenic dyskinesia, and others develop both.
[1155] In some cases, in a patient with episodic kinesigenic
dyskinesia 1, a heterozygous 1-bp duplication (649dupC) in exon 2
of the PRRT2 gene in the proline-rich domain, resulting in a
frameshift and introduction of a stop codon 7 amino acids
downstream of the insertion (Arg217ProfsTer8) can be identified. In
some cases, in a patient with episodic kinesigenic dyskinesia 1, a
heterozygous 4-bp deletion (514delTCTG) in exon 2 of the PRRT2 gene
in the proline-rich domain, resulting in a frameshift and premature
termination can be identified. In some cases, in a patient with
episodic kinesigenic dyskinesia 1, a heterozygous 1-bp deletion
(972delA) in exon 3 of the PRRT2 gene, resulting in a frameshift
and premature termination in the second transmembrane motif can be
identified. In some cases, in a patient with episodic kinesigenic
dyskinesia 1, identified a heterozygous 487C-T transition in exon 2
of the PRRT2 gene can be identified. In some cases, in a patient
with episodic kinesigenic dyskinesia 1, a heterozygous 796C-T
transition in exon 2 of the PRRT2 gene, resulting in an
arg266-to-trp (R266W) substitution in a highly conserved residue
can be identified. In some cases, in a patient with Episodic
kinesigenic dyskinesia 1, a heterozygous 1-bp deletion (649delC) in
the PRRT2 gene, resulting in a frameshift and premature termination
(Arg217GlufsTer12) can be identified. In some cases, in a patient
with Episodic kinesigenic dyskinesia 1, a heterozygous 748C-T
transition in the PRRT2 gene, resulting in a gln250-to-ter (Q250X)
substitution in the N-terminal extracellular domain can be
identified.
Benign Familial Infantile Seizuers-2
[1156] Benign familial infantile epilepsy (BFIE) is a genetic
epileptic syndrome characterized by the occurrence of afebrile
repeated seizures in healthy infants, between the third and eighth
month of life. Although BFIE cases have been reported worldwide,
prevalence and incidence remain unknown. In an Argentinian case
series, BFIE have been listed as the third most common type of
epilepsy in the first two years of life. Seizures usually occur
between 3 to 8 months of life, with clusters (8-10 a day) of
repeated and brief episodes (2-5 minutes) over a few days. They are
usually focal but can sometimes become generalized. Patients
present with motor arrest, unresponsiveness, head and/or eye
deviation to one side, staring, fluttering of eyelids, grunting,
cyanosis, diffuse hypertonia and unilateral or bilateral clonic
jerks of the limbs. During the interictal period, patients regain
full consciousness and activity. Psychomotor development is normal.
A family history of the same epilepsy is a constant finding. A
syndrome called familial infantile convulsions and choreoathetosis
has been observed in which BFIE patients present in childhood
and/or adolescence with choreoathetotic dyskinetic attacks
occurring spontaneously or following diverse stimuli (e.g.
exercise, stress). In some rare cases, BFIE has been associated
with familial or sporadic hemiplegic migraine.
[1157] BFIE is a genetically heterogeneous disease. In the majority
of cases, mutations in the proline-rich transmembrane protein 2
(PRRT2) gene located at 16p11.2 have been found. This gene encodes
a membrane protein that interacts with the presynaptic protein
SNAP-25. Mutations have also been found in the SCN2A gene (2q24.3)
encoding the brain sodium channel NaV1.2 and rarely in the KCNQ2
(20q13.33) and KCNQ3 (8q24) genes both encoding potassium channels.
Additionally, three other chromosomal loci have been identified
that are mapped to chromosome 19q, 16p and 1p.
[1158] Family history can orient the diagnosis which can be based
on electroencephalography (EEG) and video recordings. EEG can show
that partial seizures originate from the parietal-occipital region
and that the side of the hemisphere involved can vary between
episodes. Seizures can sometimes spread and involve the entire
brain. During a cluster of seizures, postictal EEG shows
lateralized occipito-parietal delta waves and spikes. Outside the
cluster, waking and sleeping interictal EEG is normal. Interictal
neurological examination and brain imaging (brain CT and/or MRI)
are normal. Genetic testing can confirm the diagnosis.
[1159] Differential diagnosis can include benign familial
neonatal-infantile seizures, an epileptic syndrome with an
intermediate onset between the neonatal and infantile age that
shares overlapping clinical characteristics with BFIE and that is
mainly due to mutations in the SCN2A gene. Other differential
diagnoses are benign non-familial infantile seizures, benign
infantile seizures associated with mild gastroenteritis and benign
infantile focal epilepsy with midline spikes and waves during sleep
(BIMSE)
[1160] BFIE is transmitted as an autosomal dominant trait with
incomplete penetrance. With anti-epileptic treatment (e.g.
carbamazepine, valproate, phenobarbital), symptoms quickly
disappear and no other type of epilepsy has been reported to
reappear.
[1161] In some cases, in patients with benign familial infantile
seizuers-2, a heterozygous 1-bp deletion (629delC) in the PRRT2
gene, resulting in a frameshift and premature termination
(Pro210GlnfsTer19) can be identified. In some cases, in patients
with benign familial infantile seizuers-2, a heterozygous 1-bp
deletion (291delC) in the PRRT2 gene, resulting in a frameshift and
premature termination (Asn98ThrfsTer17) can be identified. In some
cases, in patients with Benign familial infantile seizuers-2, a
heterozygous 1-bp deletion (c.650delG) in the PRRT2 gene, resulting
in a frameshift and premature termination (Arg217GlnfsTer12) can be
identified.
STX1B
[1162] The protein encoded by this gene belongs to a family of
proteins thought to play a role in the exocytosis of synaptic
vesicles. Vesicle exocytosis releases vesicular contents and is
important to various cellular functions. For instance, the
secretion of transmitters from neurons plays an important role in
synaptic transmission. After exocytosis, the membrane and proteins
from the vesicle are retrieved from the plasma membrane through the
process of endocytosis. Mutations in this gene have been identified
as one cause of fever-associated epilepsy syndromes. A possible
link between this gene and Parkinson's disease has also been
suggested.
[1163] Syntaxins are cellular receptors for transport vesicles. One
of these proteins, designated syntaxin 1B (STX1B), is directly
implicated in the process of calcium-dependent synaptic
transmission in rat brain. The expression of this protein is
transiently induced by long-term potentiation of synaptic responses
in the rat hippocampus. The protein may play an important role in
the excitatory pathway of synaptic transmission, which is known to
be implicated in several neurologic diseases.
Generalized Epilepsy with Febrile Seizure Plus Type 9
[1164] Generalized epilepsy with febrile seizures plus-9 is an
autosomal dominant neurologic disorder characterized by onset of
febrile and/or afebrile seizures in early childhood, usually before
age 3 years. Seizure types are variable and include generalized
tonic-clonic, atonic, myoclonic, complex partial, and absence. Most
patients have remission of seizures later in childhood with no
residual neurologic deficits, but rare patients may show mild
developmental delay or mild intellectual disabilities.
[1165] In some cases, in a patient with generalized epilepsy with
febrile seizure plus type 9, a heterozygous c.166C-T transition in
the STX1B gene, resulting in a gln56-to-ter (Q56X) substitution can
be identified.
[1166] In some cases, in a patient with generalized epilepsy with
febrile seizure plus type 9, a heterozygous complex
insertion/deletion mutation in the STX1B gene
c.133_134insGGATGTGCATTG, resulting in Lys45delinsArgMetCysIleGlu,
and c.135_136AC-GA, resulting in a leu46-to-met (L46M)
substitution) can be identified. In some cases, in a patient with
generalized epilepsy with febrile seizure plus type 9, a
heterozygous c.140C-A transversion in the STX1B gene, resulting in
a ser47-to-ter (547X) substitution can be identified In some cases,
in a patient with generalized epilepsy with febrile seizure plus
type 9, identified a heterozygous c.657T-A transversion in the
STX1B gene, resulting in a val216-to-glu (V216E) substitution at a
highly conserved residue in the SNARE motif can be identified. In
some cases, in a patient with generalized epilepsy with febrile
seizure plus type 9, a de novo heterozygous c.676G-C transversion
in the STX1B gene, resulting in a gly226-to-arg (G226R)
substitution at a highly conserved residue in the SNARE motif can
be identified.
IDUA
[1167] The IDUA gene provides instructions for producing the enzyme
alpha-L-iduronidase, which is essential for the breakdown of large
sugar molecules, for example glycosaminoglycans (GAGs). Through
hydrolysis, alpha-L-iduronidase uses water molecules to break down
unsulfated alpha-L-iduronic acid, which is present in heparan
sulfate and dermatan sulfate. Alpha-L-iduronidase can be located in
lysosomes. More than 100 mutations in the IDUA gene have been found
to cause mucopolysaccharidosis type I (MPS I). Many mutations that
cause MPS I reduce or completely eliminate the function of
alpha-L-iduronidase. It usually cannot be determined whether a
certain mutation will cause severe or attenuated MPS I; however,
people who do not produce alpha-L-iduronidase have the severe form
of this disorder.
[1168] The lack of alpha-L-iduronidase enzyme activity leads to the
accumulation of heparan sulfate and dermatan sulfate within the
lysosomes. The buildup of GAGs increases the size of the lysosomes.
The accumulated GAGs may also interfere with the functions of other
proteins inside the lysosomes and disrupt the movement of molecules
inside the cell.
Hurler-Scheie Syndrome
[1169] Hurler-Scheie syndrome is the intermediate form of
mucopolysaccharidosis type 1 (MPS1) between the two extremes Hurler
syndrome and Scheie syndrome, it is a rare lysosomal storage
disease, characterized by skeletal deformities and a delay in motor
development. The prevalence of MPS I has been estimated at
1/100,000, with Hurler-Scheie syndrome accounting for 23% of cases
or a prevalence of approximately 1/435,000. Patients with
Hurler-Scheie syndrome have normal or almost normal intelligence
but exhibit various degrees of physical impairment. Patients
present in the first years of life with musculoskeletal alterations
to different degrees including short stature, multiple dysostosis,
thoracic-lumbar kyphosis, progressive coarsening of the facial
features to different degrees, cardiomyopathy and valvular
abnormalities, neurosensorial hearing loss, enlarged tonsils and
adenoids, and nasal secretion. Hydrocephaly can occur after the age
of two. Corneal opacity is seen between two and four years of age
and requires keratoplasty to restore sight. Other manifestations
may include organomegaly, hernias and hirsutism.
[1170] Hurler-Scheie syndrome is caused by mutations in the IDUA
gene (4p16.3) leading to partial deficiency in the
alpha-L-iduronidase enzyme and lysosomal accumulation of dermatan
sulfate and heparan sulfate. Early diagnosis is difficult because
the first clinical signs are not specific. Diagnosis can be based
on detection of increased urinary secretion of heparan and dermatan
sulfate through 1,9-dimethylmethylene blue (DMB) test and
glycosaminoglycan (GAG) electrophoresis, and demonstration of
enzymatic deficiency in leukocytes or fibroblasts. Genetic testing
is available. Differential diagnoses can include the milder and
more severe forms of mucopolysaccharidosis type 1 (Scheie syndrome
and Hurler syndrome respectively), mucopolysaccharidosis typeVI and
mucopolysaccharidosis type II. Antenatal diagnosis is possible by
measurement of enzymatic activity in cultivated chorionic villus or
amniocytes and by genetic testing if the disease-causing mutation
is known. Transmission is autosomal recessive. Bone marrow or
umbilical cord blood transplant has been successful and can
preserve neurocognition, improve some aspects of the somatic
disease and increase survival. However, it is associated with many
risks and most of the positive effects occur only if the procedure
is performed in the first two years of life.
[1171] The enzyme substitute (laronidase) obtained EU marketing
authorization as an orphan drug in 2003. Given through weekly
infusions it leads to improvement of lung function and joint
mobility. Enzyme replacement therapy (ERT) can be started at
diagnosis and may be beneficial in patients awaiting hematopoietic
stem cell transplantation (HSCT). Early treatment can slow the
progression of the disease. In individual patients with MPS1 of
intermediate severity, HSCT may be considered if there is a
suitable donor. There are however no data on the efficacy of HSCT
in patients with this form of the disease.
[1172] Life expectancy for Hurler-Scheie syndrome may be reduced,
with death occurring before adolescence due to serious
cardiovascular and respiratory complications.
[1173] In some cases, in a patient with hurler scheie syndrome,
homozygosity for an arg619-to-gly (R619G) mutation due to a C-to-G
transversion at nucleotide 1943 can be identified. In some cases,
in a patient with hurler scheie syndrome, homozygosity for a
thr364-to-met (T364M) mutation in the IDUA gene can be
identified.
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[1174] In embodiments, the methods of the present invention can
exploit the presence of retained-intron-containing pre-mRNA (RIC
pre-mRNA) transcribed from the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B gene and encoding
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein, in the cell nucleus. Splicing of ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B RIC pre-mRNA species to produce mature, fully-spliced,
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B mRNA, can be induced using ASOs that stimulate
splicing out of the retained introns. The resulting mature ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B mRNA can be exported to the cytoplasm and translated,
thereby increasing the amount of ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein in the
patient's cells and alleviating symptoms of the CNS disease or
conditions caused by deficiency in ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B. This method,
described further below, is known as Targeted Augmentation of
Nuclear Gene Output (TANGO).
[1175] In some cases, the methods of the present invention can
exploit the presence of retained-intron-containing pre-mRNA (RIC
pre-mRNA) transcribed from the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B gene and encoding ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
protein, in the cell nucleus. Splicing of ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B RIC pre-mRNA species
to produce mature, fully-spliced, ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B mRNA, can be induced using
ASOs that stimulate splicing out of the retained introns. The
resulting mature ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B mRNA can be exported to the cytoplasm and
translated, thereby increasing the amount of ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein in
the patient's cells and alleviating symptoms of the CNS disease or
conditions caused by deficiency in ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B. This method, described
further below, is known as Targeted Augmentation of Nuclear Gene
Output (TANGO).
[1176] In some instances, the methods of the present invention can
exploit the presence of retained-intron-containing pre-mRNA (RIC
pre-mRNA) transcribed from the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B gene and encoding ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein, in the cell
nucleus. Splicing of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B RIC pre-mRNA species to produce mature,
fully-spliced, ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B mRNA, can be induced using ASOs that stimulate
splicing out of the retained introns. The resulting mature ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B mRNA can be
exported to the cytoplasm and translated, thereby increasing the
amount of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or
STX1B protein in the patient's cells and alleviating symptoms of
the CNS disease or conditions caused by deficiency in ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B. This method,
described further below, is known as Targeted Augmentation of
Nuclear Gene Output (TANGO).
Nuclear Transcripts
[1177] ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B gene can be analyzed for
intron-retention events. In some cases, ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B gene can be analyzed
for intron-retention events. In some cases, ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B gene can be analyzed for
intron-retention events. RNA sequencing (RNAseq), can be visualized
in the UCSC genome browser, and can show ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, MFSD8, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B transcripts
expressed in human cortical neurons (HCN) or (AST) and localized in
either the cytoplasmic or nuclear fraction. The retained-intron
containing pre-mRNA transcripts are retained in the nucleus and are
not exported out to the cytoplasm.
[1178] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 65%, about 5% to about 60%, about 5%
to about 55%, about 5% to about 50%, about 5% to about 45%, about
5% to about 40%, about 5% to about 35%, about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 10% to about 100%, about 10% to about 95%, about 10% to
about 90%, about 10% to about 85%, about 10% to about 80%, about
10% to about 75%, about 10% to about 70%, about 10% to about 65%,
about 10% to about 60%, about 10% to about 65%, about 10% to about
60%, about 10% to about 55%, about 10% to about 50%, about 10% to
about 45%, about 10% to about 40%, about 10% to about 35%, about
10% to about 30%, about 10% to about 25%, about 10% to about 20%,
about 15% to about 100%, about 15% to about 95%, about 15% to about
90%, about 15% to about 85%, about 15% to about 80%, about 15% to
about 75%, about 15% to about 70%, about 15% to about 65%, about
15% to about 60%, about 15% to about 65%, about 15% to about 60%,
about 15% to about 55%, about 15% to about 50%, about 15% to about
45%, about 15% to about 40%, about 15% to about 35%, about 15% to
about 30%, about 15% to about 25%, about 20% to about 100%, about
20% to about 95%, about 20% to about 90%, about 20% to about 85%,
about 20% to about 80%, about 20% to about 75%, about 20% to about
70%, about 20% to about 65%, about 20% to about 60%, about 20% to
about 65%, about 20% to about 60%, about 20% to about 55%, about
20% to about 50%, about 20% to about 45%, about 20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about
100%, about 25% to about 95%, about 25% to about 90%, about 25% to
about 85%, about 25% to about 80%, about 25% to about 75%, about
25% to about 70%, about 25% to about 65%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 60%, about 25% to about
55%, about 25% to about 50%, about 25% to about 45%, about 25% to
about 40%, or about 25% to about 35%, retention. In embodiments,
other ASOs useful for this purpose are identified, using, e.g.,
methods described herein.
[1179] Table 7 provides a non-limiting list of target sequences of
RIC pre-mRNA transcript of ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, and STX1B by sequence ID, and ASOs by
sequence ID, useful for increasing production of a protein by
targeting a region of a RIC pre-mRNA of ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B. In embodiments,
other ASOs useful for these purposes are identified, using, e.g.,
methods described herein.
TABLE-US-00003 TABLE 7 List of targets and ASOs that targeting a
gene disclosed herein Target Gene Sequence SEQ ID Pre-mRNA Retained
SEQ ID NO: SEQ ID NO: ASOs Intron NO: ADAR ADAR:NM_001193495 SEQ ID
NOs: 6354- 2 30656 SEQ ID SEQ ID NO: 6293 6872 NO: 6274
ADAR:NM_015841 SEQ ID NOs: 6873- 2 30656 SEQ ID NO: 6294 7391
ADAR:NM_015840 SEQ ID NOs: 7392- 2 30656 SEQ ID NO: 6295 7910
ADAR:NM_001111 SEQ ID NOs: 7911- 2 30656 SEQ ID NO: 6296 8429
ADAR:NM_001025107 SEQ ID NOs: 8430- 2 30656 SEQ ID NO: 6297 8948
ATP1A2 ATP1A2:NM_000702 SEQ ID NOs: 8949- 22 30632 SEQ ID SEQ ID
NO: 6298 9575 NO: 6275 PRICKLE2 PRICKLE2:NM_198859 SEQ ID NOs:
9576- 4 30645 SEQ ID SEQ ID NO: 6299 9825 NO: 6276 SETD5
SETD5:NM_001080517 SEQ ID NOs: 9826- 4 30659 SEQ ID SEQ ID NO: 6300
9946 NO: 6277 SETD5:NM_001292043 SEQ ID NOs: 9947- 5 30659 SEQ ID
NO: 6301 10067 EIF2B5 EIF2B5:NM_003907 SEQ ID NOs: 10068- 12 30649
SEQ ID SEQ ID NO: 6302 10159 NO: 6278 SEQ ID NOs: 10160- 13 30637
10305 PEX1 PEX1:NM_000466 SEQ ID NOs: 10306- 10 30634 SEQ ID SEQ ID
NO: 6303 10476 NO: 6279 SEQ ID NOs: 10307- 19 30643 10718 SEQ ID
NOs: 10719- 21 30653 10988 SEQ ID NOs: 10989- 14 30629 11098
PEX1:NM_001282677 SEQ ID NOs: 11099- 10 30634 SEQ ID NO: 6304 11269
SEQ ID NOs: 11100- 18 30643 11511 SEQ ID NOs: 11512- 20 30653 11781
SEQ ID NOs: 11782- 13 30629 11891 PEX1:NM_001282678 SEQ ID NOs:
11892- 10 30634 SEQ ID NO: 6305 12062 SEQ ID NOs: 11893- 19 30643
12304 SEQ ID NOs: 12305- 21 30653 12574 SEQ ID NOs: 12306- 14 30629
12684 STXBP1 STXBP 1 :NM_003165 SEQ ID NOs: 12685- 18 30627 SEQ ID
SEQ ID NO: 6306 12918 NO: 6278 SEQ ID NOs: 12919- 19 30624 13469
STXBP1:NM_001032221 SEQ ID NOs: 13470- 18 30633 SEQ ID NO: 6307
14026 EIF2B1 EIF2B1:NM_001414 SEQ ID NOs: 14027- 6 30626 SEQ ID SEQ
ID NO: 6308 14237 NO: 6279 EIF2B2 EIF2B2:NM_014239 SEQ ID NOs:
14238- 1 30631 SEQID SEQ ID NO: 6309 14326 NO: 6280 PRRT2
PRRT2:NM_001256443 SEQ ID NOs: 14327- SEQ ID SEQ ID NO: 6310 14983
1 30642 NO: 6281 PRRT2:NM_145239 SEQ ID NOs: 14984- 1 30638 SEQ ID
NO: 6311 15294 PRRT2:NM_001256442 SEQ ID NOs: 15295- 1 30638 SEQ ID
NO: 6312 15605 STX1B STX1B:NM_052874 SEQ ID NOs: 15606- 6 30658 SEQ
ID SEQ ID NO: 6313 15652 NO: 6282 SEQ ID NOs: 15653- 7 30648 15873
RAI1 RAI1:NM_030665 SEQ ID NOs: 15874- 4 30655 SEQ ID SEQ ID NO:
6314 16076 NO: 6283 TCF4 TCF4:NM_001243236 SEQ ID NOs: 30660- 10
34789 SEQ ID SEQ ID NO: 6315 30916 NO: 6284 TCF4:NM_001243235 SEQ
ID NOs: 30917- 10 34789 SEQ ID NO: 6316 31173 TCF4:NM_001243234 SEQ
ID NOs: 31174- 10 34788 SEQ ID NO: 6317 31432 TCF4:NM_001243233 SEQ
ID NOs: 31433- 13 34789 SEQ ID NO: 6318 31689 TCF4:NM_001243232 SEQ
ID NOs: 31690- 13 34788 SEQ ID NO: 6319 31948 TCF4:NM_001243231 SEQ
ID NOs: 31949- 15 34789 SEQ ID NO: 6320 32205 TCF4:NM_003199 SEQ ID
NOs: 32206- 17 34789 SEQ ID NO: 6321 32462 TCF4:NM_001306207 SEQ ID
NOs: 32463- 16 34789 SEQ ID NO: 6322 32719 TCF4:NM_001306208 SEQ ID
NOs: 32720- 13 34789 SEQ ID NO: 6323 32976 TCF4:NM_001243227 SEQ ID
NOs: 32977- 16 34788 SEQ ID NO: 6324 33235 TCF4:NM_001243228 SEQ ID
NOs: 33236- 17 34788 SEQ ID NO: 6325 33494 TCF4:NM_001243230 SEQ ID
NOs: 33495- 16 34789 SEQ ID NO: 6326 33751 TCF4:NM_001243226 SEQ ID
NOs: 33752- 18 34788 SEQ ID NO: 6327 34010 TCF4:NM_001083962 SEQ ID
NOs: 34011- 17 34788 SEQ ID NO: 6328 34269 TCF4:NM_001330605 SEQ ID
NOs: 34270- 12 34788 SEQ ID NO: 6329 34528 TCF4:NM_001330604 SEQ ID
NOs: 34529- 17 34788 SEQ ID NO: 6330 34787 NPC1 NPC1:NM_000271 SEQ
ID NOs: 19965- 15 30655 SEQ ID SEQ ID NO: 6331 20033 NO: 6285
CACNA1A CACNA1A:NM_000068 SEQ ID NOs: 20034- 36 30657 SEQ ID SEQ ID
NO: 6332 20253 NO: 6286 SEQ ID NOs: 20254- 37 30650 20469
CACNA1A:NM_023035 SEQ ID NOs: 20470- 36 30657 SEQ ID NO: 6333 20689
SEQ ID NOs: 20690- 37 30650 20905 CACNA1A:NM_001174080 SEQ ID NOs:
20906- 36 30657 SEQ ID NO: 6334 21125 SEQ ID NOs: 21126- 37 30650
21341 CACNA1A:NM_001127222 SEQ ID NOs: 21342- 36 30657 SEQ ID NO:
6335 21561 SEQ ID NOs: 21562- 37 30650 21777 CACNA1A:NM_001127221
SEQ ID NOs: 21778- 36 30647 SEQ ID NO: 6336 21983 SEQ ID NOs:
21984- 37 30639 22187 DNMT1 DNMT1:NM_001379 SEQ ID NOs: 22188- 29
30644 SEQ ID SEQ ID NO: 6337 22400 NO: 6287 SEQ ID NOs: 22401- 14
30635 22441 DNMT1:NM_001130823 SEQ ID NOs: 22442- 30 30644 SEQ ID
NO: 6338 22654 SEQ ID NOs: 22655- 15 30635 22695 DNMT1:NM_001318730
SEQ ID NOs: 22696- 29 30644 SEQ ID NO: 6339 22908 SEQ ID NOs:
22909- 14 30635 22949 DNMT1:NM_001318731 SEQ ID NOs: 22950- 30
30644 SEQ ID NO: 6340 23162 SEQ ID NOs: 23163- 15 30635 23203 NF2
NF2:NM_181830 SEQ ID NOs: 23204- 14 30646 SEQ ID SEQ ID NO: 6341
24115 NO: 6288 NF2:NM_181829 SEQ ID NOs: 24116- 15 30646 SEQ ID NO:
6342 25027 NF2:NM_181833 SEQ ID NOs: 25028- 4 30640 SEQ ID NO: 6343
25945 NF2:NM_181832 SEQ ID NOs: 25946- 16 30625 SEQ ID NO: 6344
26860 NF2:NM_181828 SEQ ID NOs: 26861- 15 30646 SEQ ID NO: 6345
27772 NF2:NM_016418 SEQ ID NOs: 27773- 16 30646 SEQ ID NO: 6346
28684 NF2:NM_000268 SEQ ID NOs: 28685- 15 30641 SEQ ID NO: 6347
29620 SHANK3 SHANK3:NM_033517 SEQ ID NOs: 29621- 16 30630 SEQ ID
SEQ ID NO: 6348 29808 NO: 6289 ARSA ARSA:NM_000487 SEQ ID NOs:
29808- 2 30651 SEQ ID SEQ ID NO: 6349 29895 NO: 6290 SEQ ID NOs:
29895- 3 30636 29971 ARSA:NM_001085425 SEQ ID NOs: 29972- 3 30651
SEQ ID NO: 6350 30058 SEQ ID NOs: 30059- 4 30636 30134
ARSA:NM_001085426 SEQ ID NOs: 30135- 3 30651 SEQ ID NO: 6351 30221
SEQ ID NOs: 30222- 4 30636 30297 ARSA:NM_001085427 SEQ ID NOs:
30298- 3 30651 SEQ ID NO: 6352 30384 SEQ ID NOs: 30385- 4 30636
30460 ARSA:NM_001085428 SEQ ID NOs: 30461- 2 30651 SEQ ID NO: 6353
30547 SEQ ID NOs: 30548- 3 30636 30623
[1180] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a ADAR genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a ADAR
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6274. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6274 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a ADAR RIC pre-mRNA sequence. In some
embodiments, the ASO targets a ADAR RIC pre-mRNA transcript
comprising a retained intron at 2, wherein the intron numbering
correspond to the mRNA sequence at NM_001193495. In some
embodiments, the ASO targets a ADAR RIC pre-mRNA transcript
comprising a retained intron at 2, wherein the intron numbering
correspond to the mRNA sequence at NM_015841. In some embodiments,
the ASO targets a ADAR RIC pre-mRNA transcript comprising a
retained intron at 2, wherein the intron numbering correspond to
the mRNA sequence at NM_015840. In some embodiments, the ASO
targets a ADAR RIC pre-mRNA transcript comprising a retained intron
at 2, wherein the intron numbering correspond to the mRNA sequence
at NM_001111. In some embodiments, the ASO targets a ADAR RIC
pre-mRNA transcript comprising a retained intron at 2, wherein the
intron numbering correspond to the mRNA sequence at
NM_001025107.
[1181] In some embodiments, the ASO targets a ADAR RIC pre-mRNA
sequence according to SEQ ID NO: 6293. In some embodiments, the ASO
targets a ADAR RIC pre-mRNA sequence according to SEQ ID NO: 6293
comprising a retained intron 2. In some embodiments, the ASO
targets a ADAR RIC pre-mRNA sequence according to SEQ ID NO: 6294.
In some embodiments, the ASO targets a ADAR RIC pre-mRNA sequence
according to SEQ ID NO: 6294 comprising a retained intron 2. In
some embodiments, the ASO targets a ADAR RIC pre-mRNA sequence
according to SEQ ID NO: 6295. In some embodiments, the ASO targets
a ADAR RIC pre-mRNA sequence according to SEQ ID NO: 62952
comprising a retained intron 2. In some embodiments, the ASO
targets a ADAR RIC pre-mRNA sequence according to SEQ ID NO: 6296.
In some embodiments, the ASO targets a ADAR RIC pre-mRNA sequence
according to SEQ ID NO: 6296 comprising a retained intron 2. In
some embodiments, the ASO targets a ADAR RIC pre-mRNA sequence
according to SEQ ID NO: 6297. In some embodiments, the ASO targets
a ADAR RIC pre-mRNA sequence according to SEQ ID NO: 6297
comprising a retained intron 2. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30656. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
6364-8948.
[1182] In some embodiments, the ASO targets exon 2 or exon 3 of a
ADAR RIC pre-mRNA comprising a retained intron 2, wherein the
intron numbering correspond to the mRNA sequence at NM_001193495.
In some embodiments, the ASO targets an exon 2 sequence upstream
(or 5') from the 5' splice site of a ADAR RIC pre-mRNA comprising
the retained intron 2. In some embodiments, the ASO targets an exon
2 sequence about 4 to about 1566 or about 14 to about 1566
nucleotides upstream (or 5') from the 5' splice site of a ADAR RIC
pre-mRNA comprising the retained intron 2. In some embodiments, the
ASO targets an exon 3 sequence downstream (or 3') from the 3'
splice site of a ADAR RIC pre-mRNA comprising the retained intron
2. In some embodiments, the ASO targets an exon 3 sequence about 2
to about 165 nucleotides downstream (or 3') from the 3' splice site
of a ADAR RIC pre-mRNA comprising the retained intron 2.
[1183] In some embodiments, the ASO targets intron 2 in a ADAR RIC
pre-mRNA comprising a retained intron 2, wherein the intron
numbering correspond to the mRNA sequence at NM_001193495. In some
embodiments, the ASO targets an intron 2 sequence downstream (or
3') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an intron 2
sequence about 6 to about 500 nucleotides downstream (or 3') from
the 5' splice site of a ADAR RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an intron 2 sequence
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an intron 2 sequence about 16 to about 499 nucleotides
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2.
[1184] In some embodiments, the ASO targets exon 2 or exon 3 of a
ADAR RIC pre-mRNA comprising a retained intron 2, wherein the
intron numbering correspond to the mRNA sequence at NM_015841. In
some embodiments, the ASO targets an exon 2 sequence upstream (or
5') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an exon 2
sequence about 4 to about 1566 or about 14 to about 1566
nucleotides upstream (or 5') from the 5' splice site of a ADAR RIC
pre-mRNA comprising the retained intron 2. In some embodiments, the
ASO targets an exon 3 sequence downstream (or 3') from the 3'
splice site of a ADAR RIC pre-mRNA comprising the retained intron
2. In some embodiments, the ASO targets an exon 3 sequence about 2
to about 165 nucleotides downstream (or 3') from the 3' splice site
of a ADAR RIC pre-mRNA comprising the retained intron 2.
[1185] In some embodiments, the ASO targets intron 2 in a ADAR RIC
pre-mRNA comprising a retained intron 2, wherein the intron
numbering correspond to the mRNA sequence at NM_015841. In some
embodiments, the ASO targets an intron 2 sequence downstream (or
3') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an intron 2
sequence about 6 to about 500 nucleotides downstream (or 3') from
the 5' splice site of a ADAR RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an intron 2 sequence
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an intron 2 sequence about 16 to about 499 nucleotides
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2.
[1186] In some embodiments, the ASO targets exon 2 or exon 3 of a
ADAR RIC pre-mRNA comprising a retained intron 2, wherein the
intron numbering correspond to the mRNA sequence at NM_015840. In
some embodiments, the ASO targets an exon 2 sequence upstream (or
5') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an exon 2
sequence about 4 to about 1566 or about 14 to about 1566
nucleotides upstream (or 5') from the 5' splice site of a ADAR RIC
pre-mRNA comprising the retained intron 2. In some embodiments, the
ASO targets an exon 3 sequence downstream (or 3') from the 3'
splice site of a ADAR RIC pre-mRNA comprising the retained intron
2. In some embodiments, the ASO targets an exon 3 sequence about 2
to about 165 nucleotides downstream (or 3') from the 3' splice site
of a ADAR RIC pre-mRNA comprising the retained intron 2.
[1187] In some embodiments, the ASO targets intron 2 in a ADAR RIC
pre-mRNA comprising a retained intron 2, wherein the intron
numbering correspond to the mRNA sequence at NM_015840. In some
embodiments, the ASO targets an intron 2 sequence downstream (or
3') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an intron 2
sequence about 6 to about 500 nucleotides downstream (or 3') from
the 5' splice site of a ADAR RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an intron 2 sequence
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an intron 2 sequence about 16 to about 499 nucleotides
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2.
[1188] In some embodiments, the ASO targets exon 2 or exon 3 of a
ADAR RIC pre-mRNA comprising a retained intron 2, wherein the
intron numbering correspond to the mRNA sequence at NM_001111. In
some embodiments, the ASO targets an exon 2 sequence upstream (or
5') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an exon 2
sequence about 4 to about 1566 or about 14 to about 1566
nucleotides upstream (or 5') from the 5' splice site of a ADAR RIC
pre-mRNA comprising the retained intron 2. In some embodiments, the
ASO targets an exon 3 sequence downstream (or 3') from the 3'
splice site of a ADAR RIC pre-mRNA comprising the retained intron
2. In some embodiments, the ASO targets an exon 3 sequence about 2
to about 165 nucleotides downstream (or 3') from the 3' splice site
of a ADAR RIC pre-mRNA comprising the retained intron 2.
[1189] In some embodiments, the ASO targets intron 2 in a ADAR RIC
pre-mRNA comprising a retained intron 2, wherein the intron
numbering correspond to the mRNA sequence at NM_001111. In some
embodiments, the ASO targets an intron 2 sequence downstream (or
3') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an intron 2
sequence about 6 to about 500 nucleotides downstream (or 3') from
the 5' splice site of a ADAR RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an intron 2 sequence
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an intron 2 sequence about 16 to about 499 nucleotides
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2.
[1190] In some embodiments, the ASO targets exon 2 or exon 3 of a
ADAR RIC pre-mRNA comprising a retained intron 2, wherein the
intron numbering correspond to the mRNA sequence at NM_001025107.
In some embodiments, the ASO targets an exon 2 sequence upstream
(or 5') from the 5' splice site of a ADAR RIC pre-mRNA comprising
the retained intron 2. In some embodiments, the ASO targets an exon
2 sequence about 4 to about 1566 or about 14 to about 1566
nucleotides upstream (or 5') from the 5' splice site of a ADAR RIC
pre-mRNA comprising the retained intron 2. In some embodiments, the
ASO targets an exon 3 sequence downstream (or 3') from the 3'
splice site of a ADAR RIC pre-mRNA comprising the retained intron
2. In some embodiments, the ASO targets an exon 3 sequence about 2
to about 165 nucleotides downstream (or 3') from the 3' splice site
of a ADAR RIC pre-mRNA comprising the retained intron 2.
[1191] In some embodiments, the ASO targets intron 2 in a ADAR RIC
pre-mRNA comprising a retained intron 2, wherein the intron
numbering correspond to the mRNA sequence at NM_001025107. In some
embodiments, the ASO targets an intron 2 sequence downstream (or
3') from the 5' splice site of a ADAR RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an intron 2
sequence about 6 to about 500 nucleotides downstream (or 3') from
the 5' splice site of a ADAR RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an intron 2 sequence
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an intron 2 sequence about 16 to about 499 nucleotides
upstream (or 5') from the 3' splice site of a ADAR RIC pre-mRNA
comprising the retained intron 2.
[1192] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a ATP1A2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
ATP1A2 genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6275. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6275 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a ATP1A2 RIC pre-mRNA
sequence. In some embodiments, the ASO targets a ATP1A2 RIC
pre-mRNA transcript comprising a retained intron at 22, wherein the
intron numbering correspond to the mRNA sequence at NM_000702.
[1193] In some embodiments, the ASO targets a ATP1A2 RIC pre-mRNA
sequence according to SEQ ID NO: 6298. In some embodiments, the ASO
targets a ATP1A2 RIC pre-mRNA sequence according to SEQ ID NO: 6298
comprising a retained intron 22. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30632. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
8949-9575.
[1194] In some embodiments, the ASO targets exon 22 or exon 23 of a
ATP1A2 RIC pre-mRNA comprising a retained intron 22, wherein the
intron numbering correspond to the mRNA sequence at NM_000702. In
some embodiments, the ASO targets an exon 22 sequence upstream (or
5') from the 5' splice site of a ATP1A2 RIC pre-mRNA comprising the
retained intron 22. In some embodiments, the ASO targets an exon 22
sequence about 4 to about 71 nucleotides upstream (or 5') from the
5' splice site of a ATP1A2 RIC pre-mRNA comprising the retained
intron 22. In some embodiments, the ASO targets an exon 23 sequence
downstream (or 3') from the 3' splice site of a ATP1A2 RIC pre-mRNA
comprising the retained intron 22. In some embodiments, the ASO
targets an exon 23 sequence about 2 to about 2271 nucleotides
downstream (or 3') from the 3' splice site of a ATP1A2 RIC pre-mRNA
comprising the retained intron 22.
[1195] In some embodiments, the ASO targets intron 22 in a ATP1A2
RIC pre-mRNA comprising a retained intron 22, wherein the intron
numbering correspond to the mRNA sequence at NM_000702. In some
embodiments, the ASO targets an intron 22 sequence downstream (or
3') from the 5' splice site of a ATP1A2 RIC pre-mRNA comprising the
retained intron 22. In some embodiments, the ASO targets an intron
22 sequence about 6 to about 498 or about 21 to about 498
nucleotides downstream (or 3') from the 5' splice site of a ATP1A2
RIC pre-mRNA comprising the retained intron 22. In some
embodiments, the ASO targets an intron 22 sequence upstream (or 5')
from the 3' splice site of a ATP1A2 RIC pre-mRNA comprising the
retained intron 22. In some embodiments, the ASO targets an intron
22 sequence about 16 to about 500 nucleotides upstream (or 5') from
the 3' splice site of a ATP1A2 RIC pre-mRNA comprising the retained
intron 22.
[1196] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PRICKLE2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
PRICKLE2 genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6276. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6276 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a PRICKLE2 RIC
pre-mRNA sequence. In some embodiments, the ASO targets a PRICKLE2
RIC pre-mRNA transcript comprising a retained intron at 4, wherein
the intron numbering correspond to the mRNA sequence at
NM_198859.
[1197] In some embodiments, the ASO targets a PRICKLE2 RIC pre-mRNA
sequence according to SEQ ID NO: 6299. In some embodiments, the ASO
targets a PRICKLE2 RIC pre-mRNA sequence according to SEQ ID NO:
6299 comprising a retained intron 4. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30645. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
9576-9825.
[1198] In some embodiments, the ASO targets exon 4 or exon 5 of a
PRICKLE2 RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_198859. In
some embodiments, the ASO targets an exon 4 sequence upstream (or
5') from the 5' splice site of a PRICKLE2 RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 119 nucleotides upstream (or 5') from
the 5' splice site of a PRICKLE2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an exon 5
sequence downstream (or 3') from the 3' splice site of a PRICKLE2
RIC pre-mRNA comprising the retained intron 4. In some embodiments,
the ASO targets an exon 5 sequence about 2 to about 187 nucleotides
downstream (or 3') from the 3' splice site of a PRICKLE2 RIC
pre-mRNA comprising the retained intron 4.
[1199] In some embodiments, the ASO targets intron 4 in a PRICKLE2
RIC pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_198859. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a PRICKLE2 RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an
intron 4 sequence about 6 to about 496 nucleotides downstream (or
3') from the 5' splice site of a PRICKLE2 RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an
intron 4 sequence upstream (or 5') from the 3' splice site of a
PRICKLE2 RIC pre-mRNA comprising the retained intron 4. In some
embodiments, the ASO targets an intron 4 sequence about 16 to about
499 nucleotides upstream (or 5') from the 3' splice site of a
PRICKLE2 RIC pre-mRNA comprising the retained intron 4.
[1200] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a SETD5 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a SETD5
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6277. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6277 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a SETD5 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a SETD5 RIC pre-mRNA transcript
comprising a retained intron at 4, wherein the intron numbering
correspond to the mRNA sequence at NM_001080517. In some
embodiments, the ASO targets a SETD5 RIC pre-mRNA transcript
comprising a retained intron at 5, wherein the intron numbering
correspond to the mRNA sequence at NM_001292043.
[1201] In some embodiments, the ASO targets a SETD5 RIC pre-mRNA
sequence according to SEQ ID NO: 6300. In some embodiments, the ASO
targets a SETD5 RIC pre-mRNA sequence according to SEQ ID NO: 6300
comprising a retained intron 4. In some embodiments, the ASO
targets a SETD5 RIC pre-mRNA sequence according to SEQ ID NO: 6301.
In some embodiments, the ASO targets a SETD5 RIC pre-mRNA sequence
according to SEQ ID NO: 6301 comprising a retained intron 5. In
some embodiments, the ASOs disclosed herein target SEQ ID NO:
30659. In some embodiments, the ASO has a sequence according to any
one of SEQ ID NOs: 9826-10067.
[1202] In some embodiments, the ASO targets exon 4 or exon 5 of a
SETD5 RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_001080517.
In some embodiments, the ASO targets an exon 4 sequence upstream
(or 5') from the 5' splice site of a SETD5 RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 89 nucleotides upstream (or 5') from
the 5' splice site of a SETD5 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 132 nucleotides
downstream (or 3') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 4.
[1203] In some embodiments, the ASO targets intron 4 in a SETD5 RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_001080517. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a SETD5 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 6 to about 204 nucleotides downstream (or 3') from
the 5' splice site of a SETD5 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 204 nucleotides
upstream (or 5') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 4.
[1204] In some embodiments, the ASO targets exon 5 or exon 6 of a
SETD5 RIC pre-mRNA comprising a retained intron 5, wherein the
intron numbering correspond to the mRNA sequence at NM_001292043.
In some embodiments, the ASO targets an exon 5 sequence upstream
(or 5') from the 5' splice site of a SETD5 RIC pre-mRNA comprising
the retained intron 5. In some embodiments, the ASO targets an exon
5 sequence about 4 to about 89 nucleotides upstream (or 5') from
the 5' splice site of a SETD5 RIC pre-mRNA comprising the retained
intron 5. In some embodiments, the ASO targets an exon 6 sequence
downstream (or 3') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 5. In some embodiments, the ASO
targets an exon 6 sequence about 2 to about 132 nucleotides
downstream (or 3') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 5.
[1205] In some embodiments, the ASO targets intron 5 in a SETD5 RIC
pre-mRNA comprising a retained intron 5, wherein the intron
numbering correspond to the mRNA sequence at NM_001292043. In some
embodiments, the ASO targets an intron 5 sequence downstream (or
3') from the 5' splice site of a SETD5 RIC pre-mRNA comprising the
retained intron 5. In some embodiments, the ASO targets an intron 5
sequence about 6 to about 204 nucleotides downstream (or 3') from
the 5' splice site of a SETD5 RIC pre-mRNA comprising the retained
intron 5. In some embodiments, the ASO targets an intron 5 sequence
upstream (or 5') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 5. In some embodiments, the ASO
targets an intron 5 sequence about 16 to about 204 nucleotides
upstream (or 5') from the 3' splice site of a SETD5 RIC pre-mRNA
comprising the retained intron 5.
[1206] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a EIF2B5 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
EIF2B5 genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6278. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6278 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a EIF2B5 RIC pre-mRNA
sequence. In some embodiments, the ASO targets a EIF2B5 RIC
pre-mRNA transcript comprising a retained intron at 12, 13 or a
combination thereof, wherein the intron numbering correspond to the
mRNA sequence at NM_003907.
[1207] In some embodiments, the ASO targets a EIF2B5 RIC pre-mRNA
sequence according to SEQ ID NO: 6302. In some embodiments, the ASO
targets a EIF2B5 RIC pre-mRNA sequence according to SEQ ID NO: 6302
comprising a retained intron 12, a retained intron 13, or a
combination thereof. In some embodiments, the ASOs disclosed herein
target SEQ ID NOs: 30637 or 30649. In some embodiments, the ASO has
a sequence according to any one of SEQ ID NOs: 10068-10305.
[1208] In some embodiments, the ASO targets exon 12 or exon 13 of a
EIF2B5 RIC pre-mRNA comprising a retained intron 12, wherein the
intron numbering correspond to the mRNA sequence at NM_003907. In
some embodiments, the ASO targets an exon 12 sequence upstream (or
5') from the 5' splice site of a EIF2B5 RIC pre-mRNA comprising the
retained intron 12. In some embodiments, the ASO targets an exon 12
sequence about 4 to about 74 nucleotides upstream (or 5') from the
5' splice site of a EIF2B5 RIC pre-mRNA comprising the retained
intron 12. In some embodiments, the ASO targets an exon 13 sequence
downstream (or 3') from the 3' splice site of a EIF2B5 RIC pre-mRNA
comprising the retained intron 12. In some embodiments, the ASO
targets an exon 13 sequence about 2 to about 105 nucleotides
downstream (or 3') from the 3' splice site of a EIF2B5 RIC pre-mRNA
comprising the retained intron 12.
[1209] In some embodiments, the ASO targets intron 12 in a EIF2B5
RIC pre-mRNA comprising a retained intron 12, wherein the intron
numbering correspond to the mRNA sequence at NM_003907. In some
embodiments, the ASO targets an intron 12 sequence downstream (or
3') from the 5' splice site of a EIF2B5 RIC pre-mRNA comprising the
retained intron 12. In some embodiments, the ASO targets an intron
12 sequence about 6 to about 161 nucleotides downstream (or 3')
from the 5' splice site of a EIF2B5 RIC pre-mRNA comprising the
retained intron 12. In some embodiments, the ASO targets an intron
12 sequence upstream (or 5') from the 3' splice site of a EIF2B5
RIC pre-mRNA comprising the retained intron 12. In some
embodiments, the ASO targets an intron 12 sequence about 16 to
about 164 nucleotides upstream (or 5') from the 3' splice site of a
EIF2B5 RIC pre-mRNA comprising the retained intron 12.
[1210] In some embodiments, the ASO targets exon 13 or exon 14 of a
EIF2B5 RIC pre-mRNA comprising a retained intron 13, wherein the
intron numbering correspond to the mRNA sequence at NM_003907. In
some embodiments, the ASO targets an exon 13 sequence upstream (or
5') from the 5' splice site of a EIF2B5 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an exon 13
sequence about 4 to about 104 nucleotides upstream (or 5') from the
5' splice site of a EIF2B5 RIC pre-mRNA comprising the retained
intron 13. In some embodiments, the ASO targets an exon 14 sequence
downstream (or 3') from the 3' splice site of a EIF2B5 RIC pre-mRNA
comprising the retained intron 13. In some embodiments, the ASO
targets an exon 14 sequence about 2 to about 107 nucleotides
downstream (or 3') from the 3' splice site of a EIF2B5 RIC pre-mRNA
comprising the retained intron 13.
[1211] In some embodiments, the ASO targets intron 13 in a EIF2B5
RIC pre-mRNA comprising a retained intron 13, wherein the intron
numbering correspond to the mRNA sequence at NM_003907. In some
embodiments, the ASO targets an intron 13 sequence downstream (or
3') from the 5' splice site of a EIF2B5 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence about 6 to about 276 nucleotides downstream (or 3')
from the 5' splice site of a EIF2B5 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence upstream (or 5') from the 3' splice site of a EIF2B5
RIC pre-mRNA comprising the retained intron 13. In some
embodiments, the ASO targets an intron 13 sequence about 16 to
about 281 nucleotides upstream (or 5') from the 3' splice site of a
EIF2B5 RIC pre-mRNA comprising the retained intron 13.
[1212] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PEX1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a PEX1
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6279. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6279 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a PEX1 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a PEX1 RIC pre-mRNA transcript
comprising a retained intron at 10, 19, 21, 14 or a combination
thereof, wherein the intron numbering correspond to the mRNA
sequence at NM_000466. In some embodiments, the ASO targets a PEX1
RIC pre-mRNA transcript comprising a retained intron at 10, 18, 20,
13 or a combination thereof, wherein the intron numbering
correspond to the mRNA sequence at NM_001282677. In some
embodiments, the ASO targets a PEX1 RIC pre-mRNA transcript
comprising a retained intron at 10, 19, 21, 14 or a combination
thereof, wherein the intron numbering correspond to the mRNA
sequence at NM_001282678.
[1213] In some embodiments, the ASO targets a PEX1 RIC pre-mRNA
sequence according to SEQ ID NO: 6303. In some embodiments, the ASO
targets a PEX1 RIC pre-mRNA sequence according to SEQ ID NO: 6303
comprising a retained intron 10, a retained intron 19, a retained
intron 21, a retained intron 14, or a combination thereof. In some
embodiments, the ASO targets a PEX1 RIC pre-mRNA sequence according
to SEQ ID NO: 6304. In some embodiments, the ASO targets a PEX1 RIC
pre-mRNA sequence according to SEQ ID NO: 6304 comprising a
retained intron 10, a retained intron 18, a retained intron 20, a
retained intron 13, or a combination thereof. In some embodiments,
the ASO targets a PEX1 RIC pre-mRNA sequence according to SEQ ID
NO: 6305. In some embodiments, the ASO targets a PEX1 RIC pre-mRNA
sequence according to SEQ ID NO: 6305 comprising a retained intron
10, a retained intron 19, a retained intron 21, a retained intron
14, or a combination thereof. In some embodiments, the ASOs
disclosed herein target SEQ ID NOs: 30629, 30653, 30643, or 30634.
In some embodiments, the ASO has a sequence according to any one of
SEQ ID NOs: 4033-641110306-12684.
[1214] In some embodiments, the ASO targets exon 10 or exon 11 of a
PEX1 RIC pre-mRNA comprising a retained intron 10, wherein the
intron numbering correspond to the mRNA sequence at NM_000466. In
some embodiments, the ASO targets an exon 10 sequence upstream (or
5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an exon 10
sequence about 4 to about 114 nucleotides upstream (or 5') from the
5' splice site of a PEX1 RIC pre-mRNA comprising the retained
intron 10. In some embodiments, the ASO targets an exon 11 sequence
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 10. In some embodiments, the ASO
targets an exon 11 sequence about 2 to about 77 nucleotides
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 10.
[1215] In some embodiments, the ASO targets intron 10 in a PEX1 RIC
pre-mRNA comprising a retained intron 10, wherein the intron
numbering correspond to the mRNA sequence at NM_000466. In some
embodiments, the ASO targets an intron 10 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence about 6 to about 336 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an intron 10 sequence about 16 to about 336
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10.
[1216] In some embodiments, the ASO targets exon 19 or exon 20 of a
PEX1 RIC pre-mRNA comprising a retained intron 19, wherein the
intron numbering correspond to the mRNA sequence at NM_000466. In
some embodiments, the ASO targets an exon 19 sequence upstream (or
5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an exon 19
sequence about 4 to about 84 nucleotides upstream (or 5') from the
5' splice site of a PEX1 RIC pre-mRNA comprising the retained
intron 19. In some embodiments, the ASO targets an exon 20 sequence
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 19. In some embodiments, the ASO
targets an exon 20 sequence about 2 to about 157 nucleotides
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 19.
[1217] In some embodiments, the ASO targets intron 19 in a PEX1 RIC
pre-mRNA comprising a retained intron 19, wherein the intron
numbering correspond to the mRNA sequence at NM_000466. In some
embodiments, the ASO targets an intron 19 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an intron
19 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an intron
19 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 19. In some embodiments,
the ASO targets an intron 19 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 19.
[1218] In some embodiments, the ASO targets exon 21 or exon 22 of a
PEX1 RIC pre-mRNA comprising a retained intron 21, wherein the
intron numbering correspond to the mRNA sequence at NM_000466. In
some embodiments, the ASO targets an exon 21 sequence upstream (or
5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 21. In some embodiments, the ASO targets an exon 21
sequence about 4 to about 214 nucleotides upstream (or 5') from the
5' splice site of a PEX1 RIC pre-mRNA comprising the retained
intron 21. In some embodiments, the ASO targets an exon 22 sequence
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 21. In some embodiments, the ASO
targets an exon 22 sequence about 2 to about 177 nucleotides
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 21.
[1219] In some embodiments, the ASO targets intron 21 in a PEX1 RIC
pre-mRNA comprising a retained intron 21, wherein the intron
numbering correspond to the mRNA sequence at NM_000466. In some
embodiments, the ASO targets an intron 21 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 21. In some embodiments, the ASO targets an intron
21 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 21. In some embodiments, the ASO targets an intron
21 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 21. In some embodiments,
the ASO targets an intron 21 sequence about 16 to about 497
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 21.
[1220] In some embodiments, the ASO targets exon 14 or exon 15 of a
PEX1 RIC pre-mRNA comprising a retained intron 14, wherein the
intron numbering correspond to the mRNA sequence at NM_000466. In
some embodiments, the ASO targets an exon 14 sequence upstream (or
5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an exon 14
sequence about 4 to about 169 nucleotides upstream (or 5') from the
5' splice site of a PEX1 RIC pre-mRNA comprising the retained
intron 14. In some embodiments, the ASO targets an exon 15 sequence
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 14. In some embodiments, the ASO
targets an exon 15 sequence about 2 to about 147 nucleotides
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 14.
[1221] In some embodiments, the ASO targets intron 14 in a PEX1 RIC
pre-mRNA comprising a retained intron 14, wherein the intron
numbering correspond to the mRNA sequence at NM_000466. In some
embodiments, the ASO targets an intron 14 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence about 6 to about 121 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 14. In some embodiments,
the ASO targets an intron 14 sequence about 16 to about 121
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 14.
[1222] In some embodiments, the ASO targets exon 10 or exon 11 of a
PEX1 RIC pre-mRNA comprising a retained intron 10, wherein the
intron numbering correspond to the mRNA sequence at NM_001282677.
In some embodiments, the ASO targets an exon 10 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 10. In some embodiments, the ASO targets an
exon 10 sequence about 4 to about 114 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an exon 11
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an exon 11 sequence about 2 to about 77 nucleotides
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 10.
[1223] In some embodiments, the ASO targets intron 10 in a PEX1 RIC
pre-mRNA comprising a retained intron 10, wherein the intron
numbering correspond to the mRNA sequence at NM_001282677. In some
embodiments, the ASO targets an intron 10 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence about 6 to about 336 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an intron 10 sequence about 16 to about 336
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10.
[1224] In some embodiments, the ASO targets exon 18 or exon 19 of a
PEX1 RIC pre-mRNA comprising a retained intron 18, wherein the
intron numbering correspond to the mRNA sequence at NM_001282677.
In some embodiments, the ASO targets an exon 18 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 18. In some embodiments, the ASO targets an
exon 18 sequence about 4 to about 84 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an exon 19
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 18. In some embodiments,
the ASO targets an exon 19 sequence about 2 to about 157
nucleotides downstream (or 3') from the 3' splice site of a PEX1
RIC pre-mRNA comprising the retained intron 18.
[1225] In some embodiments, the ASO targets intron 18 in a PEX1 RIC
pre-mRNA comprising a retained intron 18, wherein the intron
numbering correspond to the mRNA sequence at NM_001282677. In some
embodiments, the ASO targets an intron 18 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 18. In some embodiments,
the ASO targets an intron 18 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 18.
[1226] In some embodiments, the ASO targets exon 20 or exon 21 of a
PEX1 RIC pre-mRNA comprising a retained intron 20, wherein the
intron numbering correspond to the mRNA sequence at NM_001282677.
In some embodiments, the ASO targets an exon 20 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 20. In some embodiments, the ASO targets an
exon 20 sequence about 4 to about 214 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 20. In some embodiments, the ASO targets an exon 21
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 20. In some embodiments,
the ASO targets an exon 21 sequence about 2 to about 177
nucleotides downstream (or 3') from the 3' splice site of a PEX1
RIC pre-mRNA comprising the retained intron 20.
[1227] In some embodiments, the ASO targets intron 20 in a PEX1 RIC
pre-mRNA comprising a retained intron 20, wherein the intron
numbering correspond to the mRNA sequence at NM_001282677. In some
embodiments, the ASO targets an intron 20 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 20. In some embodiments, the ASO targets an intron
20 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 20. In some embodiments, the ASO targets an intron
20 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 20. In some embodiments,
the ASO targets an intron 20 sequence about 16 to about 497
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 20.
[1228] In some embodiments, the ASO targets exon 13 or exon 14 of a
PEX1 RIC pre-mRNA comprising a retained intron 13, wherein the
intron numbering correspond to the mRNA sequence at NM_001282677.
In some embodiments, the ASO targets an exon 13 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 13. In some embodiments, the ASO targets an
exon 13 sequence about 4 to about 169 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an exon 14
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an exon 14 sequence about 2 to about 147
nucleotides downstream (or 3') from the 3' splice site of a PEX1
RIC pre-mRNA comprising the retained intron 13.
[1229] In some embodiments, the ASO targets intron 13 in a PEX1 RIC
pre-mRNA comprising a retained intron 13, wherein the intron
numbering correspond to the mRNA sequence at NM_001282677. In some
embodiments, the ASO targets an intron 13 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence about 6 to about 121 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an intron 13 sequence about 16 to about 121
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 13.
[1230] In some embodiments, the ASO targets exon 10 or exon 11 of a
PEX1 RIC pre-mRNA comprising a retained intron 10, wherein the
intron numbering correspond to the mRNA sequence at NM_001282678.
In some embodiments, the ASO targets an exon 10 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 10. In some embodiments, the ASO targets an
exon 10 sequence about 4 to about 114 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an exon 11
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an exon 11 sequence about 2 to about 77 nucleotides
downstream (or 3') from the 3' splice site of a PEX1 RIC pre-mRNA
comprising the retained intron 10.
[1231] In some embodiments, the ASO targets intron 10 in a PEX1 RIC
pre-mRNA comprising a retained intron 10, wherein the intron
numbering correspond to the mRNA sequence at NM_001282678. In some
embodiments, the ASO targets an intron 10 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence about 6 to about 336 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an intron 10 sequence about 16 to about 336
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 10.
[1232] In some embodiments, the ASO targets exon 19 or exon 20 of a
PEX1 RIC pre-mRNA comprising a retained intron 19, wherein the
intron numbering correspond to the mRNA sequence at NM_001282678.
In some embodiments, the ASO targets an exon 19 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 19. In some embodiments, the ASO targets an
exon 19 sequence about 4 to about 84 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an exon 20
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 19. In some embodiments,
the ASO targets an exon 20 sequence about 2 to about 157
nucleotides downstream (or 3') from the 3' splice site of a PEX1
RIC pre-mRNA comprising the retained intron 19.
[1233] In some embodiments, the ASO targets intron 19 in a PEX1 RIC
pre-mRNA comprising a retained intron 19, wherein the intron
numbering correspond to the mRNA sequence at NM_001282678. In some
embodiments, the ASO targets an intron 19 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an intron
19 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an intron
19 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 19. In some embodiments,
the ASO targets an intron 19 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 19.
[1234] In some embodiments, the ASO targets exon 21 or exon 22 of a
PEX1 RIC pre-mRNA comprising a retained intron 21, wherein the
intron numbering correspond to the mRNA sequence at NM_001282678.
In some embodiments, the ASO targets an exon 21 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 21. In some embodiments, the ASO targets an
exon 21 sequence about 4 to about 214 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 21. In some embodiments, the ASO targets an exon 22
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 21. In some embodiments,
the ASO targets an exon 22 sequence about 2 to about 177
nucleotides downstream (or 3') from the 3' splice site of a PEX1
RIC pre-mRNA comprising the retained intron 21.
[1235] In some embodiments, the ASO targets intron 21 in a PEX1 RIC
pre-mRNA comprising a retained intron 21, wherein the intron
numbering correspond to the mRNA sequence at NM_001282678. In some
embodiments, the ASO targets an intron 21 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 21. In some embodiments, the ASO targets an intron
21 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 21. In some embodiments, the ASO targets an intron
21 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 21. In some embodiments,
the ASO targets an intron 21 sequence about 16 to about 497
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 21.
[1236] In some embodiments, the ASO targets exon 14 or exon 15 of a
PEX1 RIC pre-mRNA comprising a retained intron 14, wherein the
intron numbering correspond to the mRNA sequence at NM_001282678.
In some embodiments, the ASO targets an exon 14 sequence upstream
(or 5') from the 5' splice site of a PEX1 RIC pre-mRNA comprising
the retained intron 14. In some embodiments, the ASO targets an
exon 14 sequence about 4 to about 169 nucleotides upstream (or 5')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an exon 15
sequence downstream (or 3') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 14. In some embodiments,
the ASO targets an exon 15 sequence about 2 to about 147
nucleotides downstream (or 3') from the 3' splice site of a PEX1
RIC pre-mRNA comprising the retained intron 14.
[1237] In some embodiments, the ASO targets intron 14 in a PEX1 RIC
pre-mRNA comprising a retained intron 14, wherein the intron
numbering correspond to the mRNA sequence at NM_001282678. In some
embodiments, the ASO targets an intron 14 sequence downstream (or
3') from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence about 6 to about 121 nucleotides downstream (or 3')
from the 5' splice site of a PEX1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 14. In some embodiments,
the ASO targets an intron 14 sequence about 16 to about 121
nucleotides upstream (or 5') from the 3' splice site of a PEX1 RIC
pre-mRNA comprising the retained intron 14.
[1238] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a STXBP1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
STXBP1 genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6280. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6280 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a STXBP1 RIC pre-mRNA
sequence. In some embodiments, the ASO targets a STXBP1 RIC
pre-mRNA transcript comprising a retained intron at 18, 19 or a
combination thereof, wherein the intron numbering correspond to the
mRNA sequence at NM_003165. In some embodiments, the ASO targets a
STXBP1 RIC pre-mRNA transcript comprising a retained intron at 18,
wherein the intron numbering correspond to the mRNA sequence at
NM_001032221.
[1239] In some embodiments, the ASO targets a STXBP1 RIC pre-mRNA
sequence according to SEQ ID NO: 6306. In some embodiments, the ASO
targets a STXBP1 RIC pre-mRNA sequence according to SEQ ID NO: 6306
comprising a retained intron 18, a retained intron 19, or a
combination thereof. In some embodiments, the ASO targets a STXBP1
RIC pre-mRNA sequence according to SEQ ID NO: 6307. In some
embodiments, the ASO targets a STXBP1 RIC pre-mRNA sequence
according to SEQ ID NO: 6307 comprising a retained intron 18. In
some embodiments, the ASOs disclosed herein target SEQ ID NOs:
30627, 30624, or 30633. In some embodiments, the ASO has a sequence
according to any one of SEQ ID NOs: 12685-14026.
[1240] In some embodiments, the ASO targets exon 18 or exon 19 of a
STXBP1 RIC pre-mRNA comprising a retained intron 18, wherein the
intron numbering correspond to the mRNA sequence at NM_003165. In
some embodiments, the ASO targets an exon 18 sequence upstream (or
5') from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an exon 18
sequence about 4 to about 136 nucleotides upstream (or 5') from the
5' splice site of a STXBP1 RIC pre-mRNA comprising the retained
intron 18. In some embodiments, the ASO targets an exon 19 sequence
downstream (or 3') from the 3' splice site of a STXBP1 RIC pre-mRNA
comprising the retained intron 18. In some embodiments, the ASO
targets an exon 19 sequence about 2 to about 106 nucleotides
downstream (or 3') from the 3' splice site of a STXBP1 RIC pre-mRNA
comprising the retained intron 18.
[1241] In some embodiments, the ASO targets intron 18 in a STXBP1
RIC pre-mRNA comprising a retained intron 18, wherein the intron
numbering correspond to the mRNA sequence at NM_003165. In some
embodiments, the ASO targets an intron 18 sequence downstream (or
3') from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence upstream (or 5') from the 3' splice site of a STXBP1
RIC pre-mRNA comprising the retained intron 18. In some
embodiments, the ASO targets an intron 18 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
STXBP1 RIC pre-mRNA comprising the retained intron 18.
[1242] In some embodiments, the ASO targets exon 19 or exon 20 of a
STXBP1 RIC pre-mRNA comprising a retained intron 19, wherein the
intron numbering correspond to the mRNA sequence at NM_003165. In
some embodiments, the ASO targets an exon 19 sequence upstream (or
5') from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an exon 19
sequence about 4 to about 109 nucleotides upstream (or 5') from the
5' splice site of a STXBP1 RIC pre-mRNA comprising the retained
intron 19. In some embodiments, the ASO targets an exon 20 sequence
downstream (or 3') from the 3' splice site of a STXBP1 RIC pre-mRNA
comprising the retained intron 19. In some embodiments, the ASO
targets an exon 20 sequence about 2 to about 1922 nucleotides
downstream (or 3') from the 3' splice site of a STXBP1 RIC pre-mRNA
comprising the retained intron 19.
[1243] In some embodiments, the ASO targets intron 19 in a STXBP1
RIC pre-mRNA comprising a retained intron 19, wherein the intron
numbering correspond to the mRNA sequence at NM_003165. In some
embodiments, the ASO targets an intron 19 sequence downstream (or
3') from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an intron
19 sequence about 6 to about 499 nucleotides downstream (or 3')
from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 19. In some embodiments, the ASO targets an intron
19 sequence upstream (or 5') from the 3' splice site of a STXBP1
RIC pre-mRNA comprising the retained intron 19. In some
embodiments, the ASO targets an intron 19 sequence about 16 to
about 500 nucleotides upstream (or 5') from the 3' splice site of a
STXBP1 RIC pre-mRNA comprising the retained intron 19.
[1244] In some embodiments, the ASO targets exon 18 or exon 19 of a
STXBP1 RIC pre-mRNA comprising a retained intron 18, wherein the
intron numbering correspond to the mRNA sequence at NM_001032221.
In some embodiments, the ASO targets an exon 18 sequence upstream
(or 5') from the 5' splice site of a STXBP1 RIC pre-mRNA comprising
the retained intron 18. In some embodiments, the ASO targets an
exon 18 sequence about 4 to about 136 nucleotides upstream (or 5')
from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an exon 19
sequence downstream (or 3') from the 3' splice site of a STXBP1 RIC
pre-mRNA comprising the retained intron 18. In some embodiments,
the ASO targets an exon 19 sequence about 2 to about 1922
nucleotides downstream (or 3') from the 3' splice site of a STXBP1
RIC pre-mRNA comprising the retained intron 18.
[1245] In some embodiments, the ASO targets intron 18 in a STXBP1
RIC pre-mRNA comprising a retained intron 18, wherein the intron
numbering correspond to the mRNA sequence at NM_001032221. In some
embodiments, the ASO targets an intron 18 sequence downstream (or
3') from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a STXBP1 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence upstream (or 5') from the 3' splice site of a STXBP1
RIC pre-mRNA comprising the retained intron 18. In some
embodiments, the ASO targets an intron 18 sequence about 16 to
about 500 nucleotides upstream (or 5') from the 3' splice site of a
STXBP1 RIC pre-mRNA comprising the retained intron 18.
[1246] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a EIF2B1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
EIF2B1 genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6281. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6281 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a EIF2B1 RIC pre-mRNA
sequence. In some embodiments, the ASO targets a EIF2B1 RIC
pre-mRNA transcript comprising a retained intron at 6, wherein the
intron numbering correspond to the mRNA sequence at NM_001414.
[1247] In some embodiments, the ASO targets a EIF2B1 RIC pre-mRNA
sequence according to SEQ ID NO: 6380. In some embodiments, the ASO
targets a EIF2B1 RIC pre-mRNA sequence according to SEQ ID NO: 6380
comprising a retained intron 6. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30626. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
14027-14237.
[1248] In some embodiments, the ASO targets exon 6 or exon 7 of a
EIF2B1 RIC pre-mRNA comprising a retained intron 6, wherein the
intron numbering correspond to the mRNA sequence at NM_001414. In
some embodiments, the ASO targets an exon 6 sequence upstream (or
5') from the 5' splice site of a EIF2B1 RIC pre-mRNA comprising the
retained intron 6. In some embodiments, the ASO targets an exon 6
sequence about 4 to about 49 nucleotides upstream (or 5') from the
5' splice site of a EIF2B1 RIC pre-mRNA comprising the retained
intron 6. In some embodiments, the ASO targets an exon 7 sequence
downstream (or 3') from the 3' splice site of a EIF2B1 RIC pre-mRNA
comprising the retained intron 6. In some embodiments, the ASO
targets an exon 7 sequence about 2 to about 57 nucleotides
downstream (or 3') from the 3' splice site of a EIF2B1 RIC pre-mRNA
comprising the retained intron 6.
[1249] In some embodiments, the ASO targets intron 6 in a EIF2B1
RIC pre-mRNA comprising a retained intron 6, wherein the intron
numbering correspond to the mRNA sequence at NM_001414. In some
embodiments, the ASO targets an intron 6 sequence downstream (or
3') from the 5' splice site of a EIF2B1 RIC pre-mRNA comprising the
retained intron 6. In some embodiments, the ASO targets an intron 6
sequence about 19 to about 496 nucleotides downstream (or 3') from
the 5' splice site of a EIF2B1 RIC pre-mRNA comprising the retained
intron 6. In some embodiments, the ASO targets an intron 6 sequence
upstream (or 5') from the 3' splice site of a EIF2B1 RIC pre-mRNA
comprising the retained intron 6. In some embodiments, the ASO
targets an intron 6 sequence about 16 to about 498 nucleotides
upstream (or 5') from the 3' splice site of a EIF2B1 RIC pre-mRNA
comprising the retained intron 6.
[1250] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a EIF2B2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
EIF2B2 genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6282. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6282 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a EIF2B2 RIC pre-mRNA
sequence. In some embodiments, the ASO targets a EIF2B2 RIC
pre-mRNA transcript comprising a retained intron at 1, wherein the
intron numbering correspond to the mRNA sequence at NM_014239.
[1251] In some embodiments, the ASO targets a EIF2B2 RIC pre-mRNA
sequence according to SEQ ID NO: 6309. In some embodiments, the ASO
targets a EIF2B2 RIC pre-mRNA sequence according to SEQ ID NO: 6309
comprising a retained intron 1. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30631. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
14238-14326.
[1252] In some embodiments, the ASO targets exon 1 or exon 2 of a
EIF2B2 RIC pre-mRNA comprising a retained intron 1, wherein the
intron numbering correspond to the mRNA sequence at NM_014239. In
some embodiments, the ASO targets an exon 1 sequence upstream (or
5') from the 5' splice site of a EIF2B2 RIC pre-mRNA comprising the
retained intron 1. In some embodiments, the ASO targets an exon 1
sequence about 4 to about 219 nucleotides upstream (or 5') from the
5' splice site of a EIF2B2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an exon 2 sequence
downstream (or 3') from the 3' splice site of a EIF2B2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an exon 2 sequence about 2 to about 102 nucleotides
downstream (or 3') from the 3' splice site of a EIF2B2 RIC pre-mRNA
comprising the retained intron 1.
[1253] In some embodiments, the ASO targets intron 1 in a EIF2B2
RIC pre-mRNA comprising a retained intron 1, wherein the intron
numbering correspond to the mRNA sequence at NM_014239. In some
embodiments, the ASO targets an intron 1 sequence downstream (or
3') from the 5' splice site of a EIF2B2 RIC pre-mRNA comprising the
retained intron 1. In some embodiments, the ASO targets an intron 1
sequence about 6 to about 70 nucleotides downstream (or 3') from
the 5' splice site of a EIF2B2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an intron 1 sequence
upstream (or 5') from the 3' splice site of a EIF2B2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an intron 1 sequence about 16 to about 70 nucleotides
upstream (or 5') from the 3' splice site of a EIF2B2 RIC pre-mRNA
comprising the retained intron 1.
[1254] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PRRT2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a PRRT2
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6283. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6283 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a PRRT2 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a PRRT2 RIC pre-mRNA transcript
comprising a retained intron at 1, wherein the intron numbering
correspond to the mRNA sequence at NM_001256443. In some
embodiments, the ASO targets a PRRT2 RIC pre-mRNA transcript
comprising a retained intron at 1, wherein the intron numbering
correspond to the mRNA sequence at NM_145239. In some embodiments,
the ASO targets a PRRT2 RIC pre-mRNA transcript comprising a
retained intron at 1, wherein the intron numbering correspond to
the mRNA sequence at NM_001256442.
[1255] In some embodiments, the ASO targets a PRRT2 RIC pre-mRNA
sequence according to SEQ ID NO: 6310. In some embodiments, the ASO
targets a PRRT2 RIC pre-mRNA sequence according to SEQ ID NO: 6310
comprising a retained intron 1. In some embodiments, the ASO
targets a PRRT2 RIC pre-mRNA sequence according to SEQ ID NO: 38.
In some embodiments, the ASO targets a PRRT2 RIC pre-mRNA sequence
according to SEQ ID NO: 6311 comprising a retained intron 1. In
some embodiments, the ASO targets a PRRT2 RIC pre-mRNA sequence
according to SEQ ID NO: 6312. In some embodiments, the ASO targets
a PRRT2 RIC pre-mRNA sequence according to SEQ ID NO: 6313
comprising a retained intron 1. In some embodiments, the ASOs
disclosed herein target SEQ ID NOs: 30642 or 30638. In some
embodiments, the ASO has a sequence according to any one of SEQ ID
NOs: 14327-15605.
[1256] In some embodiments, the ASO targets exon 1 or exon 2 of a
PRRT2 RIC pre-mRNA comprising a retained intron 1, wherein the
intron numbering correspond to the mRNA sequence at NM_001256443.
In some embodiments, the ASO targets an exon 1 sequence upstream
(or 5') from the 5' splice site of a PRRT2 RIC pre-mRNA comprising
the retained intron 1. In some embodiments, the ASO targets an exon
1 sequence about 4 to about 244 nucleotides upstream (or 5') from
the 5' splice site of a PRRT2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an exon 2 sequence
downstream (or 3') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an exon 2 sequence about 2 to about 2875 nucleotides
downstream (or 3') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1.
[1257] In some embodiments, the ASO targets intron 1 in a PRRT2 RIC
pre-mRNA comprising a retained intron 1, wherein the intron
numbering correspond to the mRNA sequence at NM_001256443. In some
embodiments, the ASO targets an intron 1 sequence downstream (or
3') from the 5' splice site of a PRRT2 RIC pre-mRNA comprising the
retained intron 1. In some embodiments, the ASO targets an intron 1
sequence about 6 to about 333 nucleotides downstream (or 3') from
the 5' splice site of a PRRT2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an intron 1 sequence
upstream (or 5') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an intron 1 sequence about 16 to about 330 or about 26 to
about 330 nucleotides upstream (or 5') from the 3' splice site of a
PRRT2 RIC pre-mRNA comprising the retained intron 1.
[1258] In some embodiments, the ASO targets exon 1 or exon 2 of a
PRRT2 RIC pre-mRNA comprising a retained intron 1, wherein the
intron numbering correspond to the mRNA sequence at NM_145239. In
some embodiments, the ASO targets an exon 1 sequence upstream (or
5') from the 5' splice site of a PRRT2 RIC pre-mRNA comprising the
retained intron 1. In some embodiments, the ASO targets an exon 1
sequence about 4 to about 219 nucleotides upstream (or 5') from the
5' splice site of a PRRT2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an exon 2 sequence
downstream (or 3') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an exon 2 sequence about 2 to about 924 nucleotides
downstream (or 3') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1.
[1259] In some embodiments, the ASO targets intron 1 in a PRRT2 RIC
pre-mRNA comprising a retained intron 1, wherein the intron
numbering correspond to the mRNA sequence at NM_145239. In some
embodiments, the ASO targets an intron 1 sequence downstream (or
3') from the 5' splice site of a PRRT2 RIC pre-mRNA comprising the
retained intron 1. In some embodiments, the ASO targets an intron 1
sequence about 6 to about 346 nucleotides downstream (or 3') from
the 5' splice site of a PRRT2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an intron 1 sequence
upstream (or 5') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an intron 1 sequence about 16 to about 345 nucleotides
upstream (or 5') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1.
[1260] In some embodiments, the ASO targets exon 1 or exon 2 of a
PRRT2 RIC pre-mRNA comprising a retained intron 1, wherein the
intron numbering correspond to the mRNA sequence at NM_001256442.
In some embodiments, the ASO targets an exon 1 sequence upstream
(or 5') from the 5' splice site of a PRRT2 RIC pre-mRNA comprising
the retained intron 1. In some embodiments, the ASO targets an exon
1 sequence about 4 to about 219 nucleotides upstream (or 5') from
the 5' splice site of a PRRT2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an exon 2 sequence
downstream (or 3') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an exon 2 sequence about 2 to about 924 nucleotides
downstream (or 3') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1.
[1261] In some embodiments, the ASO targets intron 1 in a PRRT2 RIC
pre-mRNA comprising a retained intron 1, wherein the intron
numbering correspond to the mRNA sequence at NM_001256442. In some
embodiments, the ASO targets an intron 1 sequence downstream (or
3') from the 5' splice site of a PRRT2 RIC pre-mRNA comprising the
retained intron 1. In some embodiments, the ASO targets an intron 1
sequence about 6 to about 346 nucleotides downstream (or 3') from
the 5' splice site of a PRRT2 RIC pre-mRNA comprising the retained
intron 1. In some embodiments, the ASO targets an intron 1 sequence
upstream (or 5') from the 3' splice site of a PRRT2 RIC pre-mRNA
comprising the retained intron 1. In some embodiments, the ASO
targets an intron 1 sequence about 16 to about 345 or about 26 to
about 345 nucleotides upstream (or 5') from the 3' splice site of a
PRRT2 RIC pre-mRNA comprising the retained intron 1.
[1262] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a STX1B genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a STX1B
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6284. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6284 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a STX1B RIC pre-mRNA sequence. In some
embodiments, the ASO targets a STX1B RIC pre-mRNA transcript
comprising a retained intron at 6, 7 or a combination thereof,
wherein the intron numbering correspond to the mRNA sequence at
NM_052874.
[1263] In some embodiments, the ASO targets a STX1B RIC pre-mRNA
sequence according to SEQ ID NO: 6313. In some embodiments, the ASO
targets a STX1B RIC pre-mRNA sequence according to SEQ ID NO: 6313
comprising a retained intron 6, a retained intron 7, or a
combination thereof. In some embodiments, the ASOs disclosed herein
target SEQ ID NOs: 30658 or 30648. In some embodiments, the ASO has
a sequence according to any one of SEQ ID NOs: 15606-15873.
[1264] In some embodiments, the ASO targets exon 6 or exon 7 of a
STX1B RIC pre-mRNA comprising a retained intron 6, wherein the
intron numbering correspond to the mRNA sequence at NM_052874. In
some embodiments, the ASO targets an exon 6 sequence upstream (or
5') from the 5' splice site of a STX1B RIC pre-mRNA comprising the
retained intron 6. In some embodiments, the ASO targets an exon 6
sequence about 4 to about 89 nucleotides upstream (or 5') from the
5' splice site of a STX1B RIC pre-mRNA comprising the retained
intron 6. In some embodiments, the ASO targets an exon 7 sequence
downstream (or 3') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 6. In some embodiments, the ASO
targets an exon 7 sequence about 2 to about 57 nucleotides
downstream (or 3') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 6.
[1265] In some embodiments, the ASO targets intron 6 in a STX1B RIC
pre-mRNA comprising a retained intron 6, wherein the intron
numbering correspond to the mRNA sequence at NM_052874. In some
embodiments, the ASO targets an intron 6 sequence downstream (or
3') from the 5' splice site of a STX1B RIC pre-mRNA comprising the
retained intron 6. In some embodiments, the ASO targets an intron 6
sequence about 6 to about 99 nucleotides downstream (or 3') from
the 5' splice site of a STX1B RIC pre-mRNA comprising the retained
intron 6. In some embodiments, the ASO targets an intron 6 sequence
upstream (or 5') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 6. In some embodiments, the ASO
targets an intron 6 sequence about 16 to about 94 nucleotides
upstream (or 5') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 6.
[1266] In some embodiments, the ASO targets exon 7 or exon 8 of a
STX1B RIC pre-mRNA comprising a retained intron 7, wherein the
intron numbering correspond to the mRNA sequence at NM_052874. In
some embodiments, the ASO targets an exon 7 sequence upstream (or
5') from the 5' splice site of a STX1B RIC pre-mRNA comprising the
retained intron 7. In some embodiments, the ASO targets an exon 7
sequence about 4 to about 54 nucleotides upstream (or 5') from the
5' splice site of a STX1B RIC pre-mRNA comprising the retained
intron 7. In some embodiments, the ASO targets an exon 8 sequence
downstream (or 3') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 7. In some embodiments, the ASO
targets an exon 8 sequence about 2 to about 117 nucleotides
downstream (or 3') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 7.
[1267] In some embodiments, the ASO targets intron 7 in a STX1B RIC
pre-mRNA comprising a retained intron 7, wherein the intron
numbering correspond to the mRNA sequence at NM_052874. In some
embodiments, the ASO targets an intron 7 sequence downstream (or
3') from the 5' splice site of a STX1B RIC pre-mRNA comprising the
retained intron 7. In some embodiments, the ASO targets an intron 7
sequence about 6 to about 500 nucleotides downstream (or 3') from
the 5' splice site of a STX1B RIC pre-mRNA comprising the retained
intron 7. In some embodiments, the ASO targets an intron 7 sequence
upstream (or 5') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 7. In some embodiments, the ASO
targets an intron 7 sequence about 16 to about 496 nucleotides
upstream (or 5') from the 3' splice site of a STX1B RIC pre-mRNA
comprising the retained intron 7.
[1268] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a RAI1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a RAI1
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6285. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6285 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a RAI1 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a RAI1 RIC pre-mRNA transcript
comprising a retained intron at 4, wherein the intron numbering
correspond to the mRNA sequence at NM_030665.
[1269] In some embodiments, the ASO targets a RAI1 RIC pre-mRNA
sequence according to SEQ ID NO: 6314. In some embodiments, the ASO
targets a RAI1 RIC pre-mRNA sequence according to SEQ ID NO: 6314
comprising a retained intron 4. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30655. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
15874-16076.
[1270] In some embodiments, the ASO targets exon 4 or exon 5 of a
RAI1 RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_030665. In
some embodiments, the ASO targets an exon 4 sequence upstream (or
5') from the 5' splice site of a RAI1 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an exon 4
sequence about 4 to about 74 nucleotides upstream (or 5') from the
5' splice site of a RAI1 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a RAI1 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 32 nucleotides
downstream (or 3') from the 3' splice site of a RAI1 RIC pre-mRNA
comprising the retained intron 4.
[1271] In some embodiments, the ASO targets intron 4 in a RAI1 RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_030665. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a RAI1 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 6 to about 500 nucleotides downstream (or 3') from
the 5' splice site of a RAI1 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a RAI1 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 499 nucleotides
upstream (or 5') from the 3' splice site of a RAI1 RIC pre-mRNA
comprising the retained intron 4.
[1272] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a TCF4 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a TCF4
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6286. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6286 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a TCF4 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 10, wherein the intron numbering
correspond to the mRNA sequence at NM_001243236. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 10, wherein the intron numbering
correspond to the mRNA sequence at NM_001243235. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 10, wherein the intron numbering
correspond to the mRNA sequence at NM_001243234. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 13, wherein the intron numbering
correspond to the mRNA sequence at NM_001243233. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 13, wherein the intron numbering
correspond to the mRNA sequence at NM_001243232. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 15, wherein the intron numbering
correspond to the mRNA sequence at NM_001243231. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 17, wherein the intron numbering
correspond to the mRNA sequence at NM_003199. In some embodiments,
the ASO targets a TCF4 RIC pre-mRNA transcript comprising a
retained intron at 16, wherein the intron numbering correspond to
the mRNA sequence at NM_001306207. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA transcript comprising a retained intron
at 13, wherein the intron numbering correspond to the mRNA sequence
at NM_001306208. In some embodiments, the ASO targets a TCF4 RIC
pre-mRNA transcript comprising a retained intron at 16, wherein the
intron numbering correspond to the mRNA sequence at NM_001243227.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 17, wherein the intron numbering
correspond to the mRNA sequence at NM_001243228. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 16, wherein the intron numbering
correspond to the mRNA sequence at NM_001243230. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 18, wherein the intron numbering
correspond to the mRNA sequence at NM_001243226. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 17, wherein the intron numbering
correspond to the mRNA sequence at NM_001083962. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 12, wherein the intron numbering
correspond to the mRNA sequence at NM_001330605. In some
embodiments, the ASO targets a TCF4 RIC pre-mRNA transcript
comprising a retained intron at 17, wherein the intron numbering
correspond to the mRNA sequence at NM_001330604.
[1273] In some embodiments, the ASO targets a TCF4 RIC pre-mRNA
sequence according to SEQ ID NO: 6315. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6315
comprising a retained intron 10. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6316.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6316 comprising a retained intron 10. In
some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6317. In some embodiments, the ASO targets
a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6317
comprising a retained intron 10. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6318.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6318 comprising a retained intron 13. In
some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6319. In some embodiments, the ASO targets
a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6319
comprising a retained intron 13. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6320.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6320 comprising a retained intron 15. In
some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6321. In some embodiments, the ASO targets
a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6321
comprising a retained intron 17. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6322.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6322 comprising a retained intron 16. In
some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6323. In some embodiments, the ASO targets
a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6323
comprising a retained intron 13. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6324.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6324 comprising a retained intron 16. In
some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6325. In some embodiments, the ASO targets
a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6325
comprising a retained intron 17. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6326.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6326 comprising a retained intron 16. In
some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6327. In some embodiments, the ASO targets
a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6327
comprising a retained intron 18. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6328.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6328 comprising a retained intron 17. In
some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6329. In some embodiments, the ASO targets
a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6329
comprising a retained intron 12. In some embodiments, the ASO
targets a TCF4 RIC pre-mRNA sequence according to SEQ ID NO: 6330.
In some embodiments, the ASO targets a TCF4 RIC pre-mRNA sequence
according to SEQ ID NO: 6330 comprising a retained intron 17. In
some embodiments, the ASOs disclosed herein target SEQ ID NOs:
34788 or 34789. In some embodiments, the ASO has a sequence
according to any one of SEQ ID NOs: 30660-30916.
[1274] In some embodiments, the ASO targets exon 10 or exon 11 of a
TCF4 RIC pre-mRNA comprising a retained intron 10, wherein the
intron numbering correspond to the mRNA sequence at NM_001243236.
In some embodiments, the ASO targets an exon 10 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 10. In some embodiments, the ASO targets an
exon 10 sequence about 4 to about 134 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an exon 11
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an exon 11 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 10.
[1275] In some embodiments, the ASO targets intron 10 in a TCF4 RIC
pre-mRNA comprising a retained intron 10, wherein the intron
numbering correspond to the mRNA sequence at NM_001243236. In some
embodiments, the ASO targets an intron 10 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an intron 10 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10.
[1276] In some embodiments, the ASO targets exon 10 or exon 11 of a
TCF4 RIC pre-mRNA comprising a retained intron 10, wherein the
intron numbering correspond to the mRNA sequence at NM_001243235.
In some embodiments, the ASO targets an exon 10 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 10. In some embodiments, the ASO targets an
exon 10 sequence about 4 to about 134 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an exon 11
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an exon 11 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 10.
[1277] In some embodiments, the ASO targets intron 10 in a TCF4 RIC
pre-mRNA comprising a retained intron 10, wherein the intron
numbering correspond to the mRNA sequence at NM_001243235. In some
embodiments, the ASO targets an intron 10 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an intron 10 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10.
[1278] In some embodiments, the ASO targets exon 10 or exon 11 of a
TCF4 RIC pre-mRNA comprising a retained intron 10, wherein the
intron numbering correspond to the mRNA sequence at NM_001243234.
In some embodiments, the ASO targets an exon 10 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 10. In some embodiments, the ASO targets an
exon 10 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an exon 11
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an exon 11 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 10.
[1279] In some embodiments, the ASO targets intron 10 in a TCF4 RIC
pre-mRNA comprising a retained intron 10, wherein the intron
numbering correspond to the mRNA sequence at NM_001243234. In some
embodiments, the ASO targets an intron 10 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 10. In some embodiments, the ASO targets an intron
10 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10. In some embodiments,
the ASO targets an intron 10 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 10.
[1280] In some embodiments, the ASO targets exon 13 or exon 14 of a
TCF4 RIC pre-mRNA comprising a retained intron 13, wherein the
intron numbering correspond to the mRNA sequence at NM_001243233.
In some embodiments, the ASO targets an exon 13 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 13. In some embodiments, the ASO targets an
exon 13 sequence about 4 to about 134 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an exon 14
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an exon 14 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 13.
[1281] In some embodiments, the ASO targets intron 13 in a TCF4 RIC
pre-mRNA comprising a retained intron 13, wherein the intron
numbering correspond to the mRNA sequence at NM_001243233. In some
embodiments, the ASO targets an intron 13 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an intron 13 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13.
[1282] In some embodiments, the ASO targets exon 13 or exon 14 of a
TCF4 RIC pre-mRNA comprising a retained intron 13, wherein the
intron numbering correspond to the mRNA sequence at NM_001243232.
In some embodiments, the ASO targets an exon 13 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 13. In some embodiments, the ASO targets an
exon 13 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an exon 14
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an exon 14 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 13.
[1283] In some embodiments, the ASO targets intron 13 in a TCF4 RIC
pre-mRNA comprising a retained intron 13, wherein the intron
numbering correspond to the mRNA sequence at NM_001243232. In some
embodiments, the ASO targets an intron 13 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an intron 13 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13.
[1284] In some embodiments, the ASO targets exon 15 or exon 16 of a
TCF4 RIC pre-mRNA comprising a retained intron 15, wherein the
intron numbering correspond to the mRNA sequence at NM_001243231.
In some embodiments, the ASO targets an exon 15 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 15. In some embodiments, the ASO targets an
exon 15 sequence about 4 to about 134 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an exon 16
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an exon 16 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 15.
[1285] In some embodiments, the ASO targets intron 15 in a TCF4 RIC
pre-mRNA comprising a retained intron 15, wherein the intron
numbering correspond to the mRNA sequence at NM_001243231. In some
embodiments, the ASO targets an intron 15 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an intron 15 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 15.
[1286] In some embodiments, the ASO targets exon 17 or exon 18 of a
TCF4 RIC pre-mRNA comprising a retained intron 17, wherein the
intron numbering correspond to the mRNA sequence at NM_003199. In
some embodiments, the ASO targets an exon 17 sequence upstream (or
5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an exon 17
sequence about 4 to about 134 nucleotides upstream (or 5') from the
5' splice site of a TCF4 RIC pre-mRNA comprising the retained
intron 17. In some embodiments, the ASO targets an exon 18 sequence
downstream (or 3') from the 3' splice site of a TCF4 RIC pre-mRNA
comprising the retained intron 17. In some embodiments, the ASO
targets an exon 18 sequence about 2 to about 212 nucleotides
downstream (or 3') from the 3' splice site of a TCF4 RIC pre-mRNA
comprising the retained intron 17.
[1287] In some embodiments, the ASO targets intron 17 in a TCF4 RIC
pre-mRNA comprising a retained intron 17, wherein the intron
numbering correspond to the mRNA sequence at NM_003199. In some
embodiments, the ASO targets an intron 17 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17. In some embodiments,
the ASO targets an intron 17 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17.
[1288] In some embodiments, the ASO targets exon 16 or exon 17 of a
TCF4 RIC pre-mRNA comprising a retained intron 16, wherein the
intron numbering correspond to the mRNA sequence at NM_001306207.
In some embodiments, the ASO targets an exon 16 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 16. In some embodiments, the ASO targets an
exon 16 sequence about 4 to about 134 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an exon 17
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an exon 17 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 16.
[1289] In some embodiments, the ASO targets intron 16 in a TCF4 RIC
pre-mRNA comprising a retained intron 16, wherein the intron
numbering correspond to the mRNA sequence at NM_001306207. In some
embodiments, the ASO targets an intron 16 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an intron 16 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16.
[1290] In some embodiments, the ASO targets exon 13 or exon 14 of a
TCF4 RIC pre-mRNA comprising a retained intron 13, wherein the
intron numbering correspond to the mRNA sequence at NM_001306208.
In some embodiments, the ASO targets an exon 13 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 13. In some embodiments, the ASO targets an
exon 13 sequence about 4 to about 134 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an exon 14
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an exon 14 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 13.
[1291] In some embodiments, the ASO targets intron 13 in a TCF4 RIC
pre-mRNA comprising a retained intron 13, wherein the intron
numbering correspond to the mRNA sequence at NM_001306208. In some
embodiments, the ASO targets an intron 13 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 13. In some embodiments, the ASO targets an intron
13 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13. In some embodiments,
the ASO targets an intron 13 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 13.
[1292] In some embodiments, the ASO targets exon 16 or exon 17 of a
TCF4 RIC pre-mRNA comprising a retained intron 16, wherein the
intron numbering correspond to the mRNA sequence at NM_001243227.
In some embodiments, the ASO targets an exon 16 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 16. In some embodiments, the ASO targets an
exon 16 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an exon 17
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an exon 17 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 16.
[1293] In some embodiments, the ASO targets intron 16 in a TCF4 RIC
pre-mRNA comprising a retained intron 16, wherein the intron
numbering correspond to the mRNA sequence at NM_001243227. In some
embodiments, the ASO targets an intron 16 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an intron 16 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16.
[1294] In some embodiments, the ASO targets exon 17 or exon 18 of a
TCF4 RIC pre-mRNA comprising a retained intron 17, wherein the
intron numbering correspond to the mRNA sequence at NM_001243228.
In some embodiments, the ASO targets an exon 17 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 17. In some embodiments, the ASO targets an
exon 17 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an exon 18
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17. In some embodiments,
the ASO targets an exon 18 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 17.
[1295] In some embodiments, the ASO targets intron 17 in a TCF4 RIC
pre-mRNA comprising a retained intron 17, wherein the intron
numbering correspond to the mRNA sequence at NM_001243228. In some
embodiments, the ASO targets an intron 17 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17. In some embodiments,
the ASO targets an intron 17 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17.
[1296] In some embodiments, the ASO targets exon 16 or exon 17 of a
TCF4 RIC pre-mRNA comprising a retained intron 16, wherein the
intron numbering correspond to the mRNA sequence at NM_001243230.
In some embodiments, the ASO targets an exon 16 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 16. In some embodiments, the ASO targets an
exon 16 sequence about 4 to about 134 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an exon 17
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an exon 17 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 16.
[1297] In some embodiments, the ASO targets intron 16 in a TCF4 RIC
pre-mRNA comprising a retained intron 16, wherein the intron
numbering correspond to the mRNA sequence at NM_001243230. In some
embodiments, the ASO targets an intron 16 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an intron 16 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 16.
[1298] In some embodiments, the ASO targets exon 18 or exon 19 of a
TCF4 RIC pre-mRNA comprising a retained intron 18, wherein the
intron numbering correspond to the mRNA sequence at NM_001243226.
In some embodiments, the ASO targets an exon 18 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 18. In some embodiments, the ASO targets an
exon 18 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an exon 19
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 18. In some embodiments,
the ASO targets an exon 19 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 18.
[1299] In some embodiments, the ASO targets intron 18 in a TCF4 RIC
pre-mRNA comprising a retained intron 18, wherein the intron
numbering correspond to the mRNA sequence at NM_001243226. In some
embodiments, the ASO targets an intron 18 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 18. In some embodiments, the ASO targets an intron
18 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 18. In some embodiments,
the ASO targets an intron 18 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 18.
[1300] In some embodiments, the ASO targets exon 17 or exon 18 of a
TCF4 RIC pre-mRNA comprising a retained intron 17, wherein the
intron numbering correspond to the mRNA sequence at NM_001083962.
In some embodiments, the ASO targets an exon 17 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 17. In some embodiments, the ASO targets an
exon 17 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an exon 18
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17. In some embodiments,
the ASO targets an exon 18 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 17.
[1301] In some embodiments, the ASO targets intron 17 in a TCF4 RIC
pre-mRNA comprising a retained intron 17, wherein the intron
numbering correspond to the mRNA sequence at NM_001083962. In some
embodiments, the ASO targets an intron 17 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17. In some embodiments,
the ASO targets an intron 17 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17.
[1302] In some embodiments, the ASO targets exon 12 or exon 13 of a
TCF4 RIC pre-mRNA comprising a retained intron 12, wherein the
intron numbering correspond to the mRNA sequence at NM_001330605.
In some embodiments, the ASO targets an exon 12 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 12. In some embodiments, the ASO targets an
exon 12 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 12. In some embodiments, the ASO targets an exon 13
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 12. In some embodiments,
the ASO targets an exon 13 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 12.
[1303] In some embodiments, the ASO targets intron 12 in a TCF4 RIC
pre-mRNA comprising a retained intron 12, wherein the intron
numbering correspond to the mRNA sequence at NM_001330605. In some
embodiments, the ASO targets an intron 12 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 12. In some embodiments, the ASO targets an intron
12 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 12. In some embodiments, the ASO targets an intron
12 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 12. In some embodiments,
the ASO targets an intron 12 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 12.
[1304] In some embodiments, the ASO targets exon 17 or exon 18 of a
TCF4 RIC pre-mRNA comprising a retained intron 17, wherein the
intron numbering correspond to the mRNA sequence at NM_001330604.
In some embodiments, the ASO targets an exon 17 sequence upstream
(or 5') from the 5' splice site of a TCF4 RIC pre-mRNA comprising
the retained intron 17. In some embodiments, the ASO targets an
exon 17 sequence about 4 to about 144 nucleotides upstream (or 5')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an exon 18
sequence downstream (or 3') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17. In some embodiments,
the ASO targets an exon 18 sequence about 2 to about 212
nucleotides downstream (or 3') from the 3' splice site of a TCF4
RIC pre-mRNA comprising the retained intron 17.
[1305] In some embodiments, the ASO targets intron 17 in a TCF4 RIC
pre-mRNA comprising a retained intron 17, wherein the intron
numbering correspond to the mRNA sequence at NM_001330604. In some
embodiments, the ASO targets an intron 17 sequence downstream (or
3') from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence about 6 to about 496 nucleotides downstream (or 3')
from the 5' splice site of a TCF4 RIC pre-mRNA comprising the
retained intron 17. In some embodiments, the ASO targets an intron
17 sequence upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17. In some embodiments,
the ASO targets an intron 17 sequence about 16 to about 496
nucleotides upstream (or 5') from the 3' splice site of a TCF4 RIC
pre-mRNA comprising the retained intron 17.
[1306] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a NPC1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a NPC1
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6287. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6287 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a NPC1 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a NPC1 RIC pre-mRNA transcript
comprising a retained intron at 15, wherein the intron numbering
correspond to the mRNA sequence at NM_000271.
[1307] In some embodiments, the ASO targets a NPC1 RIC pre-mRNA
sequence according to SEQ ID NO: 6331. In some embodiments, the ASO
targets a NPC1 RIC pre-mRNA sequence according to SEQ ID NO: 6331
comprising a retained intron 15. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30655. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
19965-20033.
[1308] In some embodiments, the ASO targets exon 15 or exon 16 of a
NPC1 RIC pre-mRNA comprising a retained intron 15, wherein the
intron numbering correspond to the mRNA sequence at NM_000271. In
some embodiments, the ASO targets an exon 15 sequence upstream (or
5') from the 5' splice site of a NPC1 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an exon 15
sequence about 4 to about 109 nucleotides upstream (or 5') from the
5' splice site of a NPC1 RIC pre-mRNA comprising the retained
intron 15. In some embodiments, the ASO targets an exon 16 sequence
downstream (or 3') from the 3' splice site of a NPC1 RIC pre-mRNA
comprising the retained intron 15. In some embodiments, the ASO
targets an exon 16 sequence about 2 to about 122 nucleotides
downstream (or 3') from the 3' splice site of a NPC1 RIC pre-mRNA
comprising the retained intron 15.
[1309] In some embodiments, the ASO targets intron 15 in a NPC1 RIC
pre-mRNA comprising a retained intron 15, wherein the intron
numbering correspond to the mRNA sequence at NM_000271. In some
embodiments, the ASO targets an intron 15 sequence downstream (or
3') from the 5' splice site of a NPC1 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence about 6 to about 61 nucleotides downstream (or 3') from
the 5' splice site of a NPC1 RIC pre-mRNA comprising the retained
intron 15. In some embodiments, the ASO targets an intron 15
sequence upstream (or 5') from the 3' splice site of a NPC1 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an intron 15 sequence about 16 to about 61
nucleotides upstream (or 5') from the 3' splice site of a NPC1 RIC
pre-mRNA comprising the retained intron 15.
[1310] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a CACNA1A genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
CACNA1A genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6288. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6288 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a CACNA1A RIC
pre-mRNA sequence. In some embodiments, the ASO targets a CACNA1A
RIC pre-mRNA transcript comprising a retained intron at 36, 37 or a
combination thereof, wherein the intron numbering correspond to the
mRNA sequence at NM_000068. In some embodiments, the ASO targets a
CACNA1A RIC pre-mRNA transcript comprising a retained intron at 36,
37 or a combination thereof, wherein the intron numbering
correspond to the mRNA sequence at NM_023035. In some embodiments,
the ASO targets a CACNA1A RIC pre-mRNA transcript comprising a
retained intron at 36, 37 or a combination thereof, wherein the
intron numbering correspond to the mRNA sequence at NM_001174080.
In some embodiments, the ASO targets a CACNA1A RIC pre-mRNA
transcript comprising a retained intron at 36, 37 or a combination
thereof, wherein the intron numbering correspond to the mRNA
sequence at NM_001127222. In some embodiments, the ASO targets a
CACNA1A RIC pre-mRNA transcript comprising a retained intron at 36,
37 or a combination thereof, wherein the intron numbering
correspond to the mRNA sequence at NM_001127221.
[1311] In some embodiments, the ASO targets a CACNA1A RIC pre-mRNA
sequence according to SEQ ID NO: 6332. In some embodiments, the ASO
targets a CACNA1A RIC pre-mRNA sequence according to SEQ ID NO:
6332 comprising a retained intron 36, a retained intron 37, or a
combination thereof. In some embodiments, the ASO targets a CACNA1A
RIC pre-mRNA sequence according to SEQ ID NO: 6333. In some
embodiments, the ASO targets a CACNA1A RIC pre-mRNA sequence
according to SEQ ID NO: 6333 comprising a retained intron 36, a
retained intron 37, or a combination thereof. In some embodiments,
the ASO targets a CACNA1A RIC pre-mRNA sequence according to SEQ ID
NO: 6334. In some embodiments, the ASO targets a CACNA1A RIC
pre-mRNA sequence according to SEQ ID NO: 6334 comprising a
retained intron 36, a retained intron 37, or a combination thereof.
In some embodiments, the ASO targets a CACNA1A RIC pre-mRNA
sequence according to SEQ ID NO: 6335. In some embodiments, the ASO
targets a CACNA1A RIC pre-mRNA sequence according to SEQ ID NO:
6335 comprising a retained intron 36, a retained intron 37, or a
combination thereof. In some embodiments, the ASO targets a CACNA1A
RIC pre-mRNA sequence according to SEQ ID NO: 6336. In some
embodiments, the ASO targets a CACNA1A RIC pre-mRNA sequence
according to SEQ ID NO: 6336 comprising a retained intron 36, a
retained intron 37, or a combination thereof. In some embodiments,
the ASOs disclosed herein target SEQ ID NOs: 30639, 30647, 30650,
or 30657. In some embodiments, the ASO has a sequence according to
any one of SEQ ID NOs: 20034-22187.
[1312] In some embodiments, the ASO targets exon 36 or exon 37 of a
CACNA1A RIC pre-mRNA comprising a retained intron 36, wherein the
intron numbering correspond to the mRNA sequence at NM_000068. In
some embodiments, the ASO targets an exon 36 sequence upstream (or
5') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
exon 36 sequence about 4 to about 110 nucleotides upstream (or 5')
from the 5' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36. In some embodiments, the ASO targets an exon 37
sequence downstream (or 3') from the 3' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an exon 37 sequence about 2 to about
79 nucleotides downstream (or 3') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 36.
[1313] In some embodiments, the ASO targets intron 36 in a CACNA1A
RIC pre-mRNA comprising a retained intron 36, wherein the intron
numbering correspond to the mRNA sequence at NM_000068. In some
embodiments, the ASO targets an intron 36 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
intron 36 sequence about 6 to about 499 nucleotides downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
intron 36 sequence upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an intron 36 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 36.
[1314] In some embodiments, the ASO targets exon 37 or exon 38 of a
CACNA1A RIC pre-mRNA comprising a retained intron 37, wherein the
intron numbering correspond to the mRNA sequence at NM_000068. In
some embodiments, the ASO targets an exon 37 sequence upstream (or
5') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
exon 37 sequence about 4 to about 78 nucleotides upstream (or 5')
from the 5' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 37. In some embodiments, the ASO targets an exon 38
sequence downstream (or 3') from the 3' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 37. In some
embodiments, the ASO targets an exon 38 sequence about 2 to about
87 nucleotides downstream (or 3') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1315] In some embodiments, the ASO targets intron 37 in a CACNA1A
RIC pre-mRNA comprising a retained intron 37, wherein the intron
numbering correspond to the mRNA sequence at NM_000068. In some
embodiments, the ASO targets an intron 37 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence about 6 to about 500 nucleotides downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37. In some
embodiments, the ASO targets an intron 37 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1316] In some embodiments, the ASO targets exon 36 or exon 37 of a
CACNA1A RIC pre-mRNA comprising a retained intron 36, wherein the
intron numbering correspond to the mRNA sequence at NM_023035. In
some embodiments, the ASO targets an exon 36 sequence upstream (or
5') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
exon 36 sequence about 4 to about 110 nucleotides upstream (or 5')
from the 5' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36. In some embodiments, the ASO targets an exon 37
sequence downstream (or 3') from the 3' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an exon 37 sequence about 2 to about
79 nucleotides downstream (or 3') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 36.
[1317] In some embodiments, the ASO targets intron 36 in a CACNA1A
RIC pre-mRNA comprising a retained intron 36, wherein the intron
numbering correspond to the mRNA sequence at NM_023035. In some
embodiments, the ASO targets an intron 36 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
intron 36 sequence about 6 to about 499 nucleotides downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
intron 36 sequence upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an intron 36 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 36.
[1318] In some embodiments, the ASO targets exon 37 or exon 38 of a
CACNA1A RIC pre-mRNA comprising a retained intron 37, wherein the
intron numbering correspond to the mRNA sequence at NM_023035. In
some embodiments, the ASO targets an exon 37 sequence upstream (or
5') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
exon 37 sequence about 4 to about 78 nucleotides upstream (or 5')
from the 5' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 37. In some embodiments, the ASO targets an exon 38
sequence downstream (or 3') from the 3' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 37. In some
embodiments, the ASO targets an exon 38 sequence about 2 to about
87 nucleotides downstream (or 3') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1319] In some embodiments, the ASO targets intron 37 in a CACNA1A
RIC pre-mRNA comprising a retained intron 37, wherein the intron
numbering correspond to the mRNA sequence at NM_023035. In some
embodiments, the ASO targets an intron 37 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence about 6 to about 500 nucleotides downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37. In some
embodiments, the ASO targets an intron 37 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1320] In some embodiments, the ASO targets exon 36 or exon 37 of a
CACNA1A RIC pre-mRNA comprising a retained intron 36, wherein the
intron numbering correspond to the mRNA sequence at NM_001174080.
In some embodiments, the ASO targets an exon 36 sequence upstream
(or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 36. In some embodiments, the ASO
targets an exon 36 sequence about 4 to about 110 nucleotides
upstream (or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 36. In some embodiments, the ASO
targets an exon 37 sequence downstream (or 3') from the 3' splice
site of a CACNA1A RIC pre-mRNA comprising the retained intron 36.
In some embodiments, the ASO targets an exon 37 sequence about 2 to
about 79 nucleotides downstream (or 3') from the 3' splice site of
a CACNA1A RIC pre-mRNA comprising the retained intron 36.
[1321] In some embodiments, the ASO targets intron 36 in a CACNA1A
RIC pre-mRNA comprising a retained intron 36, wherein the intron
numbering correspond to the mRNA sequence at NM_001174080. In some
embodiments, the ASO targets an intron 36 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
intron 36 sequence about 6 to about 499 or about 16 to about 499
nucleotides downstream (or 3') from the 5' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an intron 36 sequence upstream (or 5')
from the 3' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36. In some embodiments, the ASO targets an intron
36 sequence about 16 to about 498 nucleotides upstream (or 5') from
the 3' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36.
[1322] In some embodiments, the ASO targets exon 37 or exon 38 of a
CACNA1A RIC pre-mRNA comprising a retained intron 37, wherein the
intron numbering correspond to the mRNA sequence at NM_001174080.
In some embodiments, the ASO targets an exon 37 sequence upstream
(or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 37. In some embodiments, the ASO
targets an exon 37 sequence about 4 to about 78 nucleotides
upstream (or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 37. In some embodiments, the ASO
targets an exon 38 sequence downstream (or 3') from the 3' splice
site of a CACNA1A RIC pre-mRNA comprising the retained intron 37.
In some embodiments, the ASO targets an exon 38 sequence about 2 to
about 87 nucleotides downstream (or 3') from the 3' splice site of
a CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1323] In some embodiments, the ASO targets intron 37 in a CACNA1A
RIC pre-mRNA comprising a retained intron 37, wherein the intron
numbering correspond to the mRNA sequence at NM_001174080. In some
embodiments, the ASO targets an intron 37 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence about 6 to about 500 nucleotides downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37. In some
embodiments, the ASO targets an intron 37 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1324] In some embodiments, the ASO targets exon 36 or exon 37 of a
CACNA1A RIC pre-mRNA comprising a retained intron 36, wherein the
intron numbering correspond to the mRNA sequence at NM_001127222.
In some embodiments, the ASO targets an exon 36 sequence upstream
(or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 36. In some embodiments, the ASO
targets an exon 36 sequence about 4 to about 110 or about 14 to
about 110 nucleotides upstream (or 5') from the 5' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an exon 37 sequence downstream (or 3')
from the 3' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36. In some embodiments, the ASO targets an exon 37
sequence about 2 to about 79 nucleotides downstream (or 3') from
the 3' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36.
[1325] In some embodiments, the ASO targets intron 36 in a CACNA1A
RIC pre-mRNA comprising a retained intron 36, wherein the intron
numbering correspond to the mRNA sequence at NM_001127222. In some
embodiments, the ASO targets an intron 36 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
intron 36 sequence about 6 to about 499 or about 16 to about 499
nucleotides downstream (or 3') from the 5' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an intron 36 sequence upstream (or 5')
from the 3' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36. In some embodiments, the ASO targets an intron
36 sequence about 16 to about 498 nucleotides upstream (or 5') from
the 3' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36.
[1326] In some embodiments, the ASO targets exon 37 or exon 38 of a
CACNA1A RIC pre-mRNA comprising a retained intron 37, wherein the
intron numbering correspond to the mRNA sequence at NM_001127222.
In some embodiments, the ASO targets an exon 37 sequence upstream
(or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 37. In some embodiments, the ASO
targets an exon 37 sequence about 4 to about 78 nucleotides
upstream (or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 37. In some embodiments, the ASO
targets an exon 38 sequence downstream (or 3') from the 3' splice
site of a CACNA1A RIC pre-mRNA comprising the retained intron 37.
In some embodiments, the ASO targets an exon 38 sequence about 2 to
about 87 nucleotides downstream (or 3') from the 3' splice site of
a CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1327] In some embodiments, the ASO targets intron 37 in a CACNA1A
RIC pre-mRNA comprising a retained intron 37, wherein the intron
numbering correspond to the mRNA sequence at NM_001127222. In some
embodiments, the ASO targets an intron 37 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence about 6 to about 500 nucleotides downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37. In some
embodiments, the ASO targets an intron 37 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1328] In some embodiments, the ASO targets exon 36 or exon 37 of a
CACNA1A RIC pre-mRNA comprising a retained intron 36, wherein the
intron numbering correspond to the mRNA sequence at NM_001127221.
In some embodiments, the ASO targets an exon 36 sequence upstream
(or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 36. In some embodiments, the ASO
targets an exon 36 sequence about 14 to about 110 nucleotides
upstream (or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 36. In some embodiments, the ASO
targets an exon 37 sequence downstream (or 3') from the 3' splice
site of a CACNA1A RIC pre-mRNA comprising the retained intron 36.
In some embodiments, the ASO targets an exon 37 sequence about 2 to
about 77 nucleotides downstream (or 3') from the 3' splice site of
a CACNA1A RIC pre-mRNA comprising the retained intron 36.
[1329] In some embodiments, the ASO targets intron 36 in a CACNA1A
RIC pre-mRNA comprising a retained intron 36, wherein the intron
numbering correspond to the mRNA sequence at NM_001127221. In some
embodiments, the ASO targets an intron 36 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 36. In some embodiments, the ASO targets an
intron 36 sequence about 6 to about 499 or about 16 to about 499
nucleotides downstream (or 3') from the 5' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 36. In some
embodiments, the ASO targets an intron 36 sequence upstream (or 5')
from the 3' splice site of a CACNA1A RIC pre-mRNA comprising the
retained intron 36. In some embodiments, the ASO targets an intron
36 sequence about 16 to about 499 or about 21 to about 499
nucleotides upstream (or 5') from the 3' splice site of a CACNA1A
RIC pre-mRNA comprising the retained intron 36.
[1330] In some embodiments, the ASO targets exon 37 or exon 38 of a
CACNA1A RIC pre-mRNA comprising a retained intron 37, wherein the
intron numbering correspond to the mRNA sequence at NM_001127221.
In some embodiments, the ASO targets an exon 37 sequence upstream
(or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 37. In some embodiments, the ASO
targets an exon 37 sequence about 4 to about 79 nucleotides
upstream (or 5') from the 5' splice site of a CACNA1A RIC pre-mRNA
comprising the retained intron 37. In some embodiments, the ASO
targets an exon 38 sequence downstream (or 3') from the 3' splice
site of a CACNA1A RIC pre-mRNA comprising the retained intron 37.
In some embodiments, the ASO targets an exon 38 sequence about 2 to
about 87 nucleotides downstream (or 3') from the 3' splice site of
a CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1331] In some embodiments, the ASO targets intron 37 in a CACNA1A
RIC pre-mRNA comprising a retained intron 37, wherein the intron
numbering correspond to the mRNA sequence at NM_001127221. In some
embodiments, the ASO targets an intron 37 sequence downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence about 6 to about 500 nucleotides downstream (or
3') from the 5' splice site of a CACNA1A RIC pre-mRNA comprising
the retained intron 37. In some embodiments, the ASO targets an
intron 37 sequence upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37. In some
embodiments, the ASO targets an intron 37 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
CACNA1A RIC pre-mRNA comprising the retained intron 37.
[1332] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a DNMT1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a DNMT1
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6289. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6289 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a DNMT1 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a DNMT1 RIC pre-mRNA transcript
comprising a retained intron at 14, 29 or a combination thereof,
wherein the intron numbering correspond to the mRNA sequence at
NM_001379. In some embodiments, the ASO targets a DNMT1 RIC
pre-mRNA transcript comprising a retained intron at 15, 30 or a
combination thereof, wherein the intron numbering correspond to the
mRNA sequence at NM_001130823. In some embodiments, the ASO targets
a DNMT1 RIC pre-mRNA transcript comprising a retained intron at 14,
29 or a combination thereof, wherein the intron numbering
correspond to the mRNA sequence at NM_001318730. In some
embodiments, the ASO targets a DNMT1 RIC pre-mRNA transcript
comprising a retained intron at 15, 30 or a combination thereof,
wherein the intron numbering correspond to the mRNA sequence at
NM_001318731.
[1333] In some embodiments, the ASO targets a DNMT1 RIC pre-mRNA
sequence according to SEQ ID NO: 6337. In some embodiments, the ASO
targets a DNMT1 RIC pre-mRNA sequence according to SEQ ID NO: 6337
comprising a retained intron 14, a retained intron 29, or a
combination thereof. In some embodiments, the ASO targets a DNMT1
RIC pre-mRNA sequence according to SEQ ID NO: 6338. In some
embodiments, the ASO targets a DNMT1 RIC pre-mRNA sequence
according to SEQ ID NO: 6338 comprising a retained intron 15, a
retained intron 30, or a combination thereof. In some embodiments,
the ASO targets a DNMT1 RIC pre-mRNA sequence according to SEQ ID
NO: 6339. In some embodiments, the ASO targets a DNMT1 RIC pre-mRNA
sequence according to SEQ ID NO: 6339 comprising a retained intron
14, a retained intron 29, or a combination thereof. In some
embodiments, the ASO targets a DNMT1 RIC pre-mRNA sequence
according to SEQ ID NO: 6340. In some embodiments, the ASO targets
a DNMT1 RIC pre-mRNA sequence according to SEQ ID NO: 6340
comprising a retained intron 15, a retained intron 30, or a
combination thereof. In some embodiments, the ASOs disclosed herein
target SEQ ID NOs: 30635 or 30644. In some embodiments, the ASO has
a sequence according to any one of SEQ ID NOs: 22188-23203.
[1334] In some embodiments, the ASO targets exon 29 or exon 30 of a
DNMT1 RIC pre-mRNA comprising a retained intron 29, wherein the
intron numbering correspond to the mRNA sequence at NM_001379. In
some embodiments, the ASO targets an exon 29 sequence upstream (or
5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an exon 29
sequence about 4 to about 173 nucleotides upstream (or 5') from the
5' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 29. In some embodiments, the ASO targets an exon 30 sequence
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 29. In some embodiments, the ASO
targets an exon 30 sequence about 2 to about 67 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 29.
[1335] In some embodiments, the ASO targets intron 29 in a DNMT1
RIC pre-mRNA comprising a retained intron 29, wherein the intron
numbering correspond to the mRNA sequence at NM_001379. In some
embodiments, the ASO targets an intron 29 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence about 6 to about 429 nucleotides downstream (or 3')
from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 29. In some embodiments,
the ASO targets an intron 29 sequence about 16 to about 433
nucleotides upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 29.
[1336] In some embodiments, the ASO targets exon 14 or exon 15 of a
DNMT1 RIC pre-mRNA comprising a retained intron 14, wherein the
intron numbering correspond to the mRNA sequence at NM_001379. In
some embodiments, the ASO targets an exon 14 sequence upstream (or
5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an exon 14
sequence about 4 to about 29 nucleotides upstream (or 5') from the
5' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 14. In some embodiments, the ASO targets an exon 15 sequence
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 14. In some embodiments, the ASO
targets an exon 15 sequence about 2 to about 62 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 14.
[1337] In some embodiments, the ASO targets intron 14 in a DNMT1
RIC pre-mRNA comprising a retained intron 14, wherein the intron
numbering correspond to the mRNA sequence at NM_001379. In some
embodiments, the ASO targets an intron 14 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence about 6 to about 61 nucleotides downstream (or 3') from
the 5' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 14. In some embodiments, the ASO targets an intron 14
sequence upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 14. In some embodiments,
the ASO targets an intron 14 sequence about 16 to about 61
nucleotides upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 14.
[1338] In some embodiments, the ASO targets exon 30 or exon 31 of a
DNMT1 RIC pre-mRNA comprising a retained intron 30, wherein the
intron numbering correspond to the mRNA sequence at NM_001130823.
In some embodiments, the ASO targets an exon 30 sequence upstream
(or 5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising
the retained intron 30. In some embodiments, the ASO targets an
exon 30 sequence about 4 to about 173 nucleotides upstream (or 5')
from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an exon 31
sequence downstream (or 3') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 30. In some embodiments,
the ASO targets an exon 31 sequence about 2 to about 67 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 30.
[1339] In some embodiments, the ASO targets intron 30 in a DNMT1
RIC pre-mRNA comprising a retained intron 30, wherein the intron
numbering correspond to the mRNA sequence at NM_001130823. In some
embodiments, the ASO targets an intron 30 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence about 6 to about 429 or about 11 to about 429
nucleotides downstream (or 3') from the 5' splice site of a DNMT1
RIC pre-mRNA comprising the retained intron 30. In some
embodiments, the ASO targets an intron 30 sequence upstream (or 5')
from the 3' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence about 16 to about 433 nucleotides upstream (or 5') from
the 3' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 30.
[1340] In some embodiments, the ASO targets exon 15 or exon 16 of a
DNMT1 RIC pre-mRNA comprising a retained intron 15, wherein the
intron numbering correspond to the mRNA sequence at NM_001130823.
In some embodiments, the ASO targets an exon 15 sequence upstream
(or 5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising
the retained intron 15. In some embodiments, the ASO targets an
exon 15 sequence about 4 to about 29 nucleotides upstream (or 5')
from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an exon 16
sequence downstream (or 3') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an exon 16 sequence about 2 to about 62 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 15.
[1341] In some embodiments, the ASO targets intron 15 in a DNMT1
RIC pre-mRNA comprising a retained intron 15, wherein the intron
numbering correspond to the mRNA sequence at NM_001130823. In some
embodiments, the ASO targets an intron 15 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence about 6 to about 61 nucleotides downstream (or 3') from
the 5' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 15. In some embodiments, the ASO targets an intron 15
sequence upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an intron 15 sequence about 16 to about 61
nucleotides upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 15.
[1342] In some embodiments, the ASO targets exon 29 or exon 30 of a
DNMT1 RIC pre-mRNA comprising a retained intron 29, wherein the
intron numbering correspond to the mRNA sequence at NM_001318730.
In some embodiments, the ASO targets an exon 29 sequence upstream
(or 5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising
the retained intron 29. In some embodiments, the ASO targets an
exon 29 sequence about 4 to about 173 nucleotides upstream (or 5')
from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an exon 30
sequence downstream (or 3') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 29. In some embodiments,
the ASO targets an exon 30 sequence about 2 to about 67 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 29.
[1343] In some embodiments, the ASO targets intron 29 in a DNMT1
RIC pre-mRNA comprising a retained intron 29, wherein the intron
numbering correspond to the mRNA sequence at NM_001318730. In some
embodiments, the ASO targets an intron 29 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence about 6 to about 429 or about 11 to about 429
nucleotides downstream (or 3') from the 5' splice site of a DNMT1
RIC pre-mRNA comprising the retained intron 29. In some
embodiments, the ASO targets an intron 29 sequence upstream (or 5')
from the 3' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 29. In some embodiments, the ASO targets an intron
29 sequence about 16 to about 433 nucleotides upstream (or 5') from
the 3' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 29.
[1344] In some embodiments, the ASO targets exon 14 or exon 15 of a
DNMT1 RIC pre-mRNA comprising a retained intron 14, wherein the
intron numbering correspond to the mRNA sequence at NM_001318730.
In some embodiments, the ASO targets an exon 14 sequence upstream
(or 5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising
the retained intron 14. In some embodiments, the ASO targets an
exon 14 sequence about 4 to about 29 nucleotides upstream (or 5')
from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an exon 15
sequence downstream (or 3') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 14. In some embodiments,
the ASO targets an exon 15 sequence about 2 to about 62 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 14.
[1345] In some embodiments, the ASO targets intron 14 in a DNMT1
RIC pre-mRNA comprising a retained intron 14, wherein the intron
numbering correspond to the mRNA sequence at NM_001318730. In some
embodiments, the ASO targets an intron 14 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence about 6 to about 61 nucleotides downstream (or 3') from
the 5' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 14. In some embodiments, the ASO targets an intron 14
sequence upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 14. In some embodiments,
the ASO targets an intron 14 sequence about 16 to about 61
nucleotides upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 14.
[1346] In some embodiments, the ASO targets exon 30 or exon 31 of a
DNMT1 RIC pre-mRNA comprising a retained intron 30, wherein the
intron numbering correspond to the mRNA sequence at NM_001318731.
In some embodiments, the ASO targets an exon 30 sequence upstream
(or 5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising
the retained intron 30. In some embodiments, the ASO targets an
exon 30 sequence about 4 to about 173 nucleotides upstream (or 5')
from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an exon 31
sequence downstream (or 3') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 30. In some embodiments,
the ASO targets an exon 31 sequence about 2 to about 67 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 30.
[1347] In some embodiments, the ASO targets intron 30 in a DNMT1
RIC pre-mRNA comprising a retained intron 30, wherein the intron
numbering correspond to the mRNA sequence at NM_001318731. In some
embodiments, the ASO targets an intron 30 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence about 6 to about 429 or about 11 to about 429
nucleotides downstream (or 3') from the 5' splice site of a DNMT1
RIC pre-mRNA comprising the retained intron 30. In some
embodiments, the ASO targets an intron 30 sequence upstream (or 5')
from the 3' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 30. In some embodiments, the ASO targets an intron
30 sequence about 16 to about 433 nucleotides upstream (or 5') from
the 3' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 30.
[1348] In some embodiments, the ASO targets exon 15 or exon 16 of a
DNMT1 RIC pre-mRNA comprising a retained intron 15, wherein the
intron numbering correspond to the mRNA sequence at NM_001318731.
In some embodiments, the ASO targets an exon 15 sequence upstream
(or 5') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising
the retained intron 15. In some embodiments, the ASO targets an
exon 15 sequence about 4 to about 29 nucleotides upstream (or 5')
from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an exon 16
sequence downstream (or 3') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an exon 16 sequence about 2 to about 62 nucleotides
downstream (or 3') from the 3' splice site of a DNMT1 RIC pre-mRNA
comprising the retained intron 15.
[1349] In some embodiments, the ASO targets intron 15 in a DNMT1
RIC pre-mRNA comprising a retained intron 15, wherein the intron
numbering correspond to the mRNA sequence at NM_001318731. In some
embodiments, the ASO targets an intron 15 sequence downstream (or
3') from the 5' splice site of a DNMT1 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence about 6 to about 61 nucleotides downstream (or 3') from
the 5' splice site of a DNMT1 RIC pre-mRNA comprising the retained
intron 15. In some embodiments, the ASO targets an intron 15
sequence upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an intron 15 sequence about 16 to about 61
nucleotides upstream (or 5') from the 3' splice site of a DNMT1 RIC
pre-mRNA comprising the retained intron 15.
[1350] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a NF2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a NF2
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6290. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6290 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a NF2 RIC pre-mRNA sequence. In some
embodiments, the ASO targets a NF2 RIC pre-mRNA transcript
comprising a retained intron at 14, wherein the intron numbering
correspond to the mRNA sequence at NM_181830. In some embodiments,
the ASO targets a NF2 RIC pre-mRNA transcript comprising a retained
intron at 15, wherein the intron numbering correspond to the mRNA
sequence at NM_181829. In some embodiments, the ASO targets a NF2
RIC pre-mRNA transcript comprising a retained intron at 4, wherein
the intron numbering correspond to the mRNA sequence at NM_181833.
In some embodiments, the ASO targets a NF2 RIC pre-mRNA transcript
comprising a retained intron at 16, wherein the intron numbering
correspond to the mRNA sequence at NM_181832. In some embodiments,
the ASO targets a NF2 RIC pre-mRNA transcript comprising a retained
intron at 15, wherein the intron numbering correspond to the mRNA
sequence at NM_181828. In some embodiments, the ASO targets a NF2
RIC pre-mRNA transcript comprising a retained intron at 16, wherein
the intron numbering correspond to the mRNA sequence at NM_016418.
In some embodiments, the ASO targets a NF2 RIC pre-mRNA transcript
comprising a retained intron at 15, wherein the intron numbering
correspond to the mRNA sequence at NM_000268.
[1351] In some embodiments, the ASO targets a NF2 RIC pre-mRNA
sequence according to SEQ ID NO: 6341. In some embodiments, the ASO
targets a NF2 RIC pre-mRNA sequence according to SEQ ID NO: 6341
comprising a retained intron 14. In some embodiments, the ASO
targets a NF2 RIC pre-mRNA sequence according to SEQ ID NO: 6342.
In some embodiments, the ASO targets a NF2 RIC pre-mRNA sequence
according to SEQ ID NO: 6342 comprising a retained intron 15. In
some embodiments, the ASO targets a NF2 RIC pre-mRNA sequence
according to SEQ ID NO: 6343. In some embodiments, the ASO targets
a NF2 RIC pre-mRNA sequence according to SEQ ID NO: 6343 comprising
a retained intron 4. In some embodiments, the ASO targets a NF2 RIC
pre-mRNA sequence according to SEQ ID NO: 6344. In some
embodiments, the ASO targets a NF2 RIC pre-mRNA sequence according
to SEQ ID NO: 6344 comprising a retained intron 16. In some
embodiments, the ASO targets a NF2 RIC pre-mRNA sequence according
to SEQ ID NO: 6345. In some embodiments, the ASO targets a NF2 RIC
pre-mRNA sequence according to SEQ ID NO: 6345 comprising a
retained intron 15. In some embodiments, the ASO targets a NF2 RIC
pre-mRNA sequence according to SEQ ID NO: 6346. In some
embodiments, the ASO targets a NF2 RIC pre-mRNA sequence according
to SEQ ID NO: 6346 comprising a retained intron 16. In some
embodiments, the ASO targets a NF2 RIC pre-mRNA sequence according
to SEQ ID NO: 6347. In some embodiments, the ASO targets a NF2 RIC
pre-mRNA sequence according to SEQ ID NO: 6347 comprising a
retained intron 15. In some embodiments, the ASOs disclosed herein
target SEQ ID NOs: 30646, 30640, 30625, or 30641. In some
embodiments, the ASO has a sequence according to any one of SEQ ID
NOs: 23204-29620.
[1352] In some embodiments, the ASO targets exon 14 or exon 15 of a
NF2 RIC pre-mRNA comprising a retained intron 14, wherein the
intron numbering correspond to the mRNA sequence at NM_181830. In
some embodiments, the ASO targets an exon 14 sequence upstream (or
5') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an exon 14
sequence about 4 to about 24 nucleotides upstream (or 5') from the
5' splice site of a NF2 RIC pre-mRNA comprising the retained intron
14. In some embodiments, the ASO targets an exon 15 sequence
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 14. In some embodiments, the ASO
targets an exon 15 sequence about 2 to about 3828 nucleotides
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 14.
[1353] In some embodiments, the ASO targets intron 14 in a NF2 RIC
pre-mRNA comprising a retained intron 14, wherein the intron
numbering correspond to the mRNA sequence at NM_181830. In some
embodiments, the ASO targets an intron 14 sequence downstream (or
3') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence about 6 to about 499 nucleotides downstream (or 3')
from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 14. In some embodiments, the ASO targets an intron
14 sequence upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 14. In some embodiments,
the ASO targets an intron 14 sequence about 16 to about 500
nucleotides upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 14.
[1354] In some embodiments, the ASO targets exon 15 or exon 16 of a
NF2 RIC pre-mRNA comprising a retained intron 15, wherein the
intron numbering correspond to the mRNA sequence at NM_181829. In
some embodiments, the ASO targets an exon 15 sequence upstream (or
5') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an exon 15
sequence about 4 to about 24 nucleotides upstream (or 5') from the
5' splice site of a NF2 RIC pre-mRNA comprising the retained intron
15. In some embodiments, the ASO targets an exon 16 sequence
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 15. In some embodiments, the ASO
targets an exon 16 sequence about 2 to about 3828 nucleotides
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 15.
[1355] In some embodiments, the ASO targets intron 15 in a NF2 RIC
pre-mRNA comprising a retained intron 15, wherein the intron
numbering correspond to the mRNA sequence at NM_181829. In some
embodiments, the ASO targets an intron 15 sequence downstream (or
3') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence about 6 to about 499 nucleotides downstream (or 3')
from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an intron 15 sequence about 16 to about 500
nucleotides upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 15.
[1356] In some embodiments, the ASO targets exon 4 or exon 5 of a
NF2 RIC pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_181833. In some
embodiments, the ASO targets an exon 4 sequence upstream (or 5')
from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an exon 4
sequence about 4 to about 64 nucleotides upstream (or 5') from the
5' splice site of a NF2 RIC pre-mRNA comprising the retained intron
4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 3828 nucleotides
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 4.
[1357] In some embodiments, the ASO targets intron 4 in a NF2 RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_181833. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 6 to about 496 nucleotides downstream (or 3') from
the 5' splice site of a NF2 RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 500 nucleotides
upstream (or 5') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 4.
[1358] In some embodiments, the ASO targets exon 16 or exon 17 of a
NF2 RIC pre-mRNA comprising a retained intron 16, wherein the
intron numbering correspond to the mRNA sequence at NM_181832. In
some embodiments, the ASO targets an exon 16 sequence upstream (or
5') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an exon 16
sequence about 4 to about 39 nucleotides upstream (or 5') from the
5' splice site of a NF2 RIC pre-mRNA comprising the retained intron
16. In some embodiments, the ASO targets an exon 17 sequence
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 16. In some embodiments, the ASO
targets an exon 17 sequence about 2 to about 3828 nucleotides
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 16.
[1359] In some embodiments, the ASO targets intron 16 in a NF2 RIC
pre-mRNA comprising a retained intron 16, wherein the intron
numbering correspond to the mRNA sequence at NM_181832. In some
embodiments, the ASO targets an intron 16 sequence downstream (or
3') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence about 6 to about 499 nucleotides downstream (or 3')
from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an intron 16 sequence about 16 to about 500
nucleotides upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 16.
[1360] In some embodiments, the ASO targets exon 15 or exon 16 of a
NF2 RIC pre-mRNA comprising a retained intron 15, wherein the
intron numbering correspond to the mRNA sequence at NM_181828. In
some embodiments, the ASO targets an exon 15 sequence upstream (or
5') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an exon 15
sequence about 4 to about 24 nucleotides upstream (or 5') from the
5' splice site of a NF2 RIC pre-mRNA comprising the retained intron
15. In some embodiments, the ASO targets an exon 16 sequence
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 15. In some embodiments, the ASO
targets an exon 16 sequence about 2 to about 3828 nucleotides
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 15.
[1361] In some embodiments, the ASO targets intron 15 in a NF2 RIC
pre-mRNA comprising a retained intron 15, wherein the intron
numbering correspond to the mRNA sequence at NM_181828. In some
embodiments, the ASO targets an intron 15 sequence downstream (or
3') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence about 6 to about 499 nucleotides downstream (or 3')
from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an intron 15 sequence about 16 to about 500
nucleotides upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 15.
[1362] In some embodiments, the ASO targets exon 16 or exon 17 of a
NF2 RIC pre-mRNA comprising a retained intron 16, wherein the
intron numbering correspond to the mRNA sequence at NM_016418. In
some embodiments, the ASO targets an exon 16 sequence upstream (or
5') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an exon 16
sequence about 4 to about 24 nucleotides upstream (or 5') from the
5' splice site of a NF2 RIC pre-mRNA comprising the retained intron
16. In some embodiments, the ASO targets an exon 17 sequence
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 16. In some embodiments, the ASO
targets an exon 17 sequence about 2 to about 3828 nucleotides
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 16.
[1363] In some embodiments, the ASO targets intron 16 in a NF2 RIC
pre-mRNA comprising a retained intron 16, wherein the intron
numbering correspond to the mRNA sequence at NM_016418. In some
embodiments, the ASO targets an intron 16 sequence downstream (or
3') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence about 6 to about 499 nucleotides downstream (or 3')
from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 16. In some embodiments,
the ASO targets an intron 16 sequence about 16 to about 500
nucleotides upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 16.
[1364] In some embodiments, the ASO targets exon 15 or exon 16 of a
NF2 RIC pre-mRNA comprising a retained intron 15, wherein the
intron numbering correspond to the mRNA sequence at NM_000268. In
some embodiments, the ASO targets an exon 15 sequence upstream (or
5') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an exon 15
sequence about 4 to about 144 nucleotides upstream (or 5') from the
5' splice site of a NF2 RIC pre-mRNA comprising the retained intron
15. In some embodiments, the ASO targets an exon 16 sequence
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 15. In some embodiments, the ASO
targets an exon 16 sequence about 2 to about 3828 nucleotides
downstream (or 3') from the 3' splice site of a NF2 RIC pre-mRNA
comprising the retained intron 15.
[1365] In some embodiments, the ASO targets intron 15 in a NF2 RIC
pre-mRNA comprising a retained intron 15, wherein the intron
numbering correspond to the mRNA sequence at NM_000268. In some
embodiments, the ASO targets an intron 15 sequence downstream (or
3') from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence about 6 to about 500 nucleotides downstream (or 3')
from the 5' splice site of a NF2 RIC pre-mRNA comprising the
retained intron 15. In some embodiments, the ASO targets an intron
15 sequence upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 15. In some embodiments,
the ASO targets an intron 15 sequence about 16 to about 500
nucleotides upstream (or 5') from the 3' splice site of a NF2 RIC
pre-mRNA comprising the retained intron 15.
[1366] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a SHANK3 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a
SHANK3 genomic sequence comprising a retained intron. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 6291. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 6291 comprising a retained intron. In some
embodiments, the ASOs disclosed herein target a SHANK3 RIC pre-mRNA
sequence. In some embodiments, the ASO targets a SHANK3 RIC
pre-mRNA transcript comprising a retained intron at 16, wherein the
intron numbering correspond to the mRNA sequence at NM_033517.
[1367] In some embodiments, the ASO targets a SHANK3 RIC pre-mRNA
sequence according to SEQ ID NO: 6348. In some embodiments, the ASO
targets a SHANK3 RIC pre-mRNA sequence according to SEQ ID NO: 6348
comprising a retained intron 16. In some embodiments, the ASOs
disclosed herein target SEQ ID NO: 30630. In some embodiments, the
ASO has a sequence according to any one of SEQ ID NOs:
29621-29808.
[1368] In some embodiments, the ASO targets exon 16 or exon 17 of a
SHANK3 RIC pre-mRNA comprising a retained intron 16, wherein the
intron numbering correspond to the mRNA sequence at NM_000214. In
some embodiments, the ASO targets an exon 16 sequence upstream (or
5') from the 5' splice site of a SHANK3 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an exon 16
sequence about 4 to about 114 nucleotides upstream (or 5') from the
5' splice site of a SHANK3 RIC pre-mRNA comprising the retained
intron 16. In some embodiments, the ASO targets an exon 17 sequence
downstream (or 3') from the 3' splice site of a SHANK3 RIC pre-mRNA
comprising the retained intron 16. In some embodiments, the ASO
targets an exon 17 sequence about 2 to about 62 or about 7 to about
62 nucleotides downstream (or 3') from the 3' splice site of a
SHANK3 RIC pre-mRNA comprising the retained intron 16.
[1369] In some embodiments, the ASO targets intron 16 in a SHANK3
RIC pre-mRNA comprising a retained intron 16, wherein the intron
numbering correspond to the mRNA sequence at NM_000214. In some
embodiments, the ASO targets an intron 16 sequence downstream (or
3') from the 5' splice site of a SHANK3 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence about 6 to about 499 nucleotides downstream (or 3')
from the 5' splice site of a SHANK3 RIC pre-mRNA comprising the
retained intron 16. In some embodiments, the ASO targets an intron
16 sequence upstream (or 5') from the 3' splice site of a SHANK3
RIC pre-mRNA comprising the retained intron 16. In some
embodiments, the ASO targets an intron 16 sequence about 16 to
about 498 nucleotides upstream (or 5') from the 3' splice site of a
SHANK3 RIC pre-mRNA comprising the retained intron 16.
[1370] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a ARSA genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript from a ARSA
genomic sequence comprising a retained intron. In some embodiments,
the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 6292. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ
ID NO: 6292 comprising a retained intron. In some embodiments, the
ASOs disclosed herein target a ARSA RIC pre-mRNA sequence. In some
embodiments, the ASO targets a ARSA RIC pre-mRNA transcript
comprising a retained intron at 2, 3 or a combination thereof,
wherein the intron numbering correspond to the mRNA sequence at
NM_000487. In some embodiments, the ASO targets a ARSA RIC pre-mRNA
transcript comprising a retained intron at 3, 4 or a combination
thereof, wherein the intron numbering correspond to the mRNA
sequence at NM_001085425. In some embodiments, the ASO targets a
ARSA RIC pre-mRNA transcript comprising a retained intron at 3, 4
or a combination thereof, wherein the intron numbering correspond
to the mRNA sequence at NM_001085426. In some embodiments, the ASO
targets a ARSA RIC pre-mRNA transcript comprising a retained intron
at 3, 4 or a combination thereof, wherein the intron numbering
correspond to the mRNA sequence at NM_001085427. In some
embodiments, the ASO targets a ARSA RIC pre-mRNA transcript
comprising a retained intron at 2, 3 or a combination thereof,
wherein the intron numbering correspond to the mRNA sequence at
NM_001085428.
[1371] In some embodiments, the ASO targets a ARSA RIC pre-mRNA
sequence according to SEQ ID NO: 6349. In some embodiments, the ASO
targets a ARSA RIC pre-mRNA sequence according to SEQ ID NO: 6349
comprising a retained intron 2, a retained intron 3, or a
combination thereof. In some embodiments, the ASO targets a ARSA
RIC pre-mRNA sequence according to SEQ ID NO: 6350. In some
embodiments, the ASO targets a ARSA RIC pre-mRNA sequence according
to SEQ ID NO: 6350 comprising a retained intron 3, a retained
intron 4, or a combination thereof. In some embodiments, the ASO
targets a ARSA RIC pre-mRNA sequence according to SEQ ID NO: 6351.
In some embodiments, the ASO targets a ARSA RIC pre-mRNA sequence
according to SEQ ID NO: 6351 comprising a retained intron 3, a
retained intron 4, or a combination thereof. In some embodiments,
the ASO targets a ARSA RIC pre-mRNA sequence according to SEQ ID
NO: 6352. In some embodiments, the ASO targets a ARSA RIC pre-mRNA
sequence according to SEQ ID NO: 6352 comprising a retained intron
3, a retained intron 4, or a combination thereof. In some
embodiments, the ASO targets a ARSA RIC pre-mRNA sequence according
to SEQ ID NO: 6353. In some embodiments, the ASO targets a ARSA RIC
pre-mRNA sequence according to SEQ ID NO: 6353 comprising a
retained intron 2, a retained intron 3, or a combination thereof.
In some embodiments, the ASOs disclosed herein target SEQ ID NOs:
30651 or 30651. In some embodiments, the ASO has a sequence
according to any one of SEQ ID NOs: 29809-30623.
[1372] In some embodiments, the ASO targets exon 2 or exon 3 of a
ARSA RIC pre-mRNA comprising a retained intron 2, wherein the
intron numbering correspond to the mRNA sequence at NM_000487. In
some embodiments, the ASO targets an exon 2 sequence upstream (or
5') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an exon 2
sequence about 4 to about 222 nucleotides upstream (or 5') from the
5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an exon 3 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an exon 3 sequence about 2 to about 202 or about 7 to about
202 nucleotides downstream (or 3') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 2.
[1373] In some embodiments, the ASO targets intron 2 in a ARSA RIC
pre-mRNA comprising a retained intron 2, wherein the intron
numbering correspond to the mRNA sequence at NM_000487. In some
embodiments, the ASO targets an intron 2 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an intron 2
sequence about 6 to about 56 nucleotides downstream (or 3') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an intron 2 sequence
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an intron 2 sequence about 16 to about 71 nucleotides
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 2.
[1374] In some embodiments, the ASO targets exon 3 or exon 4 of a
ARSA RIC pre-mRNA comprising a retained intron 3, wherein the
intron numbering correspond to the mRNA sequence at NM_000487. In
some embodiments, the ASO targets an exon 3 sequence upstream (or
5') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an exon 3
sequence about 4 to about 199 nucleotides upstream (or 5') from the
5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an exon 4 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an exon 4 sequence about 2 to about 152 nucleotides
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3.
[1375] In some embodiments, the ASO targets intron 3 in a ARSA RIC
pre-mRNA comprising a retained intron 3, wherein the intron
numbering correspond to the mRNA sequence at NM_000487. In some
embodiments, the ASO targets an intron 3 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an intron 3
sequence about 6 to about 49 nucleotides downstream (or 3') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an intron 3 sequence
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an intron 3 sequence about 16 to about 35 nucleotides
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3.
[1376] In some embodiments, the ASO targets exon 3 or exon 4 of a
ARSA RIC pre-mRNA comprising a retained intron 3, wherein the
intron numbering correspond to the mRNA sequence at NM_001085425.
In some embodiments, the ASO targets an exon 3 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 3. In some embodiments, the ASO targets an exon
3 sequence about 4 to about 222 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an exon 4 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an exon 4 sequence about 2 to about 202 or about 7 to about
202 nucleotides downstream (or 3') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 3.
[1377] In some embodiments, the ASO targets intron 3 in a ARSA RIC
pre-mRNA comprising a retained intron 3, wherein the intron
numbering correspond to the mRNA sequence at NM_001085425. In some
embodiments, the ASO targets an intron 3 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an intron 3
sequence about 6 to about 56 nucleotides downstream (or 3') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an intron 3 sequence
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an intron 3 sequence about 16 to about 71 nucleotides
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3.
[1378] In some embodiments, the ASO targets exon 4 or exon 5 of a
ARSA RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_001085425.
In some embodiments, the ASO targets an exon 4 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 199 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 152 nucleotides
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4.
[1379] In some embodiments, the ASO targets intron 4 in a ARSA RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_001085425. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 5 to about 49 or about 6 to about 49 nucleotides
downstream (or 3') from the 5' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence upstream (or 5') from the 3' splice
site of a ARSA RIC pre-mRNA comprising the retained intron 4. In
some embodiments, the ASO targets an intron 4 sequence about 16 to
about 35 nucleotides upstream (or 5') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 4.
[1380] In some embodiments, the ASO targets exon 3 or exon 4 of a
ARSA RIC pre-mRNA comprising a retained intron 3, wherein the
intron numbering correspond to the mRNA sequence at NM_001085426.
In some embodiments, the ASO targets an exon 3 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 3. In some embodiments, the ASO targets an exon
3 sequence about 4 to about 222 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an exon 4 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an exon 4 sequence about 2 to about 202 or about 7 to about
202 nucleotides downstream (or 3') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 3.
[1381] In some embodiments, the ASO targets intron 3 in a ARSA RIC
pre-mRNA comprising a retained intron 3, wherein the intron
numbering correspond to the mRNA sequence at NM_001085426. In some
embodiments, the ASO targets an intron 3 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an intron 3
sequence about 6 to about 56 nucleotides downstream (or 3') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an intron 3 sequence
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an intron 3 sequence about 16 to about 71 nucleotides
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3.
[1382] In some embodiments, the ASO targets exon 4 or exon 5 of a
ARSA RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_001085426.
In some embodiments, the ASO targets an exon 4 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 199 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 152 nucleotides
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4.
[1383] In some embodiments, the ASO targets intron 4 in a ARSA RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_001085426. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 5 to about 49 or about 6 to about 49 nucleotides
downstream (or 3') from the 5' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence upstream (or 5') from the 3' splice
site of a ARSA RIC pre-mRNA comprising the retained intron 4. In
some embodiments, the ASO targets an intron 4 sequence about 16 to
about 35 nucleotides upstream (or 5') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 4.
[1384] In some embodiments, the ASO targets exon 3 or exon 4 of a
ARSA RIC pre-mRNA comprising a retained intron 3, wherein the
intron numbering correspond to the mRNA sequence at NM_001085427.
In some embodiments, the ASO targets an exon 3 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 3. In some embodiments, the ASO targets an exon
3 sequence about 4 to about 222 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an exon 4 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an exon 4 sequence about 2 to about 202 or about 7 to about
202 nucleotides downstream (or 3') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 3.
[1385] In some embodiments, the ASO targets intron 3 in a ARSA RIC
pre-mRNA comprising a retained intron 3, wherein the intron
numbering correspond to the mRNA sequence at NM_001085427. In some
embodiments, the ASO targets an intron 3 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an intron 3
sequence about 6 to about 56 nucleotides downstream (or 3') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an intron 3 sequence
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an intron 3 sequence about 16 to about 71 nucleotides
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3.
[1386] In some embodiments, the ASO targets exon 4 or exon 5 of a
ARSA RIC pre-mRNA comprising a retained intron 4, wherein the
intron numbering correspond to the mRNA sequence at NM_001085427.
In some embodiments, the ASO targets an exon 4 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 4. In some embodiments, the ASO targets an exon
4 sequence about 4 to about 199 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 4. In some embodiments, the ASO targets an exon 5 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an exon 5 sequence about 2 to about 152 nucleotides
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4.
[1387] In some embodiments, the ASO targets intron 4 in a ARSA RIC
pre-mRNA comprising a retained intron 4, wherein the intron
numbering correspond to the mRNA sequence at NM_001085427. In some
embodiments, the ASO targets an intron 4 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 4. In some embodiments, the ASO targets an intron 4
sequence about 5 to about 49 or about 6 to about 49 nucleotides
downstream (or 3') from the 5' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence upstream (or 5') from the 3' splice
site of a ARSA RIC pre-mRNA comprising the retained intron 4. In
some embodiments, the ASO targets an intron 4 sequence about 16 to
about 35 nucleotides upstream (or 5') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 4.
[1388] In some embodiments, the ASO targets exon 2 or exon 3 of a
ARSA RIC pre-mRNA comprising a retained intron 2, wherein the
intron numbering correspond to the mRNA sequence at NM_001085428.
In some embodiments, the ASO targets an exon 2 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 2. In some embodiments, the ASO targets an exon
2 sequence about 4 to about 222 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an exon 3 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an exon 3 sequence about 2 to about 202 or about 7 to about
202 nucleotides downstream (or 3') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 2.
[1389] In some embodiments, the ASO targets intron 2 in a ARSA RIC
pre-mRNA comprising a retained intron 2, wherein the intron
numbering correspond to the mRNA sequence at NM_001085428. In some
embodiments, the ASO targets an intron 2 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 2. In some embodiments, the ASO targets an intron 2
sequence about 6 to about 56 nucleotides downstream (or 3') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 2. In some embodiments, the ASO targets an intron 2 sequence
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 2. In some embodiments, the ASO
targets an intron 2 sequence about 16 to about 71 nucleotides
upstream (or 5') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 2.
[1390] In some embodiments, the ASO targets exon 3 or exon 4 of a
ARSA RIC pre-mRNA comprising a retained intron 3, wherein the
intron numbering correspond to the mRNA sequence at NM_001085428.
In some embodiments, the ASO targets an exon 3 sequence upstream
(or 5') from the 5' splice site of a ARSA RIC pre-mRNA comprising
the retained intron 3. In some embodiments, the ASO targets an exon
3 sequence about 4 to about 199 nucleotides upstream (or 5') from
the 5' splice site of a ARSA RIC pre-mRNA comprising the retained
intron 3. In some embodiments, the ASO targets an exon 4 sequence
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an exon 4 sequence about 2 to about 152 nucleotides
downstream (or 3') from the 3' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3.
[1391] In some embodiments, the ASO targets intron 3 in a ARSA RIC
pre-mRNA comprising a retained intron 3, wherein the intron
numbering correspond to the mRNA sequence at NM_001085428. In some
embodiments, the ASO targets an intron 3 sequence downstream (or
3') from the 5' splice site of a ARSA RIC pre-mRNA comprising the
retained intron 3. In some embodiments, the ASO targets an intron 3
sequence about 5 to about 49 or about 6 to about 49 nucleotides
downstream (or 3') from the 5' splice site of a ARSA RIC pre-mRNA
comprising the retained intron 3. In some embodiments, the ASO
targets an intron 3 sequence upstream (or 5') from the 3' splice
site of a ARSA RIC pre-mRNA comprising the retained intron 3. In
some embodiments, the ASO targets an intron 3 sequence about 16 to
about 35 nucleotides upstream (or 5') from the 3' splice site of a
ARSA RIC pre-mRNA comprising the retained intron 3.
[1392] In embodiments, the targeted portion of the MFSD8 RIC
pre-mRNA is in intron 12. The MFSD8 intron numbering used herein
corresponds to the mRNA sequence at NM_152778. In embodiments, the
percent retained intron can be 42. In embodiments, hybridization of
an ASO to the targeted portion of the RIC pre-mRNA results in
enhanced splicing at the splice site (5' splice site or 3' splice
site) of retained intron 12 and subsequently increases MFSD8
protein production. It is understood that the intron numbering may
change in reference to a different MFSD8 isoform sequence. One of
skill in the art can determine the corresponding intron number in
any isoform based on an intron sequence provided herein or using
the number provided in reference to the mRNA sequence at NM_152778.
One of skill in the art also can determine the sequences of
flanking exons in any MFSD8 isoform for targeting using the methods
of the invention, based on an intron sequence provided herein or
using the intron number provided in reference to the mRNA sequence
at NM_152778.
[1393] In embodiments, the targeted portion of the IDUA RIC
pre-mRNA is in intron 3, 4, 5, 6 or 7. The IDUA intron numbering
used herein corresponds to the mRNA sequence at NM_000203. In
embodiments, the percent retained intron can be 25, 63 or 16. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 3, 4, 5, 6 or 7
and subsequently increases IDUA protein production. It is
understood that the intron numbering may change in reference to a
different IDUA isoform sequence. One of skill in the art can
determine the corresponding intron number in any isoform based on
an intron sequence provided herein or using the number provided in
reference to the mRNA sequence at NM_000203. One of skill in the
art also can determine the sequences of flanking exons in any IDUA
isoform for targeting using the methods of the invention, based on
an intron sequence provided herein or using the intron number
provided in reference to the mRNA sequence at NM_000203.
[1394] In embodiments, the targeted portion of the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
pre-mRNA is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37 or 38. In embodiments, hybridization of
an ASO to the targeted portion of the RIC pre-mRNA results in
enhanced splicing at the splice site (5' splice site or 3' splice
site) of at least one of retained introns 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, and
subsequently increases ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein production.
[1395] In embodiments, the targeted portion of the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B pre-mRNA is
in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37 or 38. In embodiments, hybridization of an ASO to
the targeted portion of the RIC pre-mRNA results in enhanced
splicing at the splice site (5' splice site or 3' splice site) of
at least one of retained introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, and subsequently
increases ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B protein production.
[1396] In some cases, the targeted portion of the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B pre-mRNA is in intron
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37 or 38. In embodiments, hybridization of an ASO to the targeted
portion of the RIC pre-mRNA results in enhanced splicing at the
splice site (5' splice site or 3' splice site) of at least one of
retained introns 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37 or 38, and subsequently increases ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein
production.
[1397] The degree of intron retention can be expressed as percent
intron retention (PIR), the percentage of transcripts in which a
given intron is retained. In brief, PIR can be calculated as the
percentage of the average number of reads mapping to the
exon-intron junctions, over the sum of the average of the
exon-intron junction reads plus the exon-exon junction reads.
[1398] PIR values for SCN1A have been reported, e.g., by
Braunschweig, et al., 2014, (see, e.g., Supplemental Table S9),
incorporated by reference herein in its entirety.
[1399] In embodiments, the methods described herein are used to
increase the production of a functional ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5-EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B protein, or any
combinations thereof. As used herein, the term "functional" refers
to the amount of activity or function of a ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B protein or
any combination thereof that is necessary to eliminate any one or
more symptoms of a treated condition. In embodiments, the methods
are used to increase the production of a partially functional
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, STX1B protein or any combination thereof. As used herein, the
term "partially functional" refers to any amount of activity or
function of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein that is less than
the amount of activity or function that is necessary to eliminate
or prevent any one or more symptoms of a disease or condition. In
some embodiments, a partially functional protein or RNA will have
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%,
at least 90%, or at least 95% less activity relative to the fully
functional protein or RNA.
[1400] In embodiments, the method is a method of increasing the
expression of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, STX1B protein or any combination
thereof by cells of a subject having a RIC pre-mRNA encoding the
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein, wherein the subject has a deficient amount
of activity of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, and wherein the
deficient amount of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein is caused by
haploinsufficiency of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein. In such an
embodiment, the subject has a first allele encoding a functional
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein, and a second allele from which the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein is not produced. In another such embodiment, the
subject has a first allele encoding a functional ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein, and a second allele encoding a nonfunctional ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein. In another such embodiment, the subject has a
first allele encoding a functional ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, and a
second allele encoding a partially functional ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein. In any of these embodiments, the antisense oligomer binds
to a targeted portion of the RIC pre-mRNA transcribed from the
first allele (encoding functional ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein),
thereby inducing constitutive splicing of the retained intron from
the RIC pre-mRNA, and causing an increase in the level of mature
mRNA encoding functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, and an
increase in the expression of the ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein in the
cells of the subject.
[1401] In embodiments, the subject has a first allele encoding a
functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B protein, and a second allele
encoding a partially functional ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, and
the antisense oligomer binds to a targeted portion of the RIC
pre-mRNA transcribed from the first allele (encoding functional
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein) or a targeted portion of the RIC pre-mRNA
transcribed from the second allele (encoding partially functional
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein), thereby inducing constitutive splicing of
the retained intron from the RIC pre-mRNA, and causing an increase
in the level of mature mRNA encoding the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein,
and an increase in the expression of functional or partially
functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B protein in the cells of the
subject.
[1402] In some instances, the subject has a first allele encoding a
functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B protein, and a second allele encoding a partially
functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B protein, and the antisense oligomer binds to a
targeted portion of the RIC pre-mRNA transcribed from the first
allele (encoding functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B protein) or a targeted portion of
the RIC pre-mRNA transcribed from the second allele (encoding
partially functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B protein), thereby inducing constitutive
splicing of the retained intron from the RIC pre-mRNA, and causing
an increase in the level of mature mRNA encoding the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
protein, and an increase in the expression of functional or
partially functional ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B protein in the cells of the subject.
[1403] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein in cells of a subject having a RIC pre-mRNA
encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B protein, wherein the subject has a
deficiency in the amount or function of a ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein.
[1404] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
protein in cells of a subject having a RIC pre-mRNA encoding
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or
STX1B protein, wherein the subject has a deficiency in the amount
or function of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B protein.
[1405] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease or condition is targeted
by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the
disease is targeted by the ASOs. For example, a disease that is the
result of a mutation or deficiency of a first protein in a
particular pathway may be ameliorated by targeting a RIC pre-mRNA
that encodes a second protein, thereby increasing production of the
second protein. In some embodiments, the function of the second
protein is able to compensate for the mutation or deficiency of the
first protein (which is causative of the disease or condition).
[1406] In embodiments, the subject has:
[1407] a. a first mutant allele from which [1408] i) the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein is produced at a reduced level compared to
production from a wild-type allele, [1409] ii) the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein is produced in a form having reduced function compared to
an equivalent wild-type protein, or [1410] iii) the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein or functional RNA is not produced; and
[1411] b. a second mutant allele from which [1412] i) the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein is produced at a reduced level compared to
production from a wild-type allele, [1413] ii) the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein is produced in a form having reduced function compared to
an equivalent wild-type protein, or [1414] iii) the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein is not produced, and
[1415] wherein the RIC pre-mRNA is transcribed from the first
allele and/or the second allele. In these embodiments, the ASO
binds to a targeted portion of the RIC pre-mRNA transcribed from
the first allele or the second allele, thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mRNA encoding ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein and an increase in the expression of the target
protein or functional RNA in the cells of the subject. In these
embodiments, the target protein or functional RNA having an
increase in expression level resulting from the constitutive
splicing of the retained intron from the RIC pre-mRNA is either in
a form having reduced function compared to the equivalent wild-type
protein (partially-functional), or having full function compared to
the equivalent wild-type protein (fully-functional).
[1416] In embodiments, the level of mRNA encoding ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B
protein or any combination thereof is increased 1.1 to 10-fold,
when compared to the amount of mRNA encoding ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
that is produced in a control cell, e.g., one that is not treated
with the antisense oligomer or one that is treated with an
antisense oligomer that does not bind to the targeted portion of
the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2,
PRRT2, IDUA, or STX1B RIC pre-mRNA.
[1417] In embodiments, the condition caused by a deficient amount
or activity of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein is not a condition
caused by alternative or aberrant splicing of the retained intron
to which the ASO is targeted. In embodiments, the condition caused
by a deficient amount or activity of the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein
is not a condition caused by alternative or aberrant splicing of
any retained intron in a RIC pre-mRNA encoding the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein. In embodiments, alternative or aberrant splicing may occur
in a pre-mRNA transcribed from the gene, however the compositions
and methods of the invention do not prevent or correct this
alternative or aberrant splicing.
[1418] In some cases, the condition caused by a deficient amount or
activity of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2,
PRRT2, or STX1B protein is not a condition caused by alternative or
aberrant splicing of the retained intron to which the ASO is
targeted. In embodiments, the condition caused by a deficient
amount or activity of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B protein is not a condition caused
by alternative or aberrant splicing of any retained intron in a RIC
pre-mRNA encoding the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B protein.
[1419] In embodiments, alternative or aberrant splicing may occur
in a pre-mRNA transcribed from the gene, however the compositions
and methods of the invention do not prevent or correct this
alternative or aberrant splicing.
[1420] In some instances, the condition caused by a deficient
amount or activity of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B protein is not a condition caused by
alternative or aberrant splicing of the retained intron to which
the ASO is targeted. In embodiments, the condition caused by a
deficient amount or activity of the ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B protein is not a condition caused
by alternative or aberrant splicing of any retained intron in a RIC
pre-mRNA encoding the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B protein. In embodiments, alternative or
aberrant splicing may occur in a pre-mRNA transcribed from the
gene, however the compositions and methods of the invention do not
prevent or correct this alternative or aberrant splicing.
[1421] In embodiments, a subject treated using the methods of the
invention expresses a partially functional ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAIL PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein
from one allele, wherein the partially functional ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein is caused by a frameshift mutation, a nonsense mutation, a
missense mutation, or a partial gene deletion. In embodiments, a
subject treated using the methods of the invention expresses a
nonfunctional ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein from one allele,
wherein the nonfunctional ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein is caused by
a frameshift mutation, a nonsense mutation, a missense mutation, a
partial gene deletion, in one allele. In embodiments, a subject
treated using the methods of the invention has a ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
whole gene deletion, in one allele.
[1422] In some cases, a subject treated using the methods of the
invention expresses a partially functional ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein from one
allele, wherein the partially functional ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein is caused by
a frameshift mutation, a nonsense mutation, a missense mutation, or
a partial gene deletion. In embodiments, a subject treated using
the methods of the invention expresses a nonfunctional ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B
protein from one allele, wherein the nonfunctional ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein is
caused by a frameshift mutation, a nonsense mutation, a missense
mutation, a partial gene deletion, in one allele. In embodiments, a
subject treated using the methods of the invention has a ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B whole
gene deletion, in one allele.
[1423] In some instances, a subject treated using the methods of
the invention expresses a partially functional ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAIL PEX1, ARSA, EIF2B5, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein from one
allele, wherein the partially functional ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1,
ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein is caused by a
frameshift mutation, a nonsense mutation, a missense mutation, or a
partial gene deletion. In embodiments, a subject treated using the
methods of the invention expresses a nonfunctional ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2,
NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B protein from one
allele, wherein the nonfunctional ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B protein is caused by a frameshift
mutation, a nonsense mutation, a missense mutation, a partial gene
deletion, in one allele. In embodiments, a subject treated using
the methods of the invention has a ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR,
STXBP1, PRICKLE2, PRRT2, or STX1B whole gene deletion, in one
allele.
Use of TANGO for Increasing Protein Expression
[1424] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) can be used in the methods of the
invention to increase expression of a ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B protein or
any combinations thereof. In these embodiments, a
retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAIL PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
STX1B protein or any combination thereof is present in the nucleus
of a cell. Cells having a ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that
comprises a retained intron, an exon flanking the 5' splice site,
and an exon flanking the 3' splice site, encoding the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein, can be contacted with antisense oligomers (ASOs)
that are complementary to a targeted portion of the RIC pre-mRNA.
Hybridization of the ASOs to the targeted portion of the RIC
pre-mRNA can result in enhanced splicing at the splice site (5'
splice site or 3' splice site) of the retained intron and
subsequently increases target protein production.
[1425] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[1426] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a RIC pre-mRNA encoding the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein.
[1427] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g. in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features (e.g.
in the case of an antisense oligomer, the presence of additional,
unrecited nucleobases).
[1428] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g. nucleobase sequence) as well as those which do not materially
affect the character or function of the claimed invention. As used
herein, the term "consisting" is used to indicate the presence of
the recited feature (e.g. nucleobase sequence) alone (so that in
the case of an antisense oligomer consisting of a specified
nucleobase sequence, the presence of additional, unrecited
nucleobases is excluded).
[1429] In embodiments, the targeted region is in a retained intron
that is the second most abundant intron in a RIC pre-mRNA encoding
the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2,
PRRT2, IDUA, or STX1B protein. For example, the second most
abundant retained intron may be targeted rather than the most
abundant retained intron due to the uniqueness of the nucleotide
sequence of the second most abundant retained intron, ease of ASO
design to target a particular nucleotide sequence, and/or amount of
increase in protein production resulting from targeting the intron
with an ASO. In embodiments, the retained intron is the second most
abundant intron in a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein in a cell, wherein the
population of RIC pre-mRNAs comprises two or more retained introns.
In embodiments, an antisense oligomer targeted to the second most
abundant intron in the population of RIC pre-mRNAs encoding the
target protein induces splicing out of two or more retained introns
in the population, including the retained intron to which the
antisense oligomer is targeted or binds. In embodiments,
fully-spliced (mature) RNA encoding the target protein is thereby
produced.
[1430] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[1431] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[1432] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for the
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B gene in the published reference genome deposited in
the NCBI repository of biological and scientific information
(operated by National Center for Biotechnology Information,
National Library of Medicine, 8600 Rockville Pike, Bethesda, Md.
USA 20894). As used herein, the "wild-type sequence" refers to NCBI
Gene ID: (ATP1A2:477; CACNA1A:773; SETD5:55209; SHANK3:85358;
NF2:4771; DNMT1:1786; TCF4:6925; RAI1:10743; PEX1:5189; ARSA:410;
EIF2B5:8893; EIF2B1:1967; EIF2B2:8892; NPC1:4864; ADAR:103;
MFSD8:256471; STXBP1:6812; PRICKLE2:166336; PRRT2:112476;
STX1B:112755; IDUA: IDUA). Also used herein, a nucleotide position
denoted with an "e" indicates the nucleotide is present in the
sequence of an exon (e.g., the exon flanking the 5' splice site or
the exon flanking the 3' splice site).
[1433] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein, resulting in increased expression of ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B.
As used herein, "contacting" or administering to cells refers to
any method of providing an ASO in immediate proximity with the
cells such that the ASO and the cells interact. A cell that is
contacted with an ASO will take up or transport the ASO into the
cell. The method involves contacting a condition or
disease-associated or condition or disease-relevant cell with any
of the ASOs described herein. In some embodiments, the ASO may be
further modified or attached (e.g., covalently attached) to another
molecule to target the ASO to a cell type, enhance contact between
the ASO and the condition or disease-associated or condition or
disease-relevant cell, or enhance uptake of the ASO.
[1434] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[1435] In embodiments, contacting a cell that expresses a ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B RIC pre-mRNA with an ASO that is complementary to a
targeted portion of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA transcript
results in a measurable increase in the amount of the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein (e.g., a target protein) encoded by the pre-mRNA.
Methods of measuring or detecting production of a protein will be
evident to one of skill in the art and include any known method,
for example, Western blotting, flow cytometry, immunofluorescence
microscopy, and ELISA.
[1436] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC
pre-mRNA transcript results in an increase in the amount of ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein produced by at least 10, 20, 30, 40, 50, 60, 80,
100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to
the amount of the protein produced by a cell in the absence of the
ASO/absence of treatment. In embodiments, the total amount of
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein produced by the cell to which the antisense
oligomer was contacted is increased about 1.1 to about 10-fold,
about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to
about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,
about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to
about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold,
about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about
8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3
to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,
about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about
9-fold, at least about 1.1-fold, at least about 1.5-fold, at least
about 2-fold, at least about 2.5-fold, at least about 3-fold, at
least about 3.5-fold, at least about 4-fold, at least about 5-fold,
or at least about 10-fold, compared to the amount of target protein
produced by an control compound. A control compound can be, for
example, an oligonucleotide that is not complementary to the
targeted portion of the RIC pre-mRNA.
[1437] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC
pre-mRNA transcript results in an increase in the amount of mRNA
encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B respectively, including the mature
mRNA encoding the target protein. In some embodiments, the amount
of mRNA encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, or the mature mRNA
encoding the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein, is increased by at
least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350,
400, 450, 500, or 1000%, compared to the amount of the protein
produced by a cell in the absence of the ASO/absence of treatment.
In embodiments, the total amount of the mRNA encoding ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein, or the mature mRNA encoding ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein produced in the cell to which the antisense oligomer was
contacted is increased about 1.1 to about 10-fold, about 1.5 to
about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold,
about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to
about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold,
about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to
about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold,
about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about
7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4
to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold,
at least about 1.1-fold, at least about 1.5-fold, at least about
2-fold, at least about 2.5-fold, at least about 3-fold, at least
about 3.5-fold, at least about 4-fold, at least about 5-fold, or at
least about 10-fold compared to the amount of mature RNA produced
in an untreated cell, e.g., an untreated cell or a cell treated
with a control compound. A control compound can be, for example, an
oligonucleotide that is not complementary to the targeted portion
of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA.
[1438] In some cases, contacting cells with an ASO that is
complementary to a targeted portion of a ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, or STX1B RIC pre-mRNA
transcript results in an increase in the amount of mRNA encoding
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, or
STX1B respectively, including the mature mRNA encoding the target
protein. In some embodiments, the amount of mRNA encoding ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, or STX1B
protein, or the mature mRNA encoding the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, or STX1B protein, is
increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200,
250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of
the protein produced by a cell in the absence of the ASO/absence of
treatment. In embodiments, the total amount of the mRNA encoding
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, or
STX1B protein, or the mature mRNA encoding ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, or STX1B protein produced in
the cell to which the antisense oligomer was contacted is increased
about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to
about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold,
about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to
about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold,
about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold compared to the
amount of mature RNA produced in an untreated cell, e.g., an
untreated cell or a cell treated with a control compound. A control
compound can be, for example, an oligonucleotide that is not
complementary to the targeted portion of the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, or STX1B RIC
pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[1439] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein in cells, for example, in a subject having a
deficiency in the amount or activity of ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAIL PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein,
by increasing the level of mRNA encoding ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein,
or the mature mRNA encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein. In
particular, the methods and compositions as described herein can
induce the constitutive splicing of a retained intron from a
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B RIC pre-mRNA transcript encoding ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein, thereby increasing the level of mRNA encoding ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B protein, or the mature mRNA encoding ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein and increasing the expression of ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
protein.
[1440] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[1441] In embodiments, constitutive splicing can correctly remove a
retained intron from a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA, wherein the
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B RIC pre-mRNA is transcribed from a wild-type gene or
allele, or a polymorphic gene or allele, that encodes a
fully-functional target protein or functional RNA, and wherein the
gene or allele does not have a mutation that causes alternative
splicing or aberrant splicing of the retained intron.
[1442] In some embodiments, constitutive splicing of a retained
intron from a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA encoding
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein correctly removes a retained intron from a
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B RIC pre-mRNA encoding ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein,
wherein the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA is transcribed from a
gene or allele from which the target gene or functional RNA is
produced at a reduced level compared to production from a wild-type
allele, and wherein the gene or allele does not have a mutation
that causes alternative splicing or aberrant splicing of the
retained intron. In these embodiments, the correct removal of the
constitutively spliced retained intron results in production of
target protein or functional RNA that is functional when compared
to an equivalent wild-type protein or functional RNA.
[1443] In other embodiments, constitutive splicing can correctly
remove a retained intron from a ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA,
wherein the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA is transcribed from a
gene or allele that encodes a target protein or functional RNA
produced in a form having reduced function compared to an
equivalent wild-type protein or functional RNA, and wherein the
gene or allele does not have a mutation that causes alternative
splicing or aberrant splicing of the retained intron. In these
embodiments, the correct removal of the constitutively spliced
retained intron results in production of partially functional
target protein, or functional RNA that is partially functional when
compared to an equivalent wild-type protein or functional RNA.
[1444] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[1445] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein or the amount of
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B protein by modulating alternative splicing or
aberrant splicing of a pre-mRNA transcribed from the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B gene. Modulation of alternative splicing or aberrant
splicing can be measured using any known method for analyzing the
sequence and length of RNA species, e.g., by RT-PCR and using
methods described elsewhere herein and in the literature. In
embodiments, modulation of alternative or aberrant splicing is
determined based on an increase or decrease in the amount of the
spliced species of interest of at least 10% or 1.1-fold. In
embodiments, modulation is determined based on an increase or
decrease at a level that is at least 10% to 100% or 1.1 to 10-fold,
as described herein regarding determining an increase in mRNA
encoding ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B protein in the methods of the
invention.
[1446] In embodiments, the method is a method wherein the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B RIC pre-mRNA can be produce by partial splicing of a
wild-type ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B pre-mRNA. In embodiments, the
method is a method wherein the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA can be
produce by partial splicing of a full-length wild-type ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B pre-mRNA. In embodiments, the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC
pre-mRNA can be produce by partial splicing of a full-length
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B pre-mRNA. In these embodiments, a full-length
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2,
IDUA, or STX1B pre-mRNA may have a polymorphism in a splice site of
the retained intron that does not impair correct splicing of the
retained intron as compared to splicing of the retained intron
having the wild-type splice site sequence.
[1447] In embodiments, the mRNA encoding ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B protein
is a full-length mature mRNA, or a wild-type mature mRNA. In these
embodiments, a full-length mature mRNA may have a polymorphism that
does not affect the activity of the target protein or the
functional RNA encoded by the mature mRNA, as compared to the
activity of ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B protein encoded by the wild-type
mature mRNA.
Antisense Oligomers
[1448] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA. As used
herein, the terms "ASO" and "antisense oligomer" are used
interchangeably and refer to an oligomer such as a polynucleotide,
comprising nucleobases, that hybridizes to a target nucleic acid
(e.g., a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA) sequence by
Watson-Crick base pairing or wobble base pairing (G-U). The ASO may
have exact sequence complementary to the target sequence or near
complementarity (e.g., sufficient complementarity to bind the
target sequence and enhancing splicing at a splice site). ASOs are
designed so that they bind (hybridize) to a target nucleic acid
(e.g., a targeted portion of a pre-mRNA transcript) and remain
hybridized under physiological conditions. Typically, if they
hybridize to a site other than the intended (targeted) nucleic acid
sequence, they hybridize to a limited number of sequences that are
not a target nucleic acid (to a few sites other than a target
nucleic acid). Design of an ASO can take into consideration the
occurrence of the nucleic acid sequence of the targeted portion of
the pre-mRNA transcript or a sufficiently similar nucleic acid
sequence in other locations in the genome or cellular pre-mRNA or
transcriptome, such that the likelihood the ASO will bind other
sites and cause "off-target" effects is limited. Any antisense
oligomers known in the art, for example in PCT Application No.
PCT/US2014/054151, published as WO 2015/035091, titled "Reducing
Nonsense-Mediated mRNA Decay," can be used to practice the methods
described herein.
[1449] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[1450] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[1451] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[1452] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and
Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by
reference in their entirety.
[1453] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine,
5,6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[1454] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081
(1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et
al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer
Drug Design 6:539 (1991); Zon et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphorothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[1455] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the invention comprises an ASO having
phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[1456] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention comprises about 5-100% Rp, at least about 5% Rp, at
least about 10% Rp, at least about 15% Rp, at least about 20% Rp,
at least about 25% Rp, at least about 30% Rp, at least about 35%
Rp, at least about 40% Rp, at least about 45% Rp, at least about
50% Rp, at least about 55% Rp, at least about 60% Rp, at least
about 65% Rp, at least about 70% Rp, at least about 75% Rp, at
least about 80% Rp, at least about 85% Rp, at least about 90% Rp,
or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
In embodiments, an ASO used in the methods of the invention
comprises about 10% to about 100% Rp, about 15% to about 100% Rp,
about 20% to about 100% Rp, about 25% to about 100% Rp, about 30%
to about 100% Rp, about 35% to about 100% Rp, about 40% to about
100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp,
about 55% to about 100% Rp, about 60% to about 100% Rp, about 65%
to about 100% Rp, about 70% to about 100% Rp, about 75% to about
100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp,
about 90% to about 100% Rp, or about 95% to about 100% Rp, about
20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about
70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp,
with the remainder Sp.
[1457] In embodiments, an ASO used in the methods of the invention
comprises about 5-100% Sp, at least about 5% Sp, at least about 10%
Sp, at least about 15% Sp, at least about 20% Sp, at least about
25% Sp, at least about 30% Sp, at least about 35% Sp, at least
about 40% Sp, at least about 45% Sp, at least about 50% Sp, at
least about 55% Sp, at least about 60% Sp, at least about 65% Sp,
at least about 70% Sp, at least about 75% Sp, at least about 80%
Sp, at least about 85% Sp, at least about 90% Sp, or at least about
95% Sp, with the remainder Rp, or about 100% Sp. In embodiments, an
ASO used in the methods of the invention comprises about 10% to
about 100% Sp, about 15% to about 100% Sp, about 20% to about 100%
Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about
35% to about 100% Sp, about 40% to about 100% Sp, about 45% to
about 100% Sp, about 50% to about 100% Sp, about 55% to about 100%
Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about
70% to about 100% Sp, about 75% to about 100% Sp, about 80% to
about 100% Sp, about 85% to about 100% Sp, about 90% to about 100%
Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about
25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about
60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
[1458] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[1459] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[1460] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[1461] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[1462] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[1463] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[1464] In other embodiments, the ASOs are complementary to (and
bind to) a targeted portion of a ATP1A2, CACNA1A, SETD5, SHANK3,
NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1,
ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA
that is downstream (in the 3' direction) of the 5' splice site of
the retained intron in a ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA (e.g.,
the direction designated by positive numbers relative to the 5'
splice site) (FIG. 1). In some embodiments, the ASOs are
complementary to a targeted portion of the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC
pre-mRNA that is within the region +6 to +4000, +6 to +3500, +6 to
+3000, +6 to +2500, +6 to +2000, +6 to +1500, +6 to +1000, +6 to
+500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 relative to
the 5' splice site of the retained intron. In some embodiments, the
ASO is not complementary to nucleotides +1 to +5 relative to the 5'
splice site (the first five nucleotides located downstream of the
5' splice site). In some embodiments, the ASOs may be complementary
to a targeted portion of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that is
within the region between nucleotides +6 and +50 relative to the 5'
splice site of the retained intron. In some aspects, the ASOs are
complementary to a targeted portion that is within the region +6 to
+90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to
+30, or +6 to +20 relative to 5' splice site of the retained
intron.
[1465] In some embodiments, the ASOs are complementary to a
targeted region of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that is
upstream (5' relative) of the 3' splice site of the retained intron
in a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2,
PRRT2, IDUA, or STX1B RIC pre-mRNA (e.g., in the direction
designated by negative numbers) (FIG. 1). In some embodiments, the
ASOs are complementary to a targeted portion of the ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
or STX1B RIC pre-mRNA that is within the region -16 to -4000, -16
to -3000, -16 to -2000, -16 to -1000, -16 to -500, -16 to -400, -16
to -300, -6 to -200, or -16 to -100 relative to the 3' splice site
of the retained intron. In some embodiments, the ASO is not
complementary to nucleotides -1 to -15 relative to the 3' splice
site (the first 15 nucleotides located upstream of the 3' splice
site). In some embodiments, the ASOs are complementary to a
targeted portion of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that is within
the region -16 to -50 relative to the 3' splice site of the
retained intron. In some aspects, the ASOs are complementary to a
targeted portion that is within the region -16 to -90, -16 to -80,
-16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30
relative to 3' splice site of the retained intron.
[1466] In embodiments, the targeted portion of the ATP1A2, CACNA1A,
SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1,
EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B
RIC pre-mRNA is within the region +100 relative to the 5' splice
site of the retained intron to -100 relative to the 3' splice site
of the retained intron.
[1467] In some embodiments, the ASOs are complementary to a
targeted portion of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that is within
the exon flanking the 5' splice site (upstream) of the retained
intron (FIG. 1). In some embodiments, the ASOs are complementary to
a targeted portion of the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that is
within the region +2e to -4e in the exon flanking the 5' splice
site of the retained intron. In some embodiments, the ASOs are not
complementary to nucleotides -1e to -3e relative to the 5' splice
site of the retained intron. In some embodiments, the ASOs are
complementary to a targeted portion of the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC
pre-mRNA that is within the region -4e to -100e, -4e to -90e, -4e
to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to -40e, -4e to
-30e, or -4e to -20e relative to the 5' splice site of the retained
intron.
[1468] In some embodiments, the ASOs are complementary to a
targeted portion of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that is within
the exon flanking the 3' splice site (downstream) of the retained
intron (FIG. 1). In some embodiments, the ASOs are complementary to
a targeted portion to the ATP1A2, CACNA1A, SETD5, SHANK3, NF2,
DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR,
MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA that is
within the region +2e to -4e in the exon flanking the 3' splice
site of the retained intron. In some embodiments, the ASOs are not
complementary to nucleotide +1e relative to the 3' splice site of
the retained intron. In some embodiments, the ASOs are
complementary to a targeted portion of the ATP1A2, CACNA1A, SETD5,
SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2,
NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, or STX1B RIC
pre-mRNA that is within the region +2e to +100e, +2e to +90e, +2e
to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to
+30e, or +2 to +20e relative to the 3' splice site of the retained
intron. The ASOs may be of any length suitable for specific binding
and effective enhancement of splicing. In some embodiments, the
ASOs consist of 8 to 50 nucleobases. For example, the ASO may be 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases
in length. In some embodiments, the ASOs consist of more than 50
nucleobases. In some embodiments, the ASO is from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40
nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25
nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40
nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25
nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40
nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25
nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40
nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25
nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35
nucleobases, or 25 to 30 nucleobases in length. In some
embodiments, the ASOs are 30 nucleotides in length. In some
embodiments, the ASOs are 29 nucleotides in length. In some
embodiments, the ASOs are 28 nucleotides in length. In some
embodiments, the ASOs are 27 nucleotides in length. In some
embodiments, the ASOs are 26 nucleotides in length. In some
embodiments, the ASOs are 25 nucleotides in length. In some
embodiments, the ASOs are 24 nucleotides in length. In some
embodiments, the ASOs are 23 nucleotides in length. In some
embodiments, the ASOs are 22 nucleotides in length. In some
embodiments, the ASOs are 21 nucleotides in length. In some
embodiments, the ASOs are 20 nucleotides in length. In some
embodiments, the ASOs are 19 nucleotides in length. In some
embodiments, the ASOs are 18 nucleotides in length. In some
embodiments, the ASOs are 17 nucleotides in length. In some
embodiments, the ASOs are 16 nucleotides in length. In some
embodiments, the ASOs are 15 nucleotides in length. In some
embodiments, the ASOs are 14 nucleotides in length. In some
embodiments, the ASOs are 13 nucleotides in length. In some
embodiments, the ASOs are 12 nucleotides in length. In some
embodiments, the ASOs are 11 nucleotides in length. In some
embodiments, the ASOs are 10 nucleotides in length.
[1469] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[1470] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[1471] In some embodiments, the nucleic acid to be targeted by an
ASO is a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1,
PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1,
PRICKLE2, PRRT2, IDUA, or STX1B RIC pre-mRNA expressed in a cell,
such as a eukaryotic cell. In some embodiments, the term "cell" may
refer to a population of cells. In some embodiments, the cell is in
a subject. In some embodiments, the cell is isolated from a
subject. In some embodiments, the cell is ex vivo. In some
embodiments, the cell is a condition or disease-relevant cell or a
cell line. In some embodiments, the cell is in vitro (e.g., in cell
culture).
Pharmaceutical Compositions
[1472] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent
or carrier.
[1473] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose. The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[1474] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[1475] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancers is a surfactant, fatty acid,
bile salt, chelating agent, or non-chelating nonsurfactant.
[1476] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[1477] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[1478] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[1479] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[1480] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[1481] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. The ASOs of the present invention may be
administered to patients parenterally, for example, by
intraperitoneal injection, intramuscular injection, subcutaneous
injection, intrathecal injection, intracerebroventricular
injection, intraperitoneal injection, or intravenous injection. In
embodiments, delivery is to the CNS. In embodiments, a fetus is
treated in utero, e.g., by administering the ASO composition to the
fetus directly or indirectly (e.g., via the mother).
[1482] The compositions of the present invention may be provided to
muscle cells by any suitable means, including direct administration
(e.g., locally by injection or topical administration at a
treatment site) or systemically (e.g., parenterally or orally),
intranasally, orally, or by intrathecal, intracerebroventricular,
inhalational, enteral, topical, intrauterine, vaginal, sublingual,
rectal, intramuscular, intrapleural, intraventricular,
intraperitoneal, ophthalmic, intravenous, or subcutaneous
means.
Methods of Identifying Additional ASOs that Enhance Splicing
[1483] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of a ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1,
ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2,
PRRT2, IDUA, or STX1B RIC pre-mRNA, specifically at the target
intron. ASOs that specifically hybridize to different nucleotides
within the target region of the pre-mRNA may be screened to
identify (determine) ASOs that improve the rate and/or extent of
splicing of the target intron. In some embodiments, the ASO may
block or interfere with the binding site(s) of a splicing
repressor(s)/silencer. Any method known in the art may be used to
identify (determine) an ASO that when hybridized to the target
region of the intron results in the desired effect (e.g., enhanced
splicing, protein or functional RNA production). These methods also
can be used for identifying ASOs that enhance splicing of the
retained intron by binding to a targeted region in an exon flanking
the retained intron, or in a non-retained intron. An example of a
method that may be used is provided below.
[1484] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site.
[1485] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1486] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[1487] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[1488] As described for the ASO walk above, the ASO micro-walk can
be perform by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1489] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. Following administration, the cells, tissues, and/or
organs of the model animals may be assessed to determine the effect
of the ASO treatment by for example evaluating splicing
(efficiency, rate, extent) and protein production by methods known
in the art and described herein. The animal models may also be any
phenotypic or behavioral indication of the disease or disease
severity.
Compositions and Methods for Treatment of Eye Diseases
[1490] Individual introns in primary transcripts of protein-coding
genes having more than one intron are spliced from the primary
transcript with different efficiencies. In most cases only the
fully spliced mRNA is exported through nuclear pores for subsequent
translation in the cytoplasm. Unspliced and partially spliced
transcripts are detectable in the nucleus. It is generally thought
that nuclear accumulation of transcripts that are not fully spliced
is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[1491] Substantial levels of partially-spliced transcripts encoding
the: ROM1 protein, deficient in Retinitis pigmentosa-7; TEAD1
protein, deficient in Sveinsson chorioretinal atrophy; RDH5
protein, deficient in Fundus Albipunctatus; NR2E3 protein,
deficient in Retinitis pigmentosa 37; PAX6 protein, deficient in
Aniridia, Coloboma of the optic nerve, Ocular coloboma, Foveal
hypoplasia-1, Bilateral optic nerve hypoplasia; CRX protein,
deficient in Cone-rod dystrophy-2, Leber congenital amaurosis-7;
FSCN2 protein, deficient in Retinitis Pigmentosa 30; ABCA4 protein,
deficient in Stargardt disease-1, Retinitis pigmentosa-19,
Age-related macular degeneration-2, Cone-rod dystrophy-3; MYOC
protein, deficient in Primary open angle glaucoma; TCF4 protein,
deficient in Fuchs endothelial corneal dystrophy-3; MFSD8 protein,
deficient Macular dystrophy with central cone involvement; CTNS
protein, deficient in Ocular nonnephropathic cystinosis; NXNL1
protein, in Leber congenital amaurosis and Bardet-biedl syndrome;
OPTN protein deficient in primary open angle glaucoma and
amyotrophic lateral sclerosis 12; RLBP1 protein deficient in
bothnia retinal dystrophy, fundus albipunctatus and retinitis
punctata albescens; RPE65 protein deficient in leber congenital
amaurosis 2 and retinitis pigmentosa 20; LRAT protein deficient in
leber congenital amaurosis 14 and retinitis pigmentosa; RDH8
protein in eye diseases with slow clearance or accumulation of all
trans retinal; RDH12 protein deficient in leber congenital
amaurosis 13 and retinitis pigmentosa; RGR protein deficient in
retinitis pigmentosa 44; CNGA3 protein deficient in achromatopsia
2; PER1 protein in jet lag, ALMS1 protein deficient in Alstrom
syndrome and IDUA protein deficient in attenuated MPS 1
(hurler-scheie syndrome and scheie syndrome) have been discovered
in the nucleus of human cells. These ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 and IDUA
pre-mRNA species comprise at least one retained intron. The present
invention provides compositions and methods for upregulating
splicing of one or more retained ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA introns
that are rate-limiting for the nuclear stages of gene expression to
increase steady-state production of fully-spliced, mature mRNA, and
thus, translated ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein levels. These
compositions and methods can utilize antisense oligomers (ASOs)
that promote constitutive splicing at intron splice sites of a
retained-intron-containing ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA pre-mRNA (RIC
pre-mRNA) that accumulates in the nucleus. Thus, in embodiments,
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein can be increased using the
methods of the invention to treat a condition caused by ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, OPTN, RLBP1, RPE65, LRAT, RDH12, RGR, CNGA3, ALMS1 or IDUA
deficiency. In embodiments, the condition is not caused by a
deficiency of the target protein but is nonetheless treated by
increasing production of the target protein using the present
methods. In certain embodiments, wherein the condition that is not
caused by a deficiency of the target protein but is nonetheless
treated by increasing production of the target protein using the
present methods, the target protein is RDH8, NXNL1, or PER1. In
related embodiments, the condition treated is an in eye diseases
with slow clearance or accumulation of all trans retinal and the
target protein is RDH8, or the condition is jet lag, and the target
protein is PER1.
[1492] In other embodiments, the methods of the invention can be
used to increase ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA production to treat a
condition in a subject in need thereof. In embodiments, the subject
has a condition in which ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA is not
necessarily deficient relative to wild-type, but where an increase
in ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA mitigates the condition nonetheless. In
embodiments, the condition can be caused by a ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA haploinsufficiency.
ROM1
[1493] ROM1 (retinal outer segment membrane protein 1) encodes Rod
outer segment membrane protein 1. This gene is a member of a
photoreceptor-specific gene family and encodes an integral membrane
protein found in the photoreceptor disk rim of the eye. This
protein can form homodimers or can heterodimerize with another
photoreceptor, retinal degeneration slow (RDS). ROM1 is essential
for disk morphogenesis, and may also function as an adhesion
molecule involved in the stabilization and compaction of outer
segment disks or in the maintenance of the curvature of the rim.
Certain defects in this gene have been associated with the
degenerative eye disease retinitis pigmentosa.
Retinitis Pigmentosa-7 (RP7)
[1494] Retinitis pigmentosa 7 (RP7) is retinal dystrophy belonging
to the group of pigmentary retinopathies. Retinitis pigmentosa is
characterized by retinal pigment deposits visible on fundus
examination and primary loss of rod photoreceptor cells followed by
secondary loss of cone photoreceptors. Patients typically have
night vision blindness and loss of midperipheral visual field. As
their condition progresses, affected individuals lose their far
peripheral visual field and eventually central vision as well. The
disease may be caused by mutations affecting distinct genetic loci,
including ROM1. A digenic form of retinitis pigmentosa 7 results
from a mutation in the PRPH2 gene and a null mutation of the ROM1
gene has been reported.
TEAD1
[1495] TEAD1 encodes Transcriptional enhancer factor TEF-1.
Transcriptional enhancer factor TEF-1 is a transcription factor
which plays a key role in the Hippo signaling pathway, a pathway
involved in organ size control and tumor suppression by restricting
proliferation and promoting apoptosis. The core of this pathway is
composed of a kinase cascade wherein MST1/MST2, in complex with its
regulatory protein SAV1, phosphorylates and activates LATS1/2 in
complex with its regulatory protein MOB1, which in turn
phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ.
TEAD1 acts by mediating gene expression of YAP1 and WWTR1/TAZ,
thereby regulating cell proliferation, migration and epithelial
mesenchymal transition (EMT) induction. TEAD1 binds specifically
and cooperatively to the SPH and GT-IIC `enhansons`
(5'-GTGGAATGT-3') and activates transcription in vivo in a
cell-specific manner. The activation function appears to be
mediated by a limiting cell-specific transcriptional intermediary
factor (TIF). TEAD1 is also involved in cardiac development, and
binds to the M-CAT motif.
Sveinsson Chorioretinal Atrophy
[1496] Sveinsson chorioretinal atrophy is caused by a mutation in
the TEA domain family member-1 gene TEAD1. Sveinsson's
chorioretinal atrophy (SCRA), also referred to as helicoid
peripapillary chorioretinal degeneration or atrophia areata, is an
autosomal dominant eye disease, characterized by symmetrical
lesions radiating from the optic disc involving the retina and the
choroid.
RDH5
[1497] RDH5 encodes 11-cis retinol dehydrogenase. This enzyme
belongs to the short-chain dehydrogenases/reductases (SDR) family.
This retinol dehydrogenase functions to catalyze the final step in
the biosynthesis of 11-cis retinaldehyde, which is the universal
chromophore of visual pigments. Mutations in this gene cause
autosomal recessive fundus albipunctatus, a rare form of night
blindness that is characterized by a delay in the regeneration of
cone and rod photopigments. Alternative splicing results in
multiple transcript variants. Read-through transcription also
exists between this gene and the neighboring upstream BLOC1S1
(biogenesis of lysosomal organelles complex-1, subunit 1) gene.
Fundus Albipunctatus
[1498] Fundus Albipuctatus is a rare form of night blindness that
is characterized by a delay in the regeneration of cone and rod
photopigments. This form of fleck retina disease is characterized
by discrete uniform white dots over the entire fundus with greatest
density in the midperiphery and no macular involvement. Night
blindness occurs. Both autosomal dominant and autosomal recessive
inheritance has been suggested.
NR2E3
[1499] NR2E3 encodes photoreceptor cell-specific nuclear receptor
(PNR), also known as NR2E3 (nuclear receptor subfamily 2, group E,
member 3). NR2E3 is a nuclear receptor of retinal photoreceptor
cells. NR2E3 is a transcriptional factor that is an activator of
rod development and repressor of cone development. NR2E3 binds the
promoter region of a number of rod- and cone-specific genes,
including rhodopsin, M- and S-opsin and rod-specific
phosphodiesterase beta subunit. NR2E3 enhances rhodopsin expression
and represses M- and S-cone opsin expression.
Retinitis Pigmentosa 37
[1500] Retinitis pigmentosa 37 is caused by homozygous or
heterozygous mutation in the NR2E3 gene. Retinitis pigmentosa 37
(RP37) is a retinal dystrophy belonging to the group of pigmentary
retinopathies. Retinitis pigmentosa is an inherited, degenerative
eye disease that causes severe vision impairment due to the
progressive degeneration of the rod photoreceptor cells in the
retina. This form of retinal dystrophy manifests initial symptoms
independent of age; thus, Retinitis pigmentosa diagnosis occurs
anywhere from early infancy to late adulthood. Patients in the
early stages of Retinitis pigmentosa first notice compromised
peripheral and dim light vision due to the decline of the rod
photoreceptors. The progressive rod degeneration is later followed
by abnormalities in the adjacent retinal pigment epithelium (RPE)
and the deterioration of cone photoreceptor cells. As peripheral
vision becomes increasingly compromised, patients experience
progressive "tunnel vision" and eventual blindness. Affected
individuals may additionally experience defective light-dark
adaptations, nyctalopia (night blindness), and the accumulation of
bone spicules in the fundus.
PAX6
[1501] PAX6 encodes Paired box protein Pax-6 also known as aniridia
type II protein (AN2) or oculorhombin. PAX6, a member of the paired
box gene family, and encodes a transcriptional regulator involved
in oculogenesis and other developmental processes. Pax6 is a
transcription factor present during embryonic development. The
encoded protein contains two different binding sites that are known
to bind DNA and function as regulators of gene transcription. PAX6
is a key regulatory gene of eye and brain development. Within the
brain, the protein is involved in development of the specialized
cells that process smell. As a transcription factor, Pax6 activates
and/or deactivates gene expression patterns to ensure for proper
development of the tissue.
Aniridia
[1502] Aniridia is caused by heterozygous mutation in the PAX6 gene
on chromosome 11p13. More than 280 mutations in the PAX6 gene have
been found to cause aniridia, which is an absence of the iris. Most
of these mutations lead to the production of an abnormally short,
nonfunctional PAX6 protein. As a result, there is less PAX6 protein
to regulate the activity of other genes.
[1503] The majority of mutations that cause aniridia occur within
the PAX6 gene, however, some disease-causing mutations occur in
neighboring regions of DNA that normally regulate the expression of
the PAX6 gene, known as regulatory regions. Mutations in PAX6 gene
regulatory regions reduce the expression of the PAX6 gene. These
mutations lead to a shortage of functional PAX6 protein, which
disrupts the formation of the eyes during development.
[1504] Aniridia is an eye disorder characterized by a complete or
partial absence of the colored part of the iris. These iris
abnormalities may cause the pupils to be abnormal or misshapen.
Aniridia can cause reduction in visual acuity and increased
sensitivity to light (photophobia). Individuals with aniridia can
also have other eye problems. Increased pressure in the eye
(glaucoma) typically appears in late childhood or early
adolescence. Clouding of the lens of the eye (cataracts), occur in
50 percent to 85 percent of people with aniridia. In about 10
percent of affected people, the optic nerves are underdeveloped.
Individuals with aniridia may also have involuntary eye movements
(nystagmus) or underdevelopment of the region at the back of the
eye responsible for sharp central vision (foveal hypoplasia). Many
of these eye problems contribute to progressive vision loss in
affected individuals. The severity of symptoms is typically the
same in both eyes. Rarely, people with aniridia have behavioral
problems, developmental delay, and problems detecting odors.
Aniridia can be inherited in an autosomal dominant pattern. In
approximately two-thirds of cases, an affected person inherits the
mutation from one affected parent. The remaining one-third of cases
result from new mutations in the gene and occur in people with no
history of the disorder in their family.
Coloboma of the Optic Nerve
[1505] Coloboma of the optic nerve is caused by mutation in the
PAX6 gene. Coloboma of the optic nerve is a congenital anomaly of
the optic disc in which there is a defect of the inferior aspect of
the optic nerve. Vision in the affected eye is impaired, the degree
of which depends on the size of the defect, and typically affects
the visual field more than visual acuity. Additionally, there is an
increased risk of serous retinal detachment, manifesting in 1/3 of
patients. If retinal detachment does occur, it is usually not
correctable and all sight is lost in the affected area of the eye,
which may or may not involve the macula.
Ocular Coloboma
[1506] Ocular coloboma is caused by heterozygous mutation in the
PAX6 gene on chromosome 11p13. Coloboma is an ocular birth defect
resulting from abnormal development of the eye during
embryogenesis. It is defined as a congenital defect in any ocular
tissue, typically presenting as absent tissue or a gap, at a site
consistent with aberrant closure of the optic fissure. Failure of
fusion can lead to coloboma of one or multiple regions of the
inferior portion of the eye affecting any part of the globe
traversed by the fissure, from the iris to the optic nerve,
including the ciliary body, retina, and choroid. Coloboma is also
frequently associated with small (microphthalmic) or absent
(anophthalmic) eyes as part of an interrelated spectrum of
developmental eye anomalies, and can affect either one or both
eyes.
Foveal Hypoplasia-1
[1507] Foveal hypoplasia-1 with or without anterior segment
anomalies and/or cataract (FVH1) is caused by heterozygous mutation
in the PAX6 gene on chromosome 11p13. Foveal hypoplasia is defined
as the lack of foveal depression with continuity of all
neurosensory retinal layers in the presumed foveal area. Foveal
hypoplasia as an isolated entity is a rare phenomenon; it is
usually described in association with other ocular disorders, such
as aniridia, microphthalmia, albinism, or achromatopsia. All
reported cases of foveal hypoplasia have been accompanied by
decreased visual acuity and nystagmus.
Bilateral Optic Nerve Hypoplasia
[1508] Bilateral optic nerve hypoplasia can be caused by mutations
in the PAX6 gene. Optic nerve hypoplasia is a medical condition
arising from the underdevelopment of the optic nerve(s). This
condition is the most common congenital optic nerve anomaly. The
optic disc appears abnormally small, because not all the optic
nerve axons have developed properly. It is often associated with
endocrinopathies (hormone deficiencies), developmental delay, and
brain malformations. The optic nerve, which is responsible for
transmitting visual signals from the retina to the brain, has
approximately 1.2 million nerve fibers in the average person. In
those diagnosed with Optic nerve hypoplasia, however, there are
noticeably fewer nerves. Optic nerve hypoplasia can be unilateral
(in one eye) or bilateral (in both eyes), although it presents most
often bilaterally (80%). Because the unilateral cases tend to have
better vision, they are typically diagnosed at a later age than
those with bilateral Optic nerve hypoplasia. Visual acuity can
range from no light perception to near-normal vision.
CRX
[1509] CRX encodes Cone-rod homeobox protein. Cone-rod homeobox
protein is a photoreceptor-specific transcription factor which
plays a role in the differentiation of photoreceptor cells. This
homeodomain protein is necessary for the maintenance of normal cone
and rod function. Cone-rod homeobox protein is a transcription
factor that binds and transactivates the sequence 5'-TAATC[CA]-3'
which is found upstream of several photoreceptor-specific genes,
including the opsin genes. CRX acts synergistically with other
transcription factors, such as NRL, RORB and RAX, to regulate
photoreceptor cell-specific gene transcription and is essential for
the maintenance of mammalian photoreceptors.
Cone-Rod Dystrophy-2 (CORD2)
[1510] Cone-rod dystrophy-2 (CORD2) is caused by heterozygous
mutation in the CRX gene on chromosome 19q13. Cone-rod dystrophy 2
(CORD2) is an inherited retinal dystrophy characterized by retinal
pigment deposits visible on fundus examination, predominantly in
the macular region, and initial loss of cone photoreceptors
followed by rod degeneration. This leads to decreased visual acuity
and sensitivity in the central visual field, followed by loss of
peripheral vision. Severe loss of vision occurs earlier than in
retinitis pigmentosa.
Leber Congenital Amaurosis-7
[1511] Leber congenital amaurosis-7 can be caused by heterozygous
or homozygous mutation in the CRX gene on chromosome 19q13. Leber
congenital amaurosis comprises a group of early-onset childhood
retinal dystrophies characterized by vision loss, nystagmus, and
severe retinal dysfunction. Patients usually present at birth with
profound vision loss and pendular nystagmus. Electroretinogram
(ERG) responses are usually nonrecordable. Other clinical findings
may include high hypermetropia, photodysphoria, oculodigital sign,
keratoconus, cataracts, and a variable appearance to the fundus
FSCN2
[1512] FSCN2 encodes Fascin-2. This gene encodes a member of the
fascin protein family. Fascins crosslink actin into filamentous
bundles within dynamic cell extensions. This family member is
proposed to play a role in photoreceptor disk morphogenesis. A
mutation in this gene results in one form of autosomal dominant
retinitis pigmentosa and macular degeneration. Multiple transcript
variants encoding different isoforms have also been found.
Retinitis Pigmentosa 30
[1513] Petinitis pigmentosa 30 is caused by mutation in the retinal
fascin gene (FSCN2) on chromosome 17q25. Retinitis pigmentosa 30
(RP30) is a retinal dystrophy belonging to the group of pigmentary
retinopathies. Retinitis pigmentosa is characterized by retinal
pigment deposits visible on fundus examination and primary loss of
rod photoreceptor cells followed by secondary loss of cone
photoreceptors. Patients typically have night vision blindness and
loss of midperipheral visual field. As their condition progresses,
they lose their far peripheral visual field and eventually central
vision as well.
ABCA4
[1514] ABCA4 encodes ATP-binding cassette, sub-family A (ABC1),
member 4, also known as ABCA4 or ABCR. ABCA4 is a member of the
ATP-binding cassette transporter gene sub-family A (ABC1) found
exclusively in multicellular eukaryotes. The ABCA4 protein is
produced in photoreceptors. The ABCA4 protein is active following
phototransduction, the process by which light entering the eye is
converted into electrical signals that are transmitted to the
brain. Phototransduction leads to the formation of potentially
toxic substances. The ABCA4 protein removes one of these
substances, called N-retinylidene-PE, from photoreceptor cells.
Stargardt Disease-1 (STGD1)
[1515] Stargardt disease-1 (STGD1) is caused by homozygous or
compound heterozygous mutation in the ABCA4 gene (601691) on
chromosome 1p22. Stargardt macular degeneration is a genetic eye
disorder that causes progressive vision loss. This disorder affects
the retina. Specifically, Stargardt macular degeneration affects a
small area near the center of the retina called the macula. The
macula is responsible for sharp central vision, which is needed for
detailed tasks such as reading, driving, and recognizing faces. In
most people with Stargardt macular degeneration, a fatty yellow
pigment (lipofuscin) builds up in cells underlying the macula. Over
time, the abnormal accumulation of this substance can damage cells
that are critical for clear central vision. In addition to central
vision loss, people with Stargardt macular degeneration have
problems with night vision that can make it difficult to navigate
in low light. Some affected individuals also have impaired color
vision. The signs and symptoms of Stargardt macular degeneration
typically appear in late childhood to early adulthood and worsen
over time.
[1516] More than 500 mutations in the ABCA4 gene have been found to
cause Stargardt macular degeneration. Most of these mutations
change single amino acids in the ABCA4 protein. A malfunctioning
ABCA4 protein cannot remove N-retinylidene-PE from photoreceptor
cells. As a result, N-retinylidene-PE combines with another
substance to produce a fatty yellow pigment called lipofuscin,
which builds up in retinal cells. The buildup of lipofuscin is
toxic to the cells of the retina and causes progressive vision loss
in people with Stargardt macular degeneration. In most cases,
Stargardt macular degeneration is caused by mutations in the ABCA4
gene.
Retinitis Pigmentosa-19 (RP19)
[1517] Retinitis Pigmentosa-19 (RP19) can be caused by homozygous
or compound heterozygous mutation in the ABCR gene (ABCA4) on
chromosome 1p22. Retinitis pigmentosa is an inherited eye disorder
characterized by progressive loss of peripheral vision and night
vision difficulties that can cause central vision loss. There are a
large number of genes linked to retinitis pigmentosa. Type 19 is
linked to a genetic defect on chromosome 1p21-p13. Retinitis
Pigmentosa-19 is characterized by night-blindness, peripheral loss
of vision, progressive retinal degeneration, tunnel vision,
progressive vision loss, decreased vision at night or in low light,
loss of central vision in advanced phases, and retinal pigment
epithelium mottling.
Age-Related Macular Degeneration-2 (ARMD2)
[1518] Age-related macular degeneration-2 (ARMD2) is conferred by
variation in the ABCA4 gene on chromosome 1p22. Age-related macular
degeneration is an eye disease that is a leading cause of vision
loss in older people in developed countries. The vision loss
usually becomes noticeable in a person's sixties or seventies and
tends to worsen over time. Age-related macular degeneration mainly
affects central vision, which is needed for detailed tasks such as
reading, driving, and recognizing faces. The vision loss in this
condition results from a gradual deterioration of light-sensing
cells in the tissue at the back of the eye that detects light and
color (the retina). Specifically, age-related macular degeneration
affects a small area near the center of the retina, called the
macula, which is responsible for central vision. Side (peripheral)
vision and night vision are generally not affected. Researchers
have described two major types of age-related macular degeneration,
known as the dry form and the wet form. The dry form is much more
common, accounting for 85 to 90 percent of all cases of age-related
macular degeneration. It is characterized by a buildup of yellowish
deposits called drusen beneath the retina and slowly progressive
vision loss. The condition typically affects vision in both eyes,
although vision loss often occurs in one eye before the other. The
wet form of age-related macular degeneration is associated with
severe vision loss that can worsen rapidly. This form of the
condition is characterized by the growth of abnormal, fragile blood
vessels underneath the macula. These vessels leak blood and fluid,
which damages the macula and makes central vision appear blurry and
distorted.
Cone-Rod Dystrophy-3 (CORD3)
[1519] Cone-rod dystrophy-3 (CORD3) is caused by homozygous or
compound heterozygous mutation in the ABCA4 on chromosome 1p22.
Cone rod dystrophies are inherited retinal dystrophies that belong
to the group of pigmentary retinopathies. The prevalence of Cone
rod dystrophie is estimated at 1 in 40,000. Cone rod dystrophies
are characterized by retinal pigment deposits, visible on fundus
examination, predominantly localized to the macular region. In
contrast to typical retinitis pigmentosa (RP), also called the rod
cone dystrophies (RCDs), resulting from the primary loss in rod
photoreceptors and later followed by the secondary loss in cone
photoreceptors, Cone rod dystrophies reflect the opposite sequence
of events. Cone rod dystrophy is characterized by primary cone
involvement or, sometimes, by concomitant loss of both cones and
rods, explaining the predominant symptoms of Cone rod dystrophies:
decreased visual acuity, color vision defects, photoaversion and
decreased sensitivity in the central visual field, later followed
by progressive loss in peripheral vision and night blindness. The
clinical course of Cone rod dystrophies is generally more severe
and rapid than that of RCDs, leading to earlier legal blindness and
disability. At end stage, however, Cone rod dystrophies do not
differ from RCDs. Cone rod dystrophies are most frequently
nonsyndromic, but they may also be part of several syndromes, such
as Bardet-Biedl syndrome and Spinocerebellar Ataxia Type 7 (SCAT).
Nonsyndromic Cone rod dystrophies are genetically heterogeneous
(ten cloned genes and three loci have been identified so far). The
four major causative genes involved in the pathogenesis of Cone rod
dystrophies are ABCA4 (which causes Stargardt disease and also 30
to 60% of autosomal recessive Cone rod dystrophies), CRX and GUCY2D
(which are responsible for many reported cases of autosomal
dominant Cone rod dystrophies), and RPGR (which causes about 2/3 of
X-linked RP and also an undetermined percentage of X-linked Cone
rod dystrophies). It is likely that highly deleterious mutations in
genes that otherwise cause RP or macular dystrophy may also lead to
Cone rod dystrophies. The diagnosis of Cone rod dystrophies is
based on clinical history, fundus examination and
electroretinogram. Moleculardiagnosis can be made for some genes,
genetic counseling is always advised. Currently, there is no
therapy that stops the evolution of the disease or restores the
vision, and the visual prognosis is poor. Management aims at
slowing down the degenerative process, treating the complications
and helping patients to cope with the social and psychological
impact of blindness.
MYOC
[1520] The MYOC gene encodes myocilin. Myocilin is found in the
trabecular meshwork and the ciliary body that regulate intraocular
pressure. It is also found in various types of muscle. Myocilin's
function is not well understood, but it may help to control the
intraocular pressure through its action in the muscle tissue of the
ciliary body. Researchers believe that myocilin functions together
with other proteins as part of a protein complex. Myocilin may
interact with a number of other proteins including a form of the
cytochrome P450 protein, the product of the CYP1B1 gene.
Primary Open Angle Glaucoma
[1521] Primary open angle glaucoma (POAG), designated GLC1A, is
caused by heterozygous mutation in the MYOC gene on chromosome 1q.
There are no symptoms associated with POAG. The pressure in the eye
slowly rises and the cornea adapts without swelling. If the cornea
were to swell, which is usually a signal that something is wrong,
symptoms would be present. But as this is not the case, this
disease often goes undetected. It is painless, and the patient
often does not realize that he or she is slowly losing vision until
the later stages of the disease. However, by the time the vision is
impaired, the damage is irreversible. Glaucoma is a group of eye
disorders in which the optic nerves connecting the eyes and the
brain are progressively damaged. This damage can lead to reduction
in side (peripheral) vision and eventual blindness. Other signs and
symptoms may include bulging eyes, excessive tearing, and abnormal
sensitivity to light (photophobia). The term "early-onset glaucoma"
may be used when the disorder appears before the age of 40. In most
people with glaucoma, the damage to the optic nerves is caused by
increased pressure within the eyes (intraocular pressure).
Intraocular pressure depends on a balance between fluid entering
and leaving the eyes. Usually glaucoma develops in older adults, in
whom the risk of developing the disorder may be affected by a
variety of medical conditions including high blood pressure
(hypertension) and diabetes mellitus, as well as family history.
The risk of early-onset glaucoma depends mainly on heredity.
Structural abnormalities that impede fluid drainage in the eye may
be present at birth and usually become apparent during the first
year of life. Such abnormalities may be part of a genetic disorder
that affects many body systems, called a syndrome. If glaucoma
appears before the age of 5 without other associated abnormalities,
it is called primary congenital glaucoma. Other individuals
experience early onset of primary open-angle glaucoma, the most
common adult form of glaucoma. If primary open-angle glaucoma
develops during childhood or early adulthood, it is called juvenile
open-angle glaucoma.
TCF4
[1522] TCF4 encodes TCF4, or sometimes referred to as
immunoglobulin transcription factor 2. TCF4 is a broadly expressed
basic helix-loop-helix (bHLH) protein that functions as a homodimer
or as a heterodimer with other bHLH proteins. These dimers bind DNA
at Ephrussi (E) box sequences. Alternative splicing produces
numerous N-terminally distinct TCF4 isoforms that differ in their
subcellular localization and transactivational capacity. TCF4
proteins act as transcription factors which will bind to the
immunoglobulin enhancer mu-E5/kappa-E2 motif. TCF4 activates
transcription by binding to the E-box (5'-CANNTG-3') found usually
on SSTR2-INR, or somatostatin receptor 2 initiator element. TCF4 is
primarily involved in neurological development of the fetus during
pregnancy by initiating neural differentiation by binding to DNA.
It is found in the central nervous system, somites, and gonadal
ridge during early development. Later in development it will be
found in the thyroid, thymus, and kidneys while in adulthood TCF4
it will be found in lymphocytes, muscles, and gastrointestinal
system.
Fuchs Endothelial Corneal Dystrophy-3 (FECD3)
[1523] Fuchs endothelial corneal dystrophy-3 (FECD3) is caused by
heterozygous intronic trinucleotide repeat expansion (CTG)n in the
TCF4 gene on chromosome 18q22. Late-onset Fuchs endothelial corneal
dystrophy (FECD) is a degenerative disorder affecting roughly 4% of
the population older than 40 years. It is distinguished from other
corneal disorders by the progressive formation of guttae, which are
microscopic refractile excrescences of the Descemet membrane, a
collagen-rich basal lamina secreted by the corneal endothelium.
From onset, it usually takes 2 decades for FECD to impair
endothelial cell function seriously, leading to stromal edema and
impaired vision. The first symptom of this condition is typically
blurred vision in the morning that usually clears during the day.
Over time, affected individuals lose visual acuity. People with
Fuchs endothelial dystrophy also become sensitive to bright lights.
Fuchs endothelial dystrophy specifically affects the front surface
of the eye called the cornea. Deposits called guttae, which are
detectable during an eye exam, form in the middle of the cornea and
eventually spread. These guttae contribute to the loss of cells in
the cornea, leading to vision problems. Tiny blisters may develop
on the cornea, which can burst and cause eye pain. The signs and
symptoms of Fuchs endothelial dystrophy usually begin in a person's
forties or fifties. A very rare early-onset variant of this
condition starts to affect vision in a person's twenties.
MFSD8
[1524] MFSD8 encodes Major facilitator superfamily domain
containing 8 also known as MFSD8. The MFSD8 protein is found in
cell lysosomes. The MFSD8 protein belongs to a large group of
related proteins called the major facilitator superfamily of
secondary active transporter proteins. Proteins in this family move
certain molecules between structures in cells or in and out of
cells. While it is likely that the MFSD8 protein transports
molecules, the specific molecules it moves are unknown. The MFSD8
protein probably transports substances across the membranes of
lysosomes.
Macular Dystrophy with Central Cone Involvement (CCMD)
[1525] Macular dystrophy with central cone involvement (CCMD) is
caused by compound heterozygous mutation in the MFSD8 gene on
chromosome 4q28. This is primarily a cone dystrophy but there is
evidence of some rod damage in older patients. A mild decrease in
central acuity is noted by individuals in the third to sixth
decades. Slight pigmentary changes and color vision abnormalities
can be documented with the onset of these symptoms and a bull's eye
maculopathy and severe atrophy of the central fovea may be present.
An enlarging central scotoma with normal periphery can sometimes be
identified. Other patients have an atrophic appearance to the
peripapillary area with a pale optic disc. Compound heterozygosity
for a missense mutation and a nonsense mutation in the MFSD8 gene
(4q28.2) has been found among members of a Dutch sibship suggesting
autosomal recessive inheritance.
CTNS
[1526] The CTNS gene encodes called cystinosin. This protein is
located in the membrane of lysosomes, which are compartments in the
cell that digest and recycle materials. Proteins digested inside
lysosomes are broken down into smaller amino acids. The amino acids
are then moved out of lysosomes by transport proteins. Cystinosin
is a transport protein that specifically moves the amino acid
cystine out of the lysosome.
Ocular Nonnephropathic Cystinosis
[1527] Ocular nonnephropathic cystinosis is caused by mutation in
the gene encoding cystinosin (CTNS) which maps to chromosome 17p13.
Ocular nonnephropathic cystinosis, a variant of the classic
nephropathic type of cystinosis, is an autosomal recessive
lysosomal storage disorder characterized by photophobia due to
corneal cystine crystals but absence of renal disease. More than 80
different mutations that are responsible for causing cystinosis
have been identified in the CTNS gene. The most common mutation is
a deletion of a large part of the CTNS gene (sometimes referred to
as the 57-kb deletion), resulting in the complete loss of
cystinosin. This deletion is responsible for approximately 50
percent of cystinosis cases in people of European descent. Other
mutations result in the production of an abnormally short protein
that cannot carry out its normal transport function. Mutations that
change very small regions of the CTNS gene may allow the
transporter protein to retain some of its usual activity, resulting
in a milder form of cystinosis.
NXNL1
[1528] NXNL1 encodes Nucleoredoxin-Like 1 (NXNL1). NXNL1 may play a
role in cone cell viability, slowing down cone degeneration. NXNL1
does not seem to play a role in degenerating rods. Diseases
associated with NXNL1 include leber congenital amaurosis and
bardet-biedl syndrome. Leber congenital amaurosis is an eye
disorder that primarily affects the retina. Individuals with this
condition typically have severe visual impairment beginning in
infancy. Other features include photophobia, involuntary movements
of the eyes (nystagmus), and extreme farsightedness. The pupils
also do not react normally to light. Additionally, the cornea may
be cone-shaped and abnormally thin (keratoconus). Franceschetti's
oculo-digital sign is characteristic of leber congenital amaurosis.
This sign consists of poking, pressing, and rubbing the eyes with a
knuckle or finger. At least 13 types of this condition have been
described, which are distinguished by their genetic cause, patterns
of vision loss, and related eye abnormalities.
[1529] Bardet-biedl syndrome is an inherited condition that affects
many parts of the body. Individuals with this condition have
progressive visual impairment due to cone-rod dystrophy, extra
fingers or toes (polydactyly), truncal obesity, decreased function
of the male gonads (hypogonadism), kidney abnormalities, and
learning difficulties. At least 14 genes are known to be associated
with bardet-biedl syndrome. This condition is usually inherited in
an autosomal recessive pattern.
OPTN\
[1530] The OPTN gene encodes the coiled-coil containing protein
optineurin. Optineurin may play a role in normal-tension glaucoma
and adult-onset primary open angle glaucoma. Optineurin interacts
with adenovirus E3-14.7K protein and may utilize tumor necrosis
factor-alpha or Fas-ligand pathways to mediate apoptosis,
inflammation or vasoconstriction. Optineurin may also function in
cellular morphogenesis and membrane trafficking, vesicle
trafficking, and transcription activation through its interactions
with the RAB8, huntingtin, and transcription factor IIIA proteins.
Alternative splicing results in multiple transcript variants
encoding the same protein.
[1531] Optineurin plays an important role in the maintenance of the
Golgi complex, in membrane trafficking, in exocytosis, through its
interaction with myosin VI and Rab8. Optineurin links myosin VI to
the Golgi complex and plays an important role in Golgi ribbon
formation. Optineurin negatively regulates the induction of IFNB in
response to RNA virus infection. Optineurin plays a neuroprotective
role in the eye and optic nerve. Optineurin is indicated as part of
the TNF-alpha signaling pathway that can shift the equilibrium
toward induction of cell death. Optineurin may act by regulating
membrane trafficking and cellular morphogenesis via a complex that
contains Rab8 and hungtingtin (HD). Optineurin mediates the
interaction of Rab8 with the probable GTPase-activating protein
TBC1D17 during Rab8-mediated endocytic trafficking, such as of
transferrin receptor (TFRC/TfR); regulates Rab8 recruitment to
tubules emanating from the endocytic recycling compartment.
Autophagy receptor that interacts directly with both the cargo to
become degraded and an autophagy modifier of the MAP1 LC3 family;
targets ubiquitin-coated bacteria (xenophagy), such as cytoplasmic
Salmonella enterica, and appears to function in the same pathway as
SQSTM1 and CALCOCO2/NDP52. Optineurin may constitute a cellular
target for adenovirus E3 14.7, an inhibitor of TNF-alpha functions,
thereby affecting cell death.
Primary Open Angle Glaucoma;
[1532] Primary open angle glaucoma (POAG) is characterized by a
specific pattern of optic nerve and visual field defects. The angle
of the anterior chamber of the eye is open, and usually the
intraocular pressure is increased. However, glaucoma can occur at
any intraocular pressure. The disease is generally asymptomatic
until the late stages, by which time significant and irreversible
optic nerve damage has already taken place. The disease is caused
by mutations affecting the OPTN gene
Amyotrophic Lateral Sclerosis 12
[1533] Amyotrophic lateral sclerosis 12 (ALS12) is a
neurodegenerative disorder affecting upper motor neurons in the
brain and lower motor neurons in the brain stem and spinal cord,
resulting in fatal paralysis. Sensory abnormalities are absent. The
pathologic hallmarks of the disease include pallor of the
corticospinal tract due to loss of motor neurons, presence of
ubiquitin-positive inclusions within surviving motor neurons, and
deposition of pathologic aggregates. The etiology of amyotrophic
lateral sclerosis is likely to be multifactorial, involving both
genetic and environmental factors. The disease is inherited in
5-10% of the cases.
[1534] In some cases, in a patient with amyotrophic lateral
sclerosis 12, heterozygous mutations in the OPTN (G538EfsX27) and
TBK1 (R117X) genes can be identified. In some cases, in a patient
with amyotrophic lateral sclerosis 12, a deletion of exon 5 can be
identified. In some cases, in a patient with amyotrophic lateral
sclerosis 12, a nonsense mutation (Q398X) can be identified. In
some cases, in a patient with amyotrophic lateral sclerosis 12,
heterozygosity for a missense mutation (E478G) within the OPTN
ubiquitin-binding domain can be identified.
RLBP1
[1535] The RLBP1 gene encodes is a 36-kD water-soluble protein
which carries 11-cis-retinaldehyde or 11-cis-retinal as physiologic
ligands. The RLBP1 protein may be a functional component of the
visual cycle. Mutations of this gene have been associated with
severe rod-cone dystrophy, Bothnia dystrophy (nonsyndromic
autosomal recessive retinitis pigmentosa) and retinitis punctata
albescens.
Bothnia Retinal Dystrophy
[1536] Bothnia retinal dystrophy (BRD) is a type of retinitis
punctata albescens. Affected individuals show night blindness from
early childhood with features consistent with retinitis punctata
albescens and macular degeneration. The disease is caused by
mutations affecting the RLBP1 gene.
Fundus Albipunctatus
[1537] Fundus albipunctatus is a retinal disorder characterized by
night blindness and delayed dark adaptation after exposure to
bright light, which typically presents during early childhood. The
fundi of affected individuals contain multiple small, white or pale
yellow dots in the retinal pigment epithelium, which may or may not
involve the macula. These dots can remain unchanged, become more
prominent, or can fade during aging; new dots may also appear. The
dark-adaptation curve of affected individuals features prolonged
recovery of cone and rod sensitivity and electroretinogram cone and
rod amplitudes are markedly reduced after 30-40 minutes of dark
adaptation; however, they may come to normal or near-normal levels
after many hours of adaptation showed that approximately 38% of
individuals with fundus albipunctatus have extensive cone
dysfunction. Mutations in the RLBP1 gene have also been reported in
fundus albipunctatus patients.
Retinitis Punctata Albescens
[1538] Retinitis punctata albescens (RPA) is a form of fleck retina
disease characterized by aggregation of white flecks posteriorly in
the retina, causing night blindness and delayed dark adaptation. It
differs from fundus albipunctatus in being progressive and evolving
to generalized atrophy of the retina. The disease is caused by
mutations affecting the RLBP1 gene.
RPE65
[1539] The RPE65 gene alos "retinal pigment epithelium-specific
protein 65 kDa." The RPE65 gene encodes the RPE65 protein that is
essential for normal vision. The RPE65 protein is produced in the
retinal pigment epithelium (RPE). The RPE supports and nourishes
the retina, which is the light-sensitive tissue that lines the back
of the eye. The RPE65 protein is involved in a multi-step process
called the visual cycle, which converts light entering the eye into
electrical signals that are transmitted to the brain. When light
hits photosensitive pigments in the retina, it changes 11-cis
retinal (a form of vitamin A) to all-trans retinal. This conversion
triggers a series of chemical reactions that create electrical
signals. The RPE65 protein then helps convert all-trans retinal
back to 11-cis retinal so the visual cycle can begin again
Leber Congenital Amaurosis 2
[1540] Leber congenital amaurosis is an eye disorder that primarily
affects the retina, which is the specialized tissue at the back of
the eye that detects light and color. People with this disorder
typically have severe visual impairment beginning in infancy. The
visual impairment tends to be stable, although it may worsen very
slowly over time.
[1541] Leber congenital amaurosis is also associated with other
vision problems, including an increased sensitivity to light
(photophobia), involuntary movements of the eyes (nystagmus), and
extreme farsightedness (hyperopia). The pupils, which usually
expand and contract in response to the amount of light entering the
eye, do not react normally to light. Instead, they expand and
contract more slowly than normal, or they may not respond to light
at all. Additionally, the clear front covering of the eye (the
cornea) may be cone-shaped and abnormally thin, a condition known
as keratoconus.
[1542] A specific behavior called Franceschetti's oculo-digital
sign is characteristic of Leber congenital amaurosis. This sign
consists of poking, pressing, and rubbing the eyes with a knuckle
or finger. Researchers suspect that this behavior may contribute to
deep-set eyes and keratoconus in affected children.
[1543] In rare cases, delayed development and intellectual
disability have been reported in people with the features of Leber
congenital amaurosis. However, it is uncertain whether these
individuals actually have Leber congenital amaurosis or another
syndrome with similar signs and symptoms. At least 13 types of
Leber congenital amaurosis have been described. The types are
distinguished by their genetic cause, patterns of vision loss, and
related eye abnormalities. Leber Congenital Amaurosis 2 is
distinguished by moderate visual impairment at infancy that
progresses to total blindness by mid to late adulthood. One of the
unique qualities of Leber Congenital Amaurosis 2 is that, even with
profound early visual impairment, retinal cells are relatively
preserved.
[1544] In some cases, in patients with leber congenital amaurosis
2, compound heterozygosity for mutations in the RPE65 gene, a 1-bp
deletion and/or a nonsense mutation can be identified.
Retinitis Pigmentosa 20
[1545] Retinitis pigmentosa 20 is retinal dystrophy belonging to
the group of pigmentary retinopathies. Retinitis pigmentosa is
characterized by retinal pigment deposits visible on fundus
examination and primary loss of rod photoreceptor cells followed by
secondary loss of cone photoreceptors. Patients typically have
night vision blindness and loss of midperipheral visual field. As
their condition progresses, they lose their far peripheral visual
field and eventually central vision as well.
LRAT
[1546] LRAT gene, or lecithin retinol acyltransferase
(phosphatidylcholine-retinol O-acyltransferase) encodes the LRAT
protein that localized to the endoplasmic reticulum, where it
catalyzes the esterification of all-trans-retinol into
all-trans-retinyl ester. This reaction is an important step in
vitamin A metabolism in the visual system. Mutations in this gene
have been associated with early-onset severe retinal dystrophy and
Leber congenital amaurosis 14. Alternative splicing results in
multiple transcript variants. The LRAT protein transfers the acyl
group from the sn-1 position of phosphatidylcholine to all-trans
retinol, producing all-trans retinyl esters. Retinyl esters are
storage forms of vitamin A. LRAT plays a critical role in vision.
It provides the all-trans retinyl ester substrates for the
isomerohydrolase which processes the esters into 11-cis-retinol in
the retinal pigment epithelium; due to a membrane-associated
alcohol dehydrogenase, 11 cis-retinol is oxidized and converted
into 11-cis-retinaldehyde which is the chromophore for rhodopsin
and the cone photopigments.
Leber Congenital Amaurosis 14;
[1547] Leber congenital amaurosis 14 (LCA14) is a severe dystrophy
of the retina, typically becoming evident in the first years of
life. Visual function is usually poor and often accompanied by
nystagmus, sluggish or near-absent pupillary responses,
photophobia, high hyperopia and keratoconus. The disease is caused
by mutations affecting the LRAT gene.
Retinitis Pigmentosa
[1548] Retinitis pigmentosa is a group of related eye disorders
that cause progressive vision loss. These disorders affect the
retina, which is the layer of light-sensitive tissue at the back of
the eye. In people with retinitis pigmentosa, vision loss occurs as
the light-sensing cells of the retina gradually deteriorate. The
first sign of retinitis pigmentosa is usually a loss of night
vision, which becomes apparent in childhood. Problems with night
vision can make it difficult to navigate in low light. Later, the
disease causes blind spots to develop in the side (peripheral)
vision. Over time, these blind spots merge to produce tunnel
vision. The disease progresses over years or decades to affect
central vision, which is needed for detailed tasks such as reading,
driving, and recognizing faces. In adulthood, many people with
retinitis pigmentosa become legally blind.
[1549] The signs and symptoms of retinitis pigmentosa are most
often limited to vision loss. When the disorder occurs by itself,
it is described as nonsyndromic. Researchers have identified
several major types of nonsyndromic retinitis pigmentosa, which are
usually distinguished by their pattern of inheritance: autosomal
dominant, autosomal recessive, or X-linked. Less commonly,
retinitis pigmentosa occurs as part of syndromes that affect other
organs and tissues in the body. These forms of the disease are
described as syndromic. The most common form of syndromic retinitis
pigmentosa is Usher syndrome, which is characterized by the
combination of vision loss and hearing loss beginning early in
life. Retinitis pigmentosa is also a feature of several other
genetic syndromes, including Bardet-Biedl syndrome; Refsum disease;
and neuropathy, ataxia, and retinitis pigmentosa (NARP).
RDH8
[1550] Retinol dehydrogenase 8 (all-trans) is an enzyme encoded by
the RDH8 gene. All-trans-retinol dehydrogenase (RDH8) is a visual
cycle enzyme that reduces all-trans-retinal to all-trans-retinol in
the presence of NADPH. It is a member of the short chain
dehydrogenase/reductase family and is located in the outer segments
of photoreceptors; hence it is also known as photoreceptor retinol
dehydrogenase. It is important in the visual cycle by beginning the
rhodopsin regeneration pathway by reducing all-trans-retinal, the
product of bleached and hydrolyzed rhodopsin.
RDH12
[1551] The RDH12 gene, Retinol dehydrogenase 12
(all-trans/9-cis/11-cis), encodes the RDH12 protein which is an
NADPH-dependent retinal reductase whose highest activity is toward
9-cis and all-trans-retinol. The encoded enzyme also plays a role
in the metabolism of short-chain aldehydes but does not exhibit
steroid dehydrogenase activity. Defects in this gene are a cause of
Leber congenital amaurosis type 13 and Retinitis Pigmentosa 53.
Leber Congenital Amaurosis13
[1552] Leber congenital amaurosis 13 (LCA13) is a severe dystrophy
of the retina, typically becoming evident in the first years of
life. Visual function is usually poor and often accompanied by
nystagmus, sluggish or near-absent pupillary responses,
photophobia, high hyperopia and keratoconus. In some cases, leber
congenital amaurosis-13 can be caused by homozygous or compound
heterozygous mutation in the photoreceptor-specific retinal
dehydrogenase gene RDH12 on chromosome 14q23.3.
Retinitis Pigmentosa 53
[1553] Retinitis pigmentosa 53 (RP53) is a retinal dystrophy
belonging to the group of pigmentary retinopathies. Retinitis
pigmentosa is characterized by retinal pigment deposits visible on
fundus examination and primary loss of rod photoreceptor cells
followed by secondary loss of cone photoreceptors. Patients
typically have night vision blindness and loss of midperipheral
visual field. As their condition progresses, they lose their far
peripheral visual field and eventually central vision as well.
RGR
[1554] The RGR gene encodes a putative retinal G-protein coupled
receptor. The gene is a member of the opsin subfamily of the 7
transmembrane, G-protein coupled receptor 1 family. Like other
opsins which bind retinaldehyde, it contains a conserved lysine
residue in the seventh transmembrane domain. The protein acts as a
photoisomerase to catalyze the conversion of all-trans-retinal to
11-cis-retinal. The reverse isomerization occurs with rhodopsin in
retinal photoreceptor cells. The protein is exclusively expressed
in tissue adjacent to retinal photoreceptor cells, the retinal
pigment epithelium and Mueller cells. This gene may be associated
with autosomal recessive and autosomal dominant retinitis
pigmentosa (arRP and adRP, respectively). Alternative splicing
results in multiple transcript variants encoding different
isoforms. Retinal G protein-coupled receptor (RGR) is a rhodopsin
homolog found exclusively in cells adjacent to the retinal
photoreceptor cells (i.e., the retinal pigment epithelium and
Muller cells). It preferentially binds all-trans retinal rather
than 11-cis retinal, which is normally found in rhodopsin. In
mammals, photons of light convert all-trans retinal within RGR to
11-cis retinal, whereas the reverse isomerization reaction occurs
in rhodopsin in photoreceptor cells.
Retinitis Pigmentosa 44
[1555] Retinitis pigmentosa 44 (RP44) is a retinal dystrophy
belonging to the group of pigmentary retinopathies. Retinitis
pigmentosa is characterized by retinal pigment deposits visible on
fundus examination and primary loss of rod photoreceptor cells
followed by secondary loss of cone photoreceptors. Patients
typically have night vision blindness and loss of midperipheral
visual field. As their condition progresses, they lose their far
peripheral visual field and eventually central vision as well. The
disease is caused by mutations affecting the RGR gene.
CNGA3
[1556] The CNGA3 or cyclic nucleotide gated channel alpha 3 gene
encodes one part (the alpha subunit) of the cone photoreceptor
cyclic nucleotide-gated (CNG) channel. These channels are found
exclusively in cones, which are located in the retina. Cones
provide vision in bright light (daylight vision), including color
vision.
[1557] CNG channels are openings in the cell membrane that
transport cations into cells. In cones, CNG channels remain open
under dark conditions, allowing cations to flow in. When light
enters the eye, it triggers the closure of these channels, stopping
the inward flow of cations. This change in cation transport alters
the cone's electrical charge, which ultimately causes
phototransduction. More than 100 mutations in the CNGA3 gene have
been found to cause the vision disorder achromatopsia. These
mutations underlie about 25 percent of cases of complete
achromatopsia, a form of the disorder characterized by a total lack
of color vision and other vision problems that are present from
early infancy. CNGA3 gene mutations have also been identified in a
few individuals with incomplete achromatopsia, a milder form of the
disorder associated with limited color vision.
[1558] The CNGA3 gene mutations that underlie complete
achromatopsia affect the production or function of the alpha
subunit. In some cases, no protein is produced. In others, the
protein is altered and does not function normally. CNG channels
assembled without the alpha subunit or with an abnormal subunit are
nonfunctional; they prevent cones from carrying out
phototransduction. In some cases, defective channels allow a huge
influx of cations into cones, which ultimately causes these cells
to undergo apoptosis. A loss of cone function underlies the lack of
color vision and other vision problems in people with complete
achromatopsia.
[1559] A few mutations in the CNGA3 gene reduce but do not
eliminate the function of CNG channels in cones. These mutations
cause incomplete achromatopsia because the partially functioning
cones can transmit some visual information to the brain. Because
these CNG channels are specific to cones, rods are generally
unaffected by this disorder.
[1560] Mutations in the CNGA3 gene have also been identified in a
small percentage of cases of progressive cone dystrophy. However,
unlike achromatopsia, progressive cone dystrophy is associated with
cones that work normally at birth but begin to malfunction in
childhood or adolescence. Over time, people with progressive cone
dystrophy develop increasing blurriness and loss of color vision.
It is unclear why some CNGA3 gene mutations cause achromatopsia and
others result in progressive cone dystrophy
Achromatopsia-2
[1561] Achromatopsia-2 is an autosomal recessive disorder and is
total colorblindness, also referred to as rod monochromacy or
complete achromatopsia, is a rare congenital autosomal recessive
disorder characterized by photophobia, reduced visual acuity,
nystagmus, and the complete inability to discriminate between
colors. Electroretinographic recordings show that in achromatopsia
the rod photoreceptor function is normal, whereas cone
photoreceptor responses are absent.
PER1
[1562] The PER1 gene encodes the period circadian protein homolog 1
protein in humans. PER1 is a master regulator of circadian rhythm
and functions in the nucleus to repress expression of the central
circadian clock genes (e.g., CLOCK). The periodicity of PER1
abundance, nuclear translocation, and transcriptional repression is
regulated by PER1 phosphorylation, ubiquitination, and proteasomal
degradation
[1563] The PER1 protein is important to the maintenance of
circadian rhythms in cells, and m ay also play a role in the
development of cancer. This gene is a member of the period family
of genes. It is expressed with a daily oscillating circadian
rhythm, or an oscillation that cycles with a period of
approximately 24 hours. PER1 is most notably expressed in the
region of the brain called the suprachiasmatic nucleus (SCN), which
is the primary circadian pacemaker in the mammalian brain. PER1 is
also expressed throughout mammalian peripheral tissues. Genes in
this family encode components of the circadian rhythms of locomotor
activity, metabolism, and behavior. Circadian expression of PER1 in
the suprachiasmatic nucleus will free-run in constant darkness,
meaning that the 24-hour period of the cycle will persist without
the aid of external light cues. Subsequently, a shift in the
light/dark cycle evokes a proportional shift of gene expression in
the suprachiasmatic nucleus. The time of gene expression is
sensitive to light, as light during a mammal's subjective night
results in a sudden increase in per expression and thus a shift in
phase in the suprachiasmatic nucleus. Alternative splicing has been
observed in this gene. In some cases, PER1 can be involved in the
effects of Jet lag.
IDUA
[1564] The IDUA gene encodes an enzyme called alpha-L-iduronidase,
which is essential for the breakdown of large sugar molecules, for
example glycosaminoglycans (GAGs). Through hydrolysis,
alpha-L-iduronidase uses water molecules to break down unsulfated
alpha-L-iduronic acid, which is present in heparan sulfate and
dermatan sulfate. Alpha-L-iduronidase can be located in lysosomes.
More than 100 mutations in the IDUA gene have been found to cause
mucopolysaccharidosis type I (MPS I). Most mutations that cause MPS
I reduce or completely eliminate the function of
alpha-L-iduronidase. It usually cannot be determined whether a
certain mutation will cause severe or attenuated MPS I; however,
people who do not produce any alpha-L-iduronidase have the severe
form of this disorder.
[1565] The lack of alpha-L-iduronidase enzyme activity leads to the
accumulation of heparan sulfate and dermatan sulfate within the
lysosomes. The buildup of GAGs increases the size of the lysosomes.
The accumulated GAGs may also interfere with the functions of other
proteins inside the lysosomes and disrupt the movement of molecules
inside the cell.
Attenuated MPS-1
[1566] Mucopolysaccharidosis type I (MPS I) is a condition that
affects many parts of the body. This disorder was once divided into
three separate syndromes: Hurler syndrome (MPS I-H), Hurler-Scheie
syndrome (MPS I-H/S), and Scheie syndrome (MPS I-S), listed from
most to least severe. Because there is so much overlap between each
of these three syndromes, MPS I is currently divided into the
severe and attenuated types.
[1567] Children with MPS I often have no signs or symptoms of the
condition at birth, although some have a soft out-pouching around
the belly-button (umbilical hernia) or lower abdomen (inguinal
hernia). People with severe MPS I generally begin to show other
signs and symptoms of the disorder within the first year of life,
while those with the attenuated form have milder features that
develop later in childhood.
[1568] Individuals with MPS I may have a large head (macrocephaly),
a buildup of fluid in the brain (hydrocephalus), heart valve
abnormalities, distinctive-looking facial features that are
described as "coarse," an enlarged liver and spleen
(hepatosplenomegaly), and a large tongue (macroglossia). Vocal
cords can also enlarge, resulting in a deep, hoarse voice. The
airway may become narrow in some people with MPS I, causing
frequent upper respiratory infections and short pauses in breathing
during sleep (sleep apnea).
[1569] People with MPS I often develop clouding of the clear
covering of the eye (cornea), which can cause significant vision
loss. Affected individuals may also have hearing loss and recurrent
ear infections.
[1570] Some individuals with MPS I have short stature and joint
deformities (contractures) that affect mobility. Most people with
the severe form of the disorder also have dysostosis multiplex,
which refers to multiple skeletal abnormalities seen on x-ray.
Carpal tunnel syndrome develops in many children with this disorder
and is characterized by numbness, tingling, and weakness in the
hand and fingers. Narrowing of the spinal canal (spinal stenosis)
in the neck can compress and damage the spinal cord.
[1571] While both forms of MPS I can affect many different organs
and tissues, people with severe MPS I experience a decline in
intellectual function and a more rapid disease progression.
Developmental delay is usually present by age 1, and severely
affected individuals eventually lose basic functional skills
(developmentally regress). Children with this form of the disorder
usually have a shortened lifespan, sometimes living only into late
childhood. Individuals with attenuated MPS I typically live into
adulthood and may or may not have a shortened lifespan. Some people
with the attenuated type have learning disabilities, while others
have no intellectual impairments. Heart disease and airway
obstruction are major causes of death in people with both types of
MPS I.
Hurler-Scheie Syndrome
[1572] Hurler-Scheie syndrome is the intermediate form of
mucopolysaccharidosis type 1 (MPS1) between the two extremes Hurler
syndrome and Scheie syndrome, it is a rare lysosomal storage
disease, characterized by skeletal deformities and a delay in motor
development. The prevalence of MPS I has been estimated at
1/100,000, with Hurler-Scheie syndrome accounting for 23% of cases
or a prevalence of approximately 1/435,000. Patients with
Hurler-Scheie syndrome have normal or almost normal intelligence
but exhibit various degrees of physical impairment. Patients
present in the first years of life with musculoskeletal alterations
to different degrees including short stature, multiple dysostosis,
thoracic-lumbar kyphosis, progressive coarsening of the facial
features to different degrees, cardiomyopathy and valvular
abnormalities, neurosensorial hearing loss, enlarged tonsils and
adenoids, and nasal secretion. Hydrocephaly can occur after the age
of two. Corneal opacity is seen between two and four years of age
and requires keratoplasty to restore sight. Other manifestations
may include organomegaly, hernias and hirsutism.
[1573] Hurler-Scheie syndrome is caused by mutations in the IDUA
gene (4p16.3) leading to partial deficiency in the
alpha-L-iduronidase enzyme and lysosomal accumulation of dermatan
sulfate and heparan sulfate. Early diagnosis is difficult because
the first clinical signs are not specific. Diagnosis can be based
on detection of increased urinary secretion of heparan and dermatan
sulfate through 1,9-dimethylmethylene blue (DMB) test and
glycosaminoglycan (GAG) electrophoresis, and demonstration of
enzymatic deficiency in leukocytes or fibroblasts. Genetic testing
is available. Differential diagnoses include the milder and more
severe forms of mucopolysaccharidosis type 1 (Scheie syndrome and
Hurler syndrome respectively), mucopolysaccharidosis typeVI and
mucopolysaccharidosis type II. Antenatal diagnosis is possible by
measurement of enzymatic activity in cultivated chorionic villus or
amniocytes and by genetic testing if the disease-causing mutation
is known. Transmission is autosomal recessive. Bone marrow or
umbilical cord blood transplant has been successful and can
preserve neurocognition, improve some aspects of the somatic
disease and increase survival. However it is associated with many
risks and most of the positive effects occur only if the procedure
is performed in the first two years of life.
[1574] The enzyme substitute (laronidase) obtained EU marketing
authorization as an orphan drug in 2003. Given through weekly
infusions it leads to improvement of lung function and joint
mobility. Enzyme replacement therapy (ERT) can be started at
diagnosis and may be beneficial in patients awaiting hematopoietic
stem cell transplantation (HSCT). Early treatment can slow the
progression of the disease. In individual patients with MPS1 of
intermediate severity, HSCT may be considered if there is a
suitable donor. There are however no data on the efficacy of HSCT
in patients with this form of the disease.
[1575] Life expectancy for Hurler-Scheie syndrome may be reduced,
with death occurring before adolescence due to serious
cardiovascular and respiratory complications.
[1576] In some cases, in a patient with hurler scheie syndrome,
homozygosity for an arg619-to-gly (R619G) mutation due to a C-to-G
transversion at nucleotide 1943 can be identified. In some cases,
in a patient with hurler scheie syndrome, homozygosity for a
thr364-to-met (T364M) mutation in the IDUA gene can be
identified.
ALMS1
[1577] The ALMS1 gene encodes the ALMS1 protein. ALMS1 gene is
located on the short (p) arm of chromosome 2 at position 13. The
ALMS1 protein may play a role in hearing, vision, regulation of
body weight, and functions of the heart, kidney, lungs, and liver.
It may also affect how the pancreas regulates insulin, a hormone
that helps control blood sugar levels.
[1578] The ALMS1 protein is present in most of the body's tissues,
usually at low levels. Within cells, this protein is located in
centrosomes. Centrosomes play a role in cell division and the
assembly of microtubules. The ALMS1 protein is also found at the
base of cilia. Based on its location within cells, the ALMS1
protein may be involved in the organization of microtubules, the
transport of various materials, and the normal function of
cilia.
Alstrom Syndrome
[1579] Alstrom syndrome is a rare condition that affects many body
systems. Many of the signs and symptoms of this condition begin in
infancy or early childhood, although some appear later in life.
Alstrom syndrome is characterized by a progressive loss of vision
and hearing, a form of heart disease that enlarges and weakens the
heart muscle (dilated cardiomyopathy), obesity, type 2 diabetes
mellitus and short stature. This disorder can also cause serious or
life-threatening medical problems involving the liver, kidneys,
bladder, and lungs. Some individuals with Alstrom syndrome have a
skin condition called acanthosis nigricans, which causes the skin
in body folds and creases to become thick, dark, and velvety. The
signs and symptoms of Alstrom syndrome vary in severity, and not
all affected individuals have all of the characteristic features of
the disorder.
[1580] More than 80 mutations in the ALMS1 gene have been
identified in people with Alstrom syndrome. 32 mutations in exon
16, 19 mutations in exon 10, and 17 mutations in exon 8 were
identified in Alstrom syndrome patients. The most common allele was
a 1-bp deletion (10775delC) identified in 12% of mutated alleles.
In some cases, in patients with Alstrom syndrome a homozygosity for
insertion of a novel 333-bp Alu Ya5 SINE retrotransposon into exon
16 of the ALMS1 gene can be identified. In some cases, in patients
with Alstrom syndrome an insertion of 19 bp in exon 16 of the ALMS1
gene, causing a frameshift resulting in early termination at codon
3530 can be identified. In some cases, in patients with Alstrom
syndrome an 8383C-T transition in the ALMS1 gene in homozygous
state, causing a nonsense change, glu2795 to ter (G2795X) can be
identified. In some cases, in patients with Alstrom syndrome a
10775delC mutation in the ALMS1 gene can be identified. In some
cases, in patients with Alstrom syndrome a deletion of 2 bp in exon
8 (2141delCT) can be identified. In some cases, in patients with
Alstrom syndrome a compound heterozygosity for a 10775delC mutation
and a trp3664-to-ter mutation in the ALMS1 gene can be identified.
In some cases, in patients with Alstrom syndrome a homozygous
8164C-T transition in the ALMS1 gene, resulting in an
arg2722-to-ter (R2722X) substitution can be identified. In some
cases, in patients with Alstrom syndrome a homozygosity for
insertion of a 333-bp Alu Ya5 element in exon 16 of the ALMS1 gene
can be identified. In some cases, in patients with Alstrom syndrome
2 alleles carrying a 10945G-T transversion in exon 16 of the ALMS1
gene, resulting in a glu3649-to-ter (E3649X) substitution can be
identified. In some cases, in patients with Alstrom syndrome a
homozygosity for the E3649X mutation in the ALMS1 gene can be
identified. Most of these mutations lead to the production of an
abnormally small version of the ALMS1 protein that does not
function properly. A lack of normally functioning ALMS1 protein in
the brain can lead to overeating. A loss of this protein in the
pancreas may cause insulin resistance. The combined effects of
overeating and insulin resistance impair the body's ability to
handle excess sugar, leading to diabetes and obesity (two common
features of Alstrom syndrome).
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[1581] In embodiments, the methods of the present invention can
exploit the presence of retained-intron-containing pre-mRNA (RIC
pre-mRNA) transcribed from the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA gene and
encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA protein, in the cell nucleus.
Splicing of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA species to
produce mature, fully-spliced, ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA mRNA, can be
induced using ASOs that stimulate splicing out of the retained
introns. The resulting mature ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA mRNA can be
exported to the cytoplasm and translated, thereby increasing the
amount of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA protein in the patient's cells and
alleviating symptoms of the eye disease or conditions caused by
deficiency in ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA. This method, described
further below, is known as Targeted Augmentation of Nuclear Gene
Output (TANGO).
Nuclear Transcripts
[1582] ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA gene can be analyzed for
intron-retention events. RNA sequencing (RNAseq), can be visualized
in the UCSC genome browser, and can show ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
transcripts expressed in ARPE-19 and localized in either the
cytoplasmic or nuclear fraction. The retained-intron containing
pre-mRNA transcripts are retained in the nucleus and are not
exported out to the cytoplasm.
[1583] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 65%, about 5% to about 60%, about 5%
to about 55%, about 5% to about 50%, about 5% to about 45%, about
5% to about 40%, about 5% to about 35%, about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 10% to about 100%, about 10% to about 95%, about 10% to
about 90%, about 10% to about 85%, about 10% to about 80%, about
10% to about 75%, about 10% to about 70%, about 10% to about 65%,
about 10% to about 60%, about 10% to about 65%, about 10% to about
60%, about 10% to about 55%, about 10% to about 50%, about 10% to
about 45%, about 10% to about 40%, about 10% to about 35%, about
10% to about 30%, about 10% to about 25%, about 10% to about 20%,
about 15% to about 100%, about 15% to about 95%, about 15% to about
90%, about 15% to about 85%, about 15% to about 80%, about 15% to
about 75%, about 15% to about 70%, about 15% to about 65%, about
15% to about 60%, about 15% to about 65%, about 15% to about 60%,
about 15% to about 55%, about 15% to about 50%, about 15% to about
45%, about 15% to about 40%, about 15% to about 35%, about 15% to
about 30%, about 15% to about 25%, about 20% to about 100%, about
20% to about 95%, about 20% to about 90%, about 20% to about 85%,
about 20% to about 80%, about 20% to about 75%, about 20% to about
70%, about 20% to about 65%, about 20% to about 60%, about 20% to
about 65%, about 20% to about 60%, about 20% to about 55%, about
20% to about 50%, about 20% to about 45%, about 20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about
100%, about 25% to about 95%, about 25% to about 90%, about 25% to
about 85%, about 25% to about 80%, about 25% to about 75%, about
25% to about 70%, about 25% to about 65%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 60%, about 25% to about
55%, about 25% to about 50%, about 25% to about 45%, about 25% to
about 40%, or about 25% to about 35%, retention. In embodiments,
other ASOs useful for this purpose are identified, using, e.g.,
methods described herein.
[1584] In some embodiments, the ROM1 intron numbering corresponds
to the mRNA sequence at NM_00327. In embodiments, the targeted
portion of the ROM1 RIC pre-mRNA is in intron 1. In embodiments,
the percent retained intron can be 32. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained intron 1 and subsequently increases
ROM1 protein production. It is understood that the intron numbering
may change in reference to a different ROM1 isoform sequence. One
of skill in the art can determine the corresponding intron number
in any isoform based on an intron sequence provided herein or using
the number provided in reference to the mRNA sequence at NM_000327.
One of skill in the art also can determine the sequences of
flanking exons in any ROM1 isoform for targeting using the methods
of the invention, based on an intron sequence provided herein or
using the intron number provided in reference to the mRNA sequence
at NM_000327.
[1585] In some embodiments, the TEAD1 intron numbering corresponds
to the mRNA sequence at NM_021961. In embodiments, the targeted
portion of the TEAD1 RIC pre-mRNA is in intron 4. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of at least one of retained intron 4 and
subsequently increases TEAD1 protein production. It is understood
that the intron numbering may change in reference to a different
TEAD1 isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_021961. One of skill in the art also can
determine the sequences of flanking exons in any TEAD1 isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_021961.
[1586] In some embodiments, the RDH5 intron numbering corresponds
to the mRNA sequence at NM_002905 or NM_001199771. In embodiments,
the targeted portion of the RDH5 RIC pre-mRNA is in intron 1 and/or
2 In embodiments, the percent retained intron can be 59%. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 1 and/or 2 and
subsequently increases RDH5 protein production. It is understood
that the intron numbering may change in reference to a different
RDH5 isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_002905 or NM_001199771. One of skill in
the art also can determine the sequences of flanking exons in any
RDH5 isoform for targeting using the methods of the invention,
based on an intron sequence provided herein or using the intron
number provided in reference to the mRNA sequence at NM_002905 or
NM_001199771.
[1587] In some embodiments, the PAX6 intron numbering corresponds
to the mRNA sequence at NM_001310160, NM_001310161, NM_001258465,
NM_000280, NM_001258464, NM_000280, NM_001258464, NM_001604,
NM_001127612, NM_001258462, NM_001310159, NM_001310158, or
NM_001258462. In embodiments, the targeted portion of the PAX6 RIC
pre-mRNA is in intron 2 and/or 3 or 1 and/or 3 and/or 4 or 3 and/or
4 and/or 5 or 4 and/or 5 and/or 6 or 4 and/or 6 and/or 7 or 2
and/or 3 and/or 4. In some embodiments, the targeted portion of the
PAX 6 RIC pre-mRNA is in intron 2 and/or 3. In some embodiments,
the targeted portion of the PAX 6 RIC pre-mRNA is in intron 1
and/or 3 and/or 4. In some embodiments, the targeted portion of the
PAX 6 RIC pre-mRNA is in intron 3 and/or 4 and/or 5. In some
embodiments, the targeted portion of the PAX 6 RIC pre-mRNA is in
intron 4 and/or 5 and/or 6. In some embodiments, the targeted
portion of the PAX 6 RIC pre-mRNA is in intron 4 and/or 6 and/or 7.
In some embodiments, the targeted portion of the PAX 6 RIC pre-mRNA
is in intron 2 and/or 3 and/or 4. In embodiments, hybridization of
an ASO to the targeted portion of the RIC pre-mRNA results in
enhanced splicing at the splice site (5' splice site or 3' splice
site) of a retained intron and subsequently increases PAX6 protein
production. It is understood that the intron numbering may change
in reference to a different PAX6 isoform sequence. One of skill in
the art can determine the corresponding intron number in any
isoform based on an intron sequence provided herein or using the
number provided in reference to the mRNA sequence at NM_001310160,
NM_001310161, NM_001258465, NM_000280, NM_001258464, NM_000280,
NM_001258464, NM_001604, NM_001127612, NM_001258462, NM_001310159,
NM_001310158, or NM_001258462. One of skill in the art also can
determine the sequences of flanking exons in any PAX6 isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_001310160, NM_001310161,
NM_001258465, NM_000280, NM_001258464, NM_000280, NM_001258464,
NM_001604, NM_001127612, NM_001258462, NM_001310159, NM_001310158,
or NM_001258462.
[1588] In some embodiments, the FSCN2 intron numbering corresponds
to the mRNA sequence at NM_012418 or NM_001077182. In embodiments,
the targeted portion of the FSCN2 RIC pre-mRNA is in intron 1
and/or 3. In embodiments, the percent retained intron can be 12 or
17. In embodiments, hybridization of an ASO to the targeted portion
of the RIC pre-mRNA results in enhanced splicing at the splice site
(5' splice site or 3' splice site) of retained intron 1 and/or 3
and subsequently increases FSCN2 protein production. It is
understood that the intron numbering may change in reference to a
different FSCN2 isoform sequence. One of skill in the art can
determine the corresponding intron number in any isoform based on
an intron sequence provided herein or using the number provided in
reference to the mRNA sequence at NM_012418 or NM_001077182. One of
skill in the art also can determine the sequences of flanking exons
in any FSCN2 isoform for targeting using the methods of the
invention, based on an intron sequence provided herein or using the
intron number provided in reference to the mRNA sequence at
NM_012418 or NM_001077182.
[1589] In embodiments, the TCF4 intron numbering corresponds to the
mRNA sequence at NM_001243236, NM_00123235, NM_001243234,
NM_001243233, NM_001243232, NM_001243231, NM_003199, NM_001306207,
NM_001306208, NM_001243227, NM_001243228, NM_001243230,
NM_001243226, NM_001083962, NM_001330605, and NM_001330604. In
embodiments, the targeted portion of the TCF4 RIC pre-mRNA is in
intron 9 or 11 or 12 or 14 or 15 or 16 or 17. In embodiments, the
targeted portion of the TCF4 RIC pre-mRNA is in intron 9. In
embodiments, the targeted portion of the TCF4 RIC pre-mRNA is in
intron 11. In embodiments, the targeted portion of the TCF4 RIC
pre-mRNA is in intron 12. In embodiments, the targeted portion of
the TCF4 RIC pre-mRNA is in intron 14. In embodiments, the targeted
portion of the TCF4 RIC pre-mRNA is in intron 15. In embodiments,
the targeted portion of the TCF4 RIC pre-mRNA is in intron 16. In
embodiments, the targeted portion of the TCF4 RIC pre-mRNA is in
intron 17. In embodiments, the percent retained intron can be 9. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of a retained intron and
subsequently increases TCF4 protein production. It is understood
that the intron numbering may change in reference to a different
TCF4 isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_001243236, NM_00123235, NM_001243234,
NM_001243233, NM_001243232, NM_001243231, NM_003199, NM_001306207,
NM_001306208, NM_001243227, NM_001243228, NM_001243230,
NM_001243226, NM_001083962, NM_001330605, and NM_001330604.
[1590] One of skill in the art also can determine the sequences of
flanking exons in any TCF4 isoform for targeting using the methods
of the invention, based on an intron sequence provided herein or
using the intron number provided in reference to the mRNA sequence
at NM_001243236, NM_00123235, NM_001243234, NM_001243233,
NM_001243232, NM_001243231, NM_003199, NM_001306207, NM_001306208,
NM_001243227, NM_001243228, NM_001243230, NM_001243226,
NM_001083962, NM_001330605, and NM_001330604.
[1591] In embodiments, the MFSD8 intron numbering corresponds to
the mRNA sequence at NM_152778. In embodiments, the targeted
portion of the MFSD8 RIC pre-mRNA is in intron 11 and/or 12. In
embodiments, and the percent retained intron can be 15 or 62. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 11 and/or 12 and
subsequently increases MFSD8 protein production. It is understood
that the intron numbering may change in reference to a different
MFSD8 isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_152778. One of skill in the art also can
determine the sequences of flanking exons in any MFSD8 isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_152778.
[1592] In embodiments, the CTNS intron numbering corresponds to the
mRNA sequence at NM_004937 or NM_001031681. In embodiments, the
targeted portion of the CTNS RIC pre-mRNA is in intron 9 and/or 10.
In embodiments, the percent retained intron can be 10 or 18. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 9 and/or 10 and
subsequently increases CTNS protein production. It is understood
that the intron numbering may change in reference to a different
CTNS isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_004937 or NM_001031681. One of skill in
the art also can determine the sequences of flanking exons in any
CTNS isoform for targeting using the methods of the invention,
based on an intron sequence provided herein or using the intron
number provided in reference to the mRNA sequence at NM_004937 or
NM_001031681.
[1593] In embodiments, the OPTN intron number corresponds to the
mRNA sequence at NM_001008211, NM_001008212, NM_001008213, or
NM_021980. In embodiments, the targeted portion of the OPTN RIC
pre-mRNA is in intron 7 or 8 or 9. In embodiments, the targeted
portion of the OPTN RIC pre-mRNA is in intron 7. In embodiments,
the targeted portion of the OPTN RIC pre-mRNA is in intron 8. In
embodiments, the targeted portion of the OPTN RIC pre-mRNA is in
intron 9. In embodiments, the percent retained intron can be 24. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 7 or 8 or 9 and
subsequently increases OPTN protein production. It is understood
that the intron numbering may change in reference to a different
OPTN isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_001008211, NM_001008212, NM_001008213,
or NM_021980. One of skill in the art also can determine the
sequences of flanking exons in any OPTN isoform for targeting using
the methods of the invention, based on an intron sequence provided
herein or using the intron number provided in reference to the mRNA
sequence at NM_001008211, NM_001008212, NM_001008213, or
NM_021980.
[1594] In embodiments, the RLBP1 intron numbering corresponds to
the mRNA sequence at NM_000326. In embodiments, the targeted
portion of the RLBP1 RIC pre-mRNA is in intron 5 and/or 2. In
embodiments, the percent retained intron can be 49. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained intron 5 and/or 2 and subsequently
increases RLBP1 protein production. It is understood that the
intron numbering may change in reference to a different RLBP1
isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_000326. One of skill in the art also can
determine the sequences of flanking exons in any RLBP1 isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_000326.
[1595] In embodiments, the RPE65 intron numbering corresponds to
the mRNA sequence at NM_000329. In embodiments, the targeted
portion of the RPE65 RIC pre-mRNA is in intron 10 and/or 9. In
embodiments, the percent retained intron can be 11 or 10. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 10 and/or 9 and
subsequently increases RPE65 protein production. It is understood
that the intron numbering may change in reference to a different
RPE65 isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_000329. One of skill in the art also can
determine the sequences of flanking exons in any RPE65 isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_000329.
[1596] In embodiments, the LRAT intron numbering corresponds to the
mRNA sequence at NM_001301645 or NM_004744. In embodiments, the
targeted portion of the LRAT RIC pre-mRNA is in intron 2. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 2 and
subsequently increases LRAT protein production. It is understood
that the intron numbering may change in reference to a different
LRAT isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_001301645 or NM_004744. One of skill in
the art also can determine the sequences of flanking exons in any
LRAT isoform for targeting using the methods of the invention,
based on an intron sequence provided herein or using the intron
number provided in reference to the mRNA sequence at NM_001301645
or NM_004744.
[1597] In embodiments, the RDH8 intron numbering corresponds to the
mRNA sequence at NM_015725. In embodiments, the targeted portion of
the RDH8 RIC pre-mRNA is in intron 4. In embodiments, hybridization
of an ASO to the targeted portion of the RIC pre-mRNA results in
enhanced splicing at the splice site (5' splice site or 3' splice
site) of retained intron 4 and subsequently increases RDH8 protein
production. It is understood that the intron numbering may change
in reference to a different RDH8 isoform sequence. One of skill in
the art can determine the corresponding intron number in any
isoform based on an intron sequence provided herein or using the
number provided in reference to the mRNA sequence at NM_015725. One
of skill in the art also can determine the sequences of flanking
exons in any RDH8 isoform for targeting using the methods of the
invention, based on an intron sequence provided herein or using the
intron number provided in reference to the mRNA sequence at
NM_015725.
[1598] In embodiments, the RDH12 intron numbering corresponds to
the mRNA sequence at NM_152443. In embodiments, the targeted
portion of the RDH12 RIC pre-mRNA is in intron 7. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained intron 7 and subsequently increases
RDH12 protein production. It is understood that the intron
numbering may change in reference to a different RDH12 isoform
sequence. One of skill in the art can determine the corresponding
intron number in any isoform based on an intron sequence provided
herein or using the number provided in reference to the mRNA
sequence at NM_152443. One of skill in the art also can determine
the sequences of flanking exons in any RDH12 isoform for targeting
using the methods of the invention, based on an intron sequence
provided herein or using the intron number provided in reference to
the mRNA sequence at NM_152443.
[1599] In embodiments, the RGR intron numbering corresponds to the
mRNA sequence at NM_002921, NM_001012722, or NM_001012720. In
embodiments, the targeted portion of the RGR RIC pre-mRNA is in
intron 1 and/or 2. In embodiments, the percent retained intron can
be 46 or 61. In embodiments, hybridization of an ASO to the
targeted portion of the RIC pre-mRNA results in enhanced splicing
at the splice site (5' splice site or 3' splice site) of retained
intron 1 and/or 2 and subsequently increases RGR protein
production. It is understood that the intron numbering may change
in reference to a different RGR isoform sequence. One of skill in
the art can determine the corresponding intron number in any
isoform based on an intron sequence provided herein or using the
number provided in reference to the mRNA sequence at NM_002921,
NM_001012722, or NM_001012720. One of skill in the art also can
determine the sequences of flanking exons in any RGR isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_002921, NM_001012722, or
NM_001012720.
[1600] In embodiments, the CNGA3 intron numbering corresponds to
the mRNA sequence at NM_001298 or NM_001079878. In embodiments, the
targeted portion of the CNGA3 RIC pre-mRNA is in intron 6 or 5. In
embodiments, the targeted portion of the CNGA3 RIC pre-mRNA is in
intron 6. In embodiment, the targeted portion of the CNGA3 RIC
pre-mRNA is in intron 5. In embodiments, the percent retained
intron can be 27. In embodiments, hybridization of an ASO to the
targeted portion of the RIC pre-mRNA results in enhanced splicing
at the splice site (5' splice site or 3' splice site) of retained
intron 6 or 5 and subsequently increases CNGA3 protein production.
It is understood that the intron numbering may change in reference
to a different CNGA3 isoform sequence. One of skill in the art can
determine the corresponding intron number in any isoform based on
an intron sequence provided herein or using the number provided in
reference to the mRNA sequence at NM_001298 or NM_001079878. One of
skill in the art also can determine the sequences of flanking exons
in any CNGA3 isoform for targeting using the methods of the
invention, based on an intron sequence provided herein or using the
intron number provided in reference to the mRNA sequence at
NM_001298 or NM_001079878.
[1601] In embodiments, the PER1 intron numbering corresponds to the
mRNA sequence at NM_002616. In embodiments, the targeted portion of
the PER1 RIC pre-mRNA is in intron 1 and/or 14. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained intron 1 and/or 14 and subsequently
increases PER1 protein production. It is understood that the intron
numbering may change in reference to a different PER1 isoform
sequence. One of skill in the art can determine the corresponding
intron number in any isoform based on an intron sequence provided
herein or using the number provided in reference to the mRNA
sequence at NM_002616. One of skill in the art also can determine
the sequences of flanking exons in any PER1 isoform for targeting
using the methods of the invention, based on an intron sequence
provided herein or using the intron number provided in reference to
the mRNA sequence at NM_002616.
[1602] In embodiments, the IDUA intron numbering corresponds to the
mRNA sequence at NM_000203 or NR_110313. In embodiments, the
targeted portion of the IDUA RIC pre-mRNA is in intron 3 and/or 4
and/or 5 and/or 6 and/or 7. In embodiments, the percent retained
intron can be 28, 29, 18, 20, or 12. In embodiments, hybridization
of an ASO to the targeted portion of the RIC pre-mRNA results in
enhanced splicing at the splice site (5' splice site or 3' splice
site) of retained intron 3 and/or 4 and/or 5 and/or 6 and/or land
subsequently increases IDUA protein production. It is understood
that the intron numbering may change in reference to a different
IDUA isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_000203 or NR_110313. One of skill in the
art also can determine the sequences of flanking exons in any IDUA
isoform for targeting using the methods of the invention, based on
an intron sequence provided herein or using the intron number
provided in reference to the mRNA sequence at NM_000203 or
NR_110313.
[1603] In embodiments, the ABCA4 intron number corresponds to the
mRNA sequence at NM_000350. In embodiments, the targeted portion of
the ABCA4 RIC pre-mRNA is in intron 40 and/or 38 and/or 36 and/or
44 and/or 39. In embodiments, hybridization of an ASO to the
targeted portion of the RIC pre-mRNA results in enhanced splicing
at the splice site (5' splice site or 3' splice site) of retained
intron 40 and/or 38 and/or 36 and/or 44 and/or 39 and subsequently
increases ABCA4 protein production. It is understood that the
intron numbering may change in reference to a different ABCA4
isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_000350. One of skill in the art also can
determine the sequences of flanking exons in any ABCA4 isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_000350.
[1604] In embodiments, the MYOC intron number corresponds to the
mRNA sequence at NM_000261. In embodiments, the targeted portion of
the MYOC RIC pre-mRNA is in intron 1 and/or 2. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained 1 and/or 2 and subsequently increases
MYOC protein production. It is understood that the intron numbering
may change in reference to a different MYOC isoform sequence. One
of skill in the art can determine the corresponding intron number
in any isoform based on an intron sequence provided herein or using
the number provided in reference to the mRNA sequence at NM_000261.
One of skill in the art also can determine the sequences of
flanking exons in any MYOC isoform for targeting using the methods
of the invention, based on an intron sequence provided herein or
using the intron number provided in reference to the mRNA sequence
at NM_000261.
[1605] In embodiments, the NR2E3 intron number corresponds to the
mRNA sequence at NM_014249 or NM_016346. In embodiments, the
targeted portion of the NR2E3 RIC pre-mRNA is in intron 1 and/or 2
and/or 3 and/or 4 and/or 5 and/or 6 and/or 7. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained 1 and/or 2 and/or 3 and/or 4 and/or 5
and/or 6 and/or 7 and subsequently increases NR2E3 protein
production. It is understood that the intron numbering may change
in reference to a different NR2E3 isoform sequence. One of skill in
the art can determine the corresponding intron number in any
isoform based on an intron sequence provided herein or using the
number provided in reference to the mRNA sequence at NM_014249 or
NM_016346. One of skill in the art also can determine the sequences
of flanking exons in any NR2E3 isoform for targeting using the
methods of the invention, based on an intron sequence provided
herein or using the intron number provided in reference to the mRNA
sequence at NM_014249 or NM_016346.
[1606] In embodiments, the NXNL1 intron number corresponds to the
mRNA sequence at NM_138454. In embodiments, the targeted portion of
the NXNL1 RIC pre-mRNA is in intron 1. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained 1 and subsequently increases NXNL1
protein production. It is understood that the intron numbering may
change in reference to a different NXNL1 isoform sequence. One of
skill in the art can determine the corresponding intron number in
any isoform based on an intron sequence provided herein or using
the number provided in reference to the mRNA sequence at NM_138454.
One of skill in the art also can determine the sequences of
flanking exons in any NXNL1 isoform for targeting using the methods
of the invention, based on an intron sequence provided herein or
using the intron number provided in reference to the mRNA sequence
at NM_138454.
[1607] In embodiments, the CRX intron number corresponds to the
mRNA sequence at NM_000554. In embodiments, the targeted portion of
the CRX RIC pre-mRNA is in intron 1 and/or 2 and/or 3. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained 1 and/or 2 and/or 3 and
subsequently increases CRX protein production. It is understood
that the intron numbering may change in reference to a different
CRX isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_000554. One of skill in the art also can
determine the sequences of flanking exons in any CRX isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_000554.
ABCA4
[1608] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from an ABCA4 genomic sequence (an ABCA4 RIC
pre-mRNA). In some embodiments, the ABCA4 genomic sequence is SEQ
ID NO: 34790. In some embodiments, the ABCA4 RIC pre-mRNA is SEQ ID
NO: 34813. In some embodiments, the ABCA4 RIC pre-mRNA transcript
comprises retained intron 40 and/or 38 and/or 36 and/or 44 and/or
39. In some embodiments, when the ABCA4 RIC pre-mRNA transcript
comprises retained intron 40, the ASOs disclosed herein target SEQ
ID NO: 61463. In some embodiments, when the ABCA4 RIC pre-mRNA
transcript comprises retained intron 38, the ASOs disclosed herein
target SEQ ID NO: 61495. In some embodiments, when the ABCA4 RIC
pre-mRNA transcript comprises retained intron 36, the ASOs
disclosed herein target SEQ ID NO: 61445. In some embodiments, when
the ABCA4 RIC pre-mRNA transcript comprises retained intron 44, the
ASOs disclosed herein target SEQ ID NO: 61470. In some embodiments,
when the ABCA4 RIC pre-mRNA transcript comprises retained intron
39, the ASOs disclosed herein target SEQ ID NO: 61453. In some
embodiments, when the ABCA4 RIC pre-mRNA transcript comprises
retained intron 40, the ASO has a sequence according to any one of
SEQ ID NOs: 34873-35104. In some embodiments, when the ABCA4 RIC
pre-mRNA transcript comprises retained intron 38, the ASO has a
sequence according to any one of SEQ ID NOs: 35105-35332. In some
embodiments, when the ABCA4 RIC pre-mRNA transcript comprises
retained intron 36, the ASO has a sequence according to any one of
SEQ ID NOs: 35333-. In some embodiments, when the ABCA4 RIC
pre-mRNA transcript comprises retained intron 44, the ASO has a
sequence according to any one of SEQ ID NOs: 35564-35805. In some
embodiments, when the ABCA4 RIC pre-mRNA transcript comprises
retained intron 39, the ASO has a sequence according to any one of
SEQ ID NOs: 35806-35915. In some embodiments, the ASOs target an
ABCA4 RIC pre-mRNA sequence.
RPE65
[1609] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a RPE65 genomic sequence (an RPE65 RIC
pre-mRNA). In some embodiments, the RPE65 genomic sequence is SEQ
ID NO: 34791. In some embodiments, the RPE65 RIC pre-mRNA is SEQ ID
NO: 34814. In some embodiments, the RPE65 RIC pre-mRNA transcript
comprises retained intron 9 and/or 10. In some embodiments, when
the RPE65 RIC pre-mRNA transcript comprises retained intron 9, the
ASOs disclosed herein target SEQ ID NO: 61480. In some embodiments,
when the RPE65 RIC pre-mRNA transcript comprises retained intron
10, the ASOs disclosed herein target SEQ ID NO: 61460. In some
embodiments, when the RPE65 RIC pre-mRNA transcript comprises
retained intron 9, the ASO has a sequence according to any one of
SEQ ID NOs: 35916-36082. In some embodiments, when the RPE65 RIC
pre-mRNA transcript comprises retained intron 10, the ASO has a
sequence according to any one of SEQ ID NOs: 36083-36317. In some
embodiments, the ASOs target an RPE65 RIC pre-mRNA sequence.
MYOC
[1610] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a MYOC genomic sequence (a MYOC RIC
pre-mRNA). In some embodiments, the MYOC genomic sequence is SEQ ID
NO: 34792. In some embodiments, the MYOC RIC pre-mRNA is SEQ ID NO:
34815. In some embodiments, the MYOC RIC pre-mRNA transcript
comprises retained intron 1 and/or 2. In some embodiments, when the
MYOC RIC pre-mRNA transcript comprises retained intron 1, the ASOs
disclosed herein target SEQ ID NO: 61458. In some embodiments, when
the MYOC RIC pre-mRNA transcript comprises retained intron 2, the
ASOs disclosed herein target SEQ ID NO: 61485. In some embodiments,
when the MYOC RIC pre-mRNA transcript comprises retained intron 1,
the ASO has a sequence according to any one of SEQ ID NOs:
36318-36644. In some embodiments, when the MYOC RIC pre-mRNA
transcript comprises retained intron 2, the ASO has a sequence
according to any one of SEQ ID NOs: 36645-37107. In some
embodiments, the ASOs target a MYOC RIC pre-mRNA sequence.
CNGA3
[1611] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a CNGA3 genomic sequence (a CNGA3 RIC
pre-mRNA). In some embodiments, the CNGA3 genomic sequence is SEQ
ID NO: 34793. In some embodiments, the CNGA3 RIC pre-mRNA is SEQ ID
NO: 34816 or 34817. In some embodiments, the CNGA3 RIC pre-mRNA
transcript comprises retained intron 6 and/or 5. In some
embodiments, when the CNGA3 RIC pre-mRNA transcript comprises
retained intron 6, the ASOs disclosed herein target SEQ ID NO:
61500. In some embodiments, when the CNGA3 RIC pre-mRNA transcript
comprises retained intron 5, the ASOs disclosed herein target SEQ
ID NO: 61500. In some embodiments, when the CNGA3 RIC pre-mRNA
transcript comprises retained intron 6, the ASO has a sequence
according to any one of SEQ ID NOs: 37108-37333. In some
embodiments, when the CNGA3 RIC pre-mRNA transcript comprises
retained intron 5, the ASO has a sequence according to any one of
SEQ ID NOs: 37334-37559. In some embodiments, the ASOs target a
CNGA3 RIC pre-mRNA sequence.
MFSD8
[1612] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a MFSD8 genomic sequence (an MFSD8 RIC
pre-mRNA). In some embodiments, the MFSD8 genomic sequence is SEQ
ID NO: 34794. In some embodiments, the MFSD8 RIC pre-mRNA is SEQ ID
NO: 34818. In some embodiments, the MFSD8 RIC pre-mRNA transcript
comprises retained intron 11 and/or 12. In some embodiments, when
the MFSD8 RIC pre-mRNA transcript comprises retained intron 11, the
ASOs disclosed herein target SEQ ID NO: 61492. In some embodiments,
when the MFSD8 RIC pre-mRNA transcript comprises retained intron
12, the ASOs disclosed herein target SEQ ID NO: 61497. In some
embodiments, when the MFSD8 RIC pre-mRNA transcript comprises
retained intron 11, the ASO has a sequence according to any one of
SEQ ID NOs: 37560-37641. In some embodiments, when the MFSD8 RIC
pre-mRNA transcript comprises retained intron 12, the ASO has a
sequence according to any one of SEQ ID NOs: 37642-38420. In some
embodiments, the ASOs target an MFSD8 RIC pre-mRNA sequence.
IDUA
[1613] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a IDUA genomic sequence (an IDUA RIC
pre-mRNA). In some embodiments, the IDUA genomic sequence is SEQ ID
NO: 34795. In some embodiments, the IDUA RIC pre-mRNA is SEQ ID NO:
34819 or 34820. In some embodiments, the IDUA RIC pre-mRNA
transcript comprises retained intron 3 and/or 4 and/or 5 and/or 6
and/or 7. In some embodiments, when the IDUA RIC pre-mRNA
transcript comprises retained intron 3, the ASOs disclosed herein
target SEQ ID NO: 61457. In some embodiments, when the IDUA RIC
pre-mRNA transcript comprises retained intron 4, the ASOs disclosed
herein target SEQ ID NO: 61468. In some embodiments, when the IDUA
RIC pre-mRNA transcript comprises retained intron 5, the ASOs
disclosed herein target SEQ ID NO: 61489. In some embodiments, when
the IDUA RIC pre-mRNA transcript comprises retained intron 6, the
ASOs disclosed herein target SEQ ID NO: 61444. In some embodiments,
when the IDUA RIC pre-mRNA transcript comprises retained intron 7,
the ASOs disclosed herein target SEQ ID NO: 61452. In some
embodiments, when the IDUA RIC pre-mRNA transcript comprises
retained intron 3, the ASO has a sequence according to any one of
SEQ ID NOs: 38421-38486 or 38827-38893. In some embodiments, when
the IDUA RIC pre-mRNA transcript comprises retained intron 4, the
ASO has a sequence according to any one of SEQ ID NOs: 38487-38602
or 38893-39008. In some embodiments, when the IDUA RIC pre-mRNA
transcript comprises retained intron 5, the ASO has a sequence
according to any one of SEQ ID NOs: 38603-38668 or 39009-39074. In
some embodiments, when the IDUA RIC pre-mRNA transcript comprises
retained intron 6, the ASO has a sequence according to any one of
SEQ ID NOs: 38669-38741 or 39075-39147. In some embodiments, when
the IDUA RIC pre-mRNA transcript comprises retained intron 7, the
ASO has a sequence according to any one of SEQ ID NOs: 38742-38826
or 39147-39232. In some embodiments, the ASOs target an IDUA RIC
pre-mRNA sequence.
LRAT
[1614] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a LRAT genomic sequence (an LRAT RIC
pre-mRNA). In some embodiments, the LRAT genomic sequence is SEQ ID
NO: 34796. In some embodiments, the LRAT RIC pre-mRNA is SEQ ID NO:
34821 or 34822. In some embodiments, the LRAT RIC pre-mRNA
transcript comprises retained intron 2. In some embodiments, when
the LRAT RIC pre-mRNA transcript comprises retained intron 2, the
ASOs disclosed herein target SEQ ID NO: 61474. In some embodiments,
when the LRAT RIC pre-mRNA transcript comprises retained intron 2,
the ASO has a sequence according to any one of SEQ ID NOs:
39233-41436. In some embodiments, the ASOs target an LRAT RIC
pre-mRNA sequence.
[1615] OPTN
[1616] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a OPTN genomic sequence (an OPTN RIC
pre-mRNA). In some embodiments, the OPTN genomic sequence is SEQ ID
NO: 34797. In some embodiments, the OPTN RIC pre-mRNA is SEQ ID NO:
34823, 34824, 34825, or 34826. In some embodiments, the OPTN RIC
pre-mRNA transcript comprises retained intron 9 or 8 or 7. In some
embodiments, when the OPTN RIC pre-mRNA transcript comprises
retained intron 9, the ASOs disclosed herein target SEQ ID NO:
61503. In some embodiments, when the OPTN RIC pre-mRNA transcript
comprises retained intron 8, the ASOs disclosed herein target SEQ
ID NO: 61503. In some embodiments, when the OPTN RIC pre-mRNA
transcript comprises retained intron 7, the ASOs disclosed herein
target SEQ ID NO: 61503. In some embodiments, when the OPTN RIC
pre-mRNA transcript comprises retained intron 9, the ASO has a
sequence according to any one of SEQ ID NOs: 41437-41669 or
41903-42135. In some embodiments, when the OPTN RIC pre-mRNA
transcript comprises retained intron 8, the ASO has a sequence
according to any one of SEQ ID NOs: 41670-41903. In some
embodiments, when the OPTN RIC pre-mRNA transcript comprises
retained intron 7, the ASO has a sequence according to any one of
SEQ ID NOs: 42136-42368. In some embodiments, the ASOs target an
OPTN RIC pre-mRNA sequence.
RGR
[1617] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a RGR genomic sequence (an RGR RIC
pre-mRNA). In some embodiments, the RGR genomic sequence is SEQ ID
NO: 34798. In some embodiments, the RGR RIC pre-mRNA is SEQ ID NO:
34827, 34828, or 34829. In some embodiments, the RGR RIC pre-mRNA
transcript comprises retained intron 1 and/or 2. In some
embodiments, when the RGR RIC pre-mRNA transcript comprises
retained intron 1, the ASOs disclosed herein target SEQ ID NO:
61446. In some embodiments, when the RGR RIC pre-mRNA transcript
comprises retained intron 2, the ASOs disclosed herein target SEQ
ID NO: 61476 or 61472. In some embodiments, when the RGR RIC
pre-mRNA transcript comprises retained intron 1, the ASO has a
sequence according to any one of SEQ ID NOs: 42369-42595,
42830-43056, or 43289-43515. In some embodiments, when the RGR RIC
pre-mRNA transcript comprises retained intron 2, the ASO has a
sequence according to any one of SEQ ID NOs: 42596-42829,
43057-43288, or 43516-43747. In some embodiments, the ASOs target
an RGR RIC pre-mRNA sequence.
TEAD1
[1618] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a TEAD1 genomic sequence (a TEAD1 RIC
pre-mRNA). In some embodiments, the TEAD1 genomic sequence is SEQ
ID NO: 34799. In some embodiments, the TEAD1 RIC pre-mRNA is SEQ ID
NO: 34830. In some embodiments, the TEAD1 RIC pre-mRNA transcript
comprises retained intron 4. In some embodiments, when the TEAD1
RIC pre-mRNA transcript comprises retained intron 4, the ASOs
disclosed herein target SEQ ID NO: 61461. In some embodiments, when
the TEAD1 RIC pre-mRNA transcript comprises retained intron 4, the
ASO has a sequence according to any one of SEQ ID NOs: 43748-43952.
In some embodiments, the ASOs target a TEAD1 RIC pre-mRNA
sequence.
PAX6
[1619] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PAX6 genomic sequence (a PAX6 RIC
pre-mRNA). In some embodiments, the PAX6 genomic sequence is SEQ ID
NO: 34800. In some embodiments, the PAX6 RIC pre-mRNA is SEQ ID NO:
34831, 34832, 34833, 34834, 34835, 34836, 34837, 34838, 34839,
34840, or 34841. In some embodiments, the PAX6 RIC pre-mRNA
transcript comprises retained intron 2 and/or 3 or 1 and/or 3
and/or 4 or 3 and/or 4 and/or 5 or 4 and/or 5 and/or 6 or 4 and/or
6 and/or 7 or 2 and/or 3 and/or 4. In some embodiments, when the
PAX6 RIC pre-mRNA transcript comprises retained intron 1, the ASOs
disclosed herein target SEQ ID NO: 61496. In some embodiments, when
the PAX6 RIC pre-mRNA transcript comprises retained intron 2, the
ASOs disclosed herein target SEQ ID NO: 61486 or 61483. In some
embodiments, when the PAX6 RIC pre-mRNA transcript comprises
retained intron 3, the ASOs disclosed herein target SEQ ID NO:
61466, 61467, 61483, or 61448. In some embodiments, when the PAX6
RIC pre-mRNA transcript comprises retained intron 4, the ASOs
disclosed herein target SEQ ID NO: 61502, 61448, or 61483. In some
embodiments, when the PAX6 RIC pre-mRNA transcript comprises
retained intron 5, the ASOs disclosed herein target SEQ ID NO:
61502 or 61448. In some embodiments, when the PAX6 RIC pre-mRNA
transcript comprises retained intron 6, the ASOs disclosed herein
target SEQ ID NO: 61502 or 61467. In some embodiments, when the
PAX6 RIC pre-mRNA transcript comprises retained intron 7, the ASOs
disclosed herein target SEQ ID NO: 61502. In some embodiments, when
the PAX6 RIC pre-mRNA transcript comprises retained intron 1, the
ASO has a sequence according to any one of SEQ ID NOs: 44297-44564.
In some embodiments, when the PAX6 RIC pre-mRNA transcript
comprises retained intron 2, the ASO has a sequence according to
any one of SEQ ID NOs: 43955-44088 or 48374-48574. In some
embodiments, when the PAX6 RIC pre-mRNA transcript comprises
retained intron 3, the ASO has a sequence according to any one of
SEQ ID NOs: 44089-44302, 44565-44637, 44850-45049, or 48578-48809.
In some embodiments, when the PAX6 RIC pre-mRNA transcript
comprises retained intron 4, the ASO has a sequence according to
any one of SEQ ID NOs: 44638-44849, 45050-45281, 45493-45692,
46136-46335, 46779-46978, 47257-47456, 47900-48099, 48810-49220, or
49499-49698. In some embodiments, when the PAX6 RIC pre-mRNA
transcript comprises retained intron 5, the ASO has a sequence
according to any one of SEQ ID NOs: 45282-45492, 45693-45924,
46336-46567, or 47457-47688. In some embodiments, when the PAX6 RIC
pre-mRNA transcript comprises retained intron 6, the ASO has a
sequence according to any one of SEQ ID NOs: 45925-46135,
46568-46778, 46979-47045, 47689-47899, 48100-48166, 49221-49287, or
49699-49765. In some embodiments, when the PAX6 RIC pre-mRNA
transcript comprises retained intron 7, the ASO has a sequence
according to any one of SEQ ID NOs: 47046-47256, 48167-48377,
49288-49498, or 49766-49976. In some embodiments, the ASOs target a
PAX6 RIC pre-mRNA sequence.
ROM1
[1620] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a ROM1 genomic sequence (a ROM1 RIC
pre-mRNA). In some embodiments, the ROM1 genomic sequence is SEQ ID
NO: 34801. In some embodiments, the ROM1 RIC pre-mRNA is SEQ ID NO:
34842. In some embodiments, the ROM1 RIC pre-mRNA transcript
comprises retained intron 1. In some embodiments, when the ROM1 RIC
pre-mRNA transcript comprises retained intron 1, the ASOs disclosed
herein target SEQ ID NO: 61454. In some embodiments, when the ROM1
RIC pre-mRNA transcript comprises retained intron 1, the ASO has a
sequence according to any one of SEQ ID NOs: 49969-50275. In some
embodiments, the ASOs target a ROM1 RIC pre-mRNA sequence.
RDH5
[1621] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a RDH5 genomic sequence (an RDH5 RIC
pre-mRNA). In some embodiments, the RDH5 genomic sequence is SEQ ID
NO: 34802. In some embodiments, the RDH5 RIC pre-mRNA is SEQ ID NO:
34843 or 34844. In some embodiments, the RDH5 RIC pre-mRNA
transcript comprises retained intron 1 and/or 2. In some
embodiments, when the RDH5 RIC pre-mRNA transcript comprises
retained intron 1, the ASOs disclosed herein target SEQ ID NO:
61493 or 61498. In some embodiments, when the RDH5 RIC pre-mRNA
transcript comprises retained intron 2, the ASOs disclosed herein
target SEQ ID NO: 61455 or 61473. In some embodiments, when the
RDH5 RIC pre-mRNA transcript comprises retained intron 1, the ASO
has a sequence according to any one of SEQ ID NOs: 50276-50489 or
50276-50846. In some embodiments, when the RDH5 RIC pre-mRNA
transcript comprises retained intron 2, the ASO has a sequence
according to any one of SEQ ID NOs: 50490-50633 or 50847-50991. In
some embodiments, the ASOs target an RDH5 RIC pre-mRNA
sequence.
RDH12
[1622] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a RDH12 genomic sequence (an RDH12 RIC
pre-mRNA). In some embodiments, the RDH12 genomic sequence is SEQ
ID NO: 34803. In some embodiments, the RDH12 RIC pre-mRNA is SEQ ID
NO: 34845. In some embodiments, the RDH12 RIC pre-mRNA transcript
comprises retained intron 7. In some embodiments, when the RDH12
RIC pre-mRNA transcript comprises retained intron 7, the ASOs
disclosed herein target SEQ ID NO: 61482. In some embodiments, when
the RDH12 RIC pre-mRNA transcript comprises retained intron 7, the
ASO has a sequence according to any one of SEQ ID NOs: 50992-51247.
In some embodiments, the ASOs target an RDH12 RIC pre-mRNA
sequence.
NR2E3
[1623] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a NR2E3 genomic sequence (an NR2E3 RIC
pre-mRNA). In some embodiments, the NR2E3 genomic sequence is SEQ
ID NO: 34804. In some embodiments, the NR2E3 RIC pre-mRNA is SEQ ID
NO: 34846 or 34846. In some embodiments, the NR2E3 RIC pre-mRNA
transcript comprises retained intron 1 and/or 2 and/or 3 and/or 4
and/or 5 and/or 6 and/or 7. In some embodiments, when the NR2E3 RIC
pre-mRNA transcript comprises retained intron 1, the ASOs disclosed
herein target SEQ ID NO: 61491. In some embodiments, when the NR2E3
RIC pre-mRNA transcript comprises retained intron 2, the ASOs
disclosed herein target SEQ ID NO: 61449. In some embodiments, when
the NR2E3 RIC pre-mRNA transcript comprises retained intron 3, the
ASOs disclosed herein target SEQ ID NO: 61494. In some embodiments,
when the NR2E3 RIC pre-mRNA transcript comprises retained intron 4,
the ASOs disclosed herein target SEQ ID NO: 61487. In some
embodiments, when the NR2E3 RIC pre-mRNA transcript comprises
retained intron 5, the ASOs disclosed herein target SEQ ID NO:
61447. In some embodiments, when the NR2E3 RIC pre-mRNA transcript
comprises retained intron 6, the ASOs disclosed herein target SEQ
ID NO: 61465. In some embodiments, when the NR2E3 RIC pre-mRNA
transcript comprises retained intron 7, the ASOs disclosed herein
target SEQ ID NO: 61501 or 61490. In some embodiments, when the
NR2E3 RIC pre-mRNA transcript comprises retained intron 1, the ASO
has a sequence according to any one of SEQ ID NOs: 51256-16661 or
52052-52254. In some embodiments, when the NR2E3 RIC pre-mRNA
transcript comprises retained intron 2, the ASO has a sequence
according to any one of SEQ ID NOs: 51459-16272 or 52255-52320. In
some embodiments, when the NR2E3 RIC pre-mRNA transcript comprises
retained intron 3, the ASO has a sequence according to any one of
SEQ ID NOs: 51525-51595 or 52321-52391. In some embodiments, when
the NR2E3 RIC pre-mRNA transcript comprises retained intron 4, the
ASO has a sequence according to any one of SEQ ID NOs: 51596-51687
or 52392-52483. In some embodiments, when the NR2E3 RIC pre-mRNA
transcript comprises retained intron 5, the ASO has a sequence
according to any one of SEQ ID NOs: 51688-51924 or 52484-52720. In
some embodiments, when the NR2E3 RIC pre-mRNA transcript comprises
retained intron 6, the ASO has a sequence according to any one of
SEQ ID NOs: 51925-51966 or 52721-52762. In some embodiments, when
the NR2E3 RIC pre-mRNA transcript comprises retained intron 7, the
ASO has a sequence according to any one of SEQ ID NOs: 51967-52051
or 52763-53006. In some embodiments, the ASOs target an NR2E3 RIC
pre-mRNA sequence.
RLBP1
[1624] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a RLBP1 genomic sequence (an RLBP1 RIC
pre-mRNA). In some embodiments, the RLBP1 genomic sequence is SEQ
ID NO: 34805. In some embodiments, the RLBP1 RIC pre-mRNA is SEQ ID
NO: 34848. In some embodiments, the RLBP1 RIC pre-mRNA transcript
comprises retained intron 2 and/or 5 In some embodiments, when the
RLBP1 RIC pre-mRNA transcript comprises retained intron 2, the ASOs
disclosed herein target SEQ ID NO: 61462. In some embodiments, when
the RLBP1 RIC pre-mRNA transcript comprises retained intron 5, the
ASOs disclosed herein target SEQ ID NO: 61456. In some embodiments,
when the RLBP1 RIC pre-mRNA transcript comprises retained intron 2,
the ASO has a sequence according to any one of SEQ ID NOs:
52999-53172. In some embodiments, when the RLBP1 RIC pre-mRNA
transcript comprises retained intron 5, the ASO has a sequence
according to any one of SEQ ID NOs: 53173-53427. In some
embodiments, the ASOs target an RLBP1 RIC pre-mRNA sequence.
CTNS
[1625] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a CTNS genomic sequence (a CTNS RIC
pre-mRNA). In some embodiments, the CTNS genomic sequence is SEQ ID
NO: 34806. In some embodiments, the CTNS RIC pre-mRNA is SEQ ID NO:
34849 or 34850. In some embodiments, the CTNS RIC pre-mRNA
transcript comprises retained intron 9 and/or 10. In some
embodiments, when the CTNS RIC pre-mRNA transcript comprises
retained intron 9, the ASOs disclosed herein target SEQ ID NO:
61479. In some embodiments, when the CTNS RIC pre-mRNA transcript
comprises retained intron 10, the ASOs disclosed herein target SEQ
ID NO: 61481. In some embodiments, when the CTNS RIC pre-mRNA
transcript comprises retained intron 9, the ASO has a sequence
according to any one of SEQ ID NOs: 53436-53658 or 53884-54106. In
some embodiments, when the CTNS RIC pre-mRNA transcript comprises
retained intron 10, the ASO has a sequence according to any one of
SEQ ID NOs: 53659-53883 or 54107-54331. In some embodiments, the
ASOs target a CTNS RIC pre-mRNA sequence.
PER1
[1626] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a PER1 genomic sequence (a PER1 RIC
pre-mRNA). In some embodiments, the PER1 genomic sequence is SEQ ID
NO: 34807. In some embodiments, the PER1 RIC pre-mRNA is SEQ ID NO:
34851. In some embodiments, the PER1 RIC pre-mRNA transcript
comprises retained intron 1 and/or 14. In some embodiments, when
the PER1 RIC pre-mRNA transcript comprises retained intron 1, the
ASOs disclosed herein target SEQ ID NO: 61471. In some embodiments,
when the PER1 RIC pre-mRNA transcript comprises retained intron 14,
the ASOs disclosed herein target SEQ ID NO: 61499. In some
embodiments, when the PER1 RIC pre-mRNA transcript comprises
retained intron 1, the ASO has a sequence according to any one of
SEQ ID NOs: 54324-54571. In some embodiments, when the PER1 RIC
pre-mRNA transcript comprises retained intron 14, the ASO has a
sequence according to any one of SEQ ID NOs: 54572-54634. In some
embodiments, the ASOs target a PER1 RIC pre-mRNA sequence.
FSCN2
[1627] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a FSCN2 genomic sequence (an FSCN2 RIC
pre-mRNA). In some embodiments, the FSCN2 genomic sequence is SEQ
ID NO: 34808. In some embodiments, the FSCN2 RIC pre-mRNA is SEQ ID
NO: 34852 or 34853. In some embodiments, the FSCN2 RIC pre-mRNA
transcript comprises retained intron 1 and/or 3. In some
embodiments, when the FSCN2 RIC pre-mRNA transcript comprises
retained intron 1, the ASOs disclosed herein target SEQ ID NO:
61459. In some embodiments, when the FSCN2 RIC pre-mRNA transcript
comprises retained intron 3, the ASOs disclosed herein target SEQ
ID NO: 61451 or 61450. In some embodiments, when the FSCN2 RIC
pre-mRNA transcript comprises retained intron 1, the ASO has a
sequence according to any one of SEQ ID NOs: 54635-55021 or
55137-55523. In some embodiments, when the FSCN2 RIC pre-mRNA
transcript comprises retained intron 3, the ASO has a sequence
according to any one of SEQ ID NOs: 55022-55136 or 55524-55638. In
some embodiments, the ASOs target an FSCN2 RIC pre-mRNA
sequence.
TCF4
[1628] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a TCF4 genomic sequence (a TCF4 RIC
pre-mRNA). In some embodiments, the TCF4 genomic sequence is SEQ ID
NO: 34809. In some embodiments, the TCF4 RIC pre-mRNA is SEQ ID NO:
34854, 34855, 34856, 34857, 34858, 34859, 34860, 34861, 34862,
34863, 34864, 34865, 34866, 34867, 34868, or 34869. In some
embodiments, the TCF4 RIC pre-mRNA transcript comprises retained
intron 9 or 12 or 14 or 16 or 15 or 17 or 11.
[1629] In some embodiments, when the TCF4 RIC pre-mRNA transcript
comprises retained intron 9, the ASOs disclosed herein target SEQ
ID NO: 61477. In some embodiments, when the TCF4 RIC pre-mRNA
transcript comprises retained intron 12, the ASOs disclosed herein
target SEQ ID NO: 61477 or 61478. In some embodiments, when the
TCF4 RIC pre-mRNA transcript comprises retained intron 14, the ASOs
disclosed herein target SEQ ID NO: 61477. In some embodiments, when
the TCF4 RIC pre-mRNA transcript comprises retained intron 16, the
ASOs disclosed herein target SEQ ID NO: 61477 or 61478. In some
embodiments, when the TCF4 RIC pre-mRNA transcript comprises
retained intron 15, the ASOs disclosed herein target SEQ ID NO:
61477 or 61478. In some embodiments, when the TCF4 RIC pre-mRNA
transcript comprises retained intron 17, the ASOs disclosed herein
target SEQ ID NO: 61478. In some embodiments, when the TCF4 RIC
pre-mRNA transcript comprises retained intron 11, the ASOs
disclosed herein target SEQ ID NO: 61478. In some embodiments, when
the TCF4 RIC pre-mRNA transcript comprises retained intron 9, the
ASO has a sequence according to any one of SEQ ID NOs: 55649-56366.
In some embodiments, when the TCF4 RIC pre-mRNA transcript
comprises retained intron 12, the ASO has a sequence according to
any one of SEQ ID NOs: 56367-56852 or 57579-57820. In some
embodiments, when the TCF4 RIC pre-mRNA transcript comprises
retained intron 14, the ASO has a sequence according to any one of
SEQ ID NOs: 56853-57094. In some embodiments, when the TCF4 RIC
pre-mRNA transcript comprises retained intron 16, the ASO has a
sequence according to any one of SEQ ID NOs: 57095-57336,
58065-58308, 58795-59038, or 59283-59526. In some embodiments, when
the TCF4 RIC pre-mRNA transcript comprises retained intron 15, the
ASO has a sequence according to any one of SEQ ID NOs: 57337-57578,
57821-58064, or 58299-58550. In some embodiments, when the TCF4 RIC
pre-mRNA transcript comprises retained intron 17, the ASO has a
sequence according to any one of SEQ ID NOs: 58551-58794. In some
embodiments, when the TCF4 RIC pre-mRNA transcript comprises
retained intron 11, the ASO has a sequence according to any one of
SEQ ID NOs: 59039-59282. In some embodiments, the ASOs target a
TCF4 RIC pre-mRNA sequence.
RDH8
[1630] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a RDH8 genomic sequence (an RDH8 RIC
pre-mRNA). In some embodiments, the RDH8 genomic sequence is SEQ ID
NO: 34810. In some embodiments, the RDH8 RIC pre-mRNA is SEQ ID NO:
34870. In some embodiments, the RDH8 RIC pre-mRNA transcript
comprises retained intron 4. In some embodiments, when the RDH8 RIC
pre-mRNA transcript comprises retained intron 4, the ASOs disclosed
herein target SEQ ID NO: 61469. In some embodiments, when the RDH8
RIC pre-mRNA transcript comprises retained intron 4, the ASO has a
sequence according to any one of SEQ ID NOs: 59526-59662. In some
embodiments, the ASOs target an RDH8 RIC pre-mRNA sequence.
NXNL1
[1631] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a NXNL1 genomic sequence (an NXNL1 RIC
pre-mRNA). In some embodiments, the NXNL1 genomic sequence is SEQ
ID NO: 34811. In some embodiments, the NXNL1 RIC pre-mRNA is SEQ ID
NO: 34871. In some embodiments, the NXNL1 RIC pre-mRNA transcript
comprises retained intron 1. In some embodiments, when the NXNL1
RIC pre-mRNA transcript comprises retained intron 1, the ASOs
disclosed herein target SEQ ID NO: 61488. In some embodiments, when
the NXNL1 RIC pre-mRNA transcript comprises retained intron 1, the
ASO has a sequence according to any one of SEQ ID NOs: 59663-60020.
In some embodiments, the ASOs target an NXNL1 RIC pre-mRNA
sequence.
CRX
[1632] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a CRX genomic sequence (a CRX RIC
pre-mRNA). In some embodiments, the CRX genomic sequence is SEQ ID
NO: 34812. In some embodiments, the CRX RIC pre-mRNA is SEQ ID NO:
34872. In some embodiments, the CRX RIC pre-mRNA transcript
comprises retained intron 1 and/or 2 and/or or 3. In some
embodiments, when the CRX RIC pre-mRNA transcript comprises
retained intron 1, the ASOs disclosed herein target SEQ ID NO:
61464. In some embodiments, when the CRX RIC pre-mRNA transcript
comprises retained intron 2, the ASOs disclosed herein target SEQ
ID NO: 61484. In some embodiments, when the CRX RIC pre-mRNA
transcript comprises retained intron 3, the ASOs disclosed herein
target SEQ ID NO: 61475. In some embodiments, when the CRX RIC
pre-mRNA transcript comprises retained intron 1, the ASO has a
sequence according to any one of SEQ ID NOs: 60021-60254. In some
embodiments, when the CRX RIC pre-mRNA transcript comprises
retained intron 2, the ASO has a sequence according to any one of
SEQ ID NOs: 60255-60484. In some embodiments, when the CRX RIC
pre-mRNA transcript comprises retained intron 3, the ASO has a
sequence according to any one of SEQ ID NOs: 60485-61443. In some
embodiments, the ASOs target a CRX RIC pre-mRNA sequence.
[1633] In some embodiments the RIC pre-mRNA transcript comprises a
retained intron. In some embodiments, the ASO targets an exon
flanking the 5' splice site. In some embodiments, the ASO targets
the retained intron. In some embodiments, the ASO targets an exon
flanking the 3' splice site. A subsequent example of ASO targeting
is provided below using TEAD1.
[1634] In some embodiments, the ASO targets exon 4 of TEAD1 RIC
pre-mRNA comprising a retained intron 4. In some embodiments, the
ASO targets an exon 4 sequence upstream (or 5') from the 5' splice
site of a TEAD1 RIC pre-mRNA comprising a retained intron 4. In
some embodiments, the ASO targets an exon sequence about 4 to about
44 nucleotides upstream (or 5') from the 5' splice site of a TEAD1
RIC pre-mRNA comprising a retained intron 4. In some embodiments,
the ASO has a sequence according to any one of SEQ ID NOs:
43748-43756.
[1635] In some embodiments, the ASO targets intron 4 in a TEAD1 RIC
pre-mRNA comprising a retained intron 4. In some embodiments, the
ASO targets an intron 4 sequence downstream (or 3') from the 5'
splice site of a TEAD1 RIC pre-mRNA comprising a retained intron 4.
In some embodiments, the ASO targets an intron 4 sequence about 6
to about 496 nucleotides downstream (or 3') from the 5' splice site
of a TEAD1 RIC pre-mRNA comprising a retained intron 4. In some
embodiments, the ASO has a sequence according to any one of SEQ ID
NOs: 8968-9064.
[1636] In some embodiments, the ASO targets an intron 4 sequence
upstream (or 5') from the 3' splice site of a TEAD1 RIC pre-mRNA
comprising a retained intron 4. In some embodiments, the ASO
targets an intron 4 sequence about 16 to about 499 nucleotides
upstream (or 5') from the 3' splice site of a TEAD1 RIC pre-mRNA a
comprising retained intron 4. In some embodiments, the ASO has a
sequence according to any one of SEQ ID NOs: 43854-43943.
[1637] In some embodiments, the ASO targets exon 5 in a TEAD1 RIC
pre-mRNA comprising a retained intron 4. In some embodiments, the
ASO targets an exon 5 sequence downstream (or 3') from the 3'
splice site of a TEAD1 RIC pre-mRNA comprising a retained intron 4.
In some embodiments, the ASO targets an exon 5 sequence about 2 to
about 42 nucleotides downstream (or 3') from the 3' splice site of
a TEAD1 RIC pre-mRNA comprising a retained intron 4. In some
embodiments, the ASO has a sequence according to any one of SEQ ID
NOs: 44044-43952.
[1638] In embodiments, the targeted portion of the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA pre-mRNA is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of at least one of retained introns
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25, and subsequently increases ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein production.
[1639] The degree of intron retention can be expressed as percent
intron retention (PIR), the percentage of transcripts in which a
given intron is retained. In brief, PIR can be calculated as the
percentage of the average number of reads mapping to the
exon-intron junctions, over the sum of the average of the
exon-intron junction reads plus the exon-exon junction reads.
[1640] PIR values for SCN1A have been reported, e.g., by
Braunschweig, et al., 2014, (see, e.g., Supplemental Table S9),
incorporated by reference herein in its entirety.
[1641] In embodiments, the methods described herein are used to
increase the production of a functional ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein. As used herein, the term "functional" refers to the amount
of activity or function of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein that is
necessary to eliminate any one or more symptoms of a treated
condition. In embodiments, the methods are used to increase the
production of a partially functional ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein. As used herein, the term "partially functional" refers to
any amount of activity or function of the ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein that is less than the amount of activity or function that
is necessary to eliminate or prevent any one or more symptoms of a
disease or condition. In some embodiments, a partially functional
protein or RNA will have at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
75%, at least 80%, 85%, at least 90%, or at least 95% less activity
relative to the fully functional protein or RNA.
[1642] In embodiments, the method is a method of increasing the
expression of the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein by cells of a
subject having a RIC pre-mRNA encoding the ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein, wherein the subject has a deficient amount of
activity of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, and wherein the
deficient amount of the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein is caused by
haploinsufficiency of the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein. In such
an embodiment, the subject has a first allele encoding a functional
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein, and a second allele from which
the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein is not produced. In another such
embodiment, the subject has a first allele encoding a functional
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein, and a second allele encoding a
nonfunctional ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein. In another such
embodiment, the subject has a first allele encoding a functional
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein, and a second allele encoding a
partially functional ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein. In any of
these embodiments, the antisense oligomer binds to a targeted
portion of the RIC pre-mRNA transcribed from the first allele
(encoding functional ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein), thereby
inducing constitutive splicing of the retained intron from the RIC
pre-mRNA, and causing an increase in the level of mature mRNA
encoding functional ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, and an
increase in the expression of the ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein
in the cells of the subject
[1643] In embodiments, the subject has a first allele encoding a
functional ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA protein, and a second allele
encoding a partially functional ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein,
and the antisense oligomer binds to a targeted portion of the RIC
pre-mRNA transcribed from the first allele (encoding functional
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein) or a targeted portion of the
RIC pre-mRNA transcribed from the second allele (encoding partially
functional ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding the
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein, and an increase in the
expression of functional or partially functional ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein in the cells of the subject.
[1644] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA protein in cells of a subject having a RIC
pre-mRNA encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, wherein the
subject has a deficiency in the amount or function of a ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA protein.
[1645] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease or condition is targeted
by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the
disease is targeted by the ASOs. For example, a disease that is the
result of a mutation or deficiency of a first protein in a
particular pathway may be ameliorated by targeting a RIC pre-mRNA
that encodes a second protein, thereby increasing production of the
second protein. In some embodiments, the function of the second
protein is able to compensate for the mutation or deficiency of the
first protein (which is causative of the disease or condition).
[1646] In embodiments, the subject has:
[1647] a. a first mutant allele from which [1648] i) the ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA protein is produced at a reduced level compared
to production from a wild-type allele, [1649] ii) the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein is produced in a form having reduced function
compared to an equivalent wild-type protein, or [1650] iii) the
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein or functional RNA is not
produced; and
[1651] b. a second mutant allele from which [1652] i) the ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA protein is produced at a reduced level compared
to production from a wild-type allele, [1653] ii) the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein is produced in a form having reduced function
compared to an equivalent wild-type protein, or [1654] iii) the
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein is not produced, and
[1655] wherein the RIC pre-mRNA is transcribed from the first
allele and/or the second allele. In these embodiments, the ASO
binds to a targeted portion of the RIC pre-mRNA transcribed from
the first allele or the second allele, thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein and an increase in the expression of the
target protein or functional RNA in the cells of the subject. In
these embodiments, the target protein or functional RNA having an
increase in expression level resulting from the constitutive
splicing of the retained intron from the RIC pre-mRNA is either in
a form having reduced function compared to the equivalent wild-type
protein (partially-functional), or having full function compared to
the equivalent wild-type protein (fully-functional).
[1656] In embodiments, the level of mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein is increased 1.1 to 10-fold, when compared to
the amount of mRNA encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA that is produced
in a control cell, e.g., one that is not treated with the antisense
oligomer or one that is treated with an antisense oligomer that
does not bind to the targeted portion of the ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA RIC pre-mRNA.
[1657] In embodiments, the condition caused by a deficient amount
or activity of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein is not a condition
caused by alternative or aberrant splicing of the retained intron
to which the ASO is targeted. In embodiments, the condition caused
by a deficient amount or activity of the ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein is not a condition caused by alternative or aberrant
splicing of any retained intron in a RIC pre-mRNA encoding the
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein. In embodiments, alternative or
aberrant splicing may occur in a pre-mRNA transcribed from the
gene, however the compositions and methods of the invention do not
prevent or correct this alternative or aberrant splicing.
[1658] In embodiments, a subject treated using the methods of the
invention expresses a partially functional ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein from one allele, wherein the partially functional
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein is caused by a frameshift
mutation, a nonsense mutation, a missense mutation, or a partial
gene deletion. In embodiments, a subject treated using the methods
of the invention expresses a nonfunctional ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein from one allele, wherein the nonfunctional ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA protein is caused by a frameshift mutation, a
nonsense mutation, a missense mutation, a partial gene deletion, in
one allele. In embodiments, a subject treated using the methods of
the invention has a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA whole gene deletion,
in one allele.
Use of TANGO for Increasing Protein Expression
[1659] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) can be used in the methods of the
invention to increase expression of a ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein. In these embodiments, a retained-intron-containing
pre-mRNA (RIC pre-mRNA) encoding ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein
is present in the nucleus of a cell. Cells having a ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA RIC pre-mRNA that comprises a retained intron, an exon
flanking the 5' splice site, and an exon flanking the 3' splice
site, encoding the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, can be
contacted with antisense oligomers (ASOs) that are complementary to
a targeted portion of the RIC pre-mRNA. Hybridization of the ASOs
to the targeted portion of the RIC pre-mRNA can result in enhanced
splicing at the splice site (5' splice site or 3' splice site) of
the retained intron and subsequently increases target protein
production.
[1660] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[1661] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a RIC pre-mRNA encoding the ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein.
[1662] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g. in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features (e.g.
in the case of an antisense oligomer, the presence of additional,
unrecited nucleobases).
[1663] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g. nucleobase sequence) as well as those which do not materially
affect the character or function of the claimed invention. As used
herein, the term "consisting" is used to indicate the presence of
the recited feature (e.g. nucleobase sequence) alone (so that in
the case of an antisense oligomer consisting of a specified
nucleobase sequence, the presence of additional, unrecited
nucleobases is excluded).
[1664] In embodiments, the targeted region is in a retained intron
that is the second most abundant intron in a RIC pre-mRNA encoding
the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein. For example, the second most
abundant retained intron may be targeted rather than the most
abundant retained intron due to the uniqueness of the nucleotide
sequence of the second most abundant retained intron, ease of ASO
design to target a particular nucleotide sequence, and/or amount of
increase in protein production resulting from targeting the intron
with an ASO. In embodiments, the retained intron is the second most
abundant intron in a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein in a cell, wherein the
population of RIC pre-mRNAs comprises two or more retained introns.
In embodiments, an antisense oligomer targeted to the second most
abundant intron in the population of RIC pre-mRNAs encoding the
target protein induces splicing out of two or more retained introns
in the population, including the retained intron to which the
antisense oligomer is targeted or binds. In embodiments,
fully-spliced (mature) RNA encoding the target protein is thereby
produced.
[1665] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[1666] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[1667] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for the
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA gene in the published reference genome
deposited in the NCBI repository of biological and scientific
information (operated by National Center for Biotechnology
Information, National Library of Medicine, 8600 Rockville Pike,
Bethesda, Md. USA 20894). As used herein, the "wild-type sequence"
refers to NCBI Gene ID: (ROM1:6094; TEAD1:7003; RDH5:5959;
NR2E3:10002; PAX6:5080; CRX: 1406; FSCN2: 25794; ABCA4:24;
MYOC:4653; TCF4:6925; MFSD8:256471; CTNS:1497; NXNL1:115861). Also
used herein, a nucleotide position denoted with an "e" indicates
the nucleotide is present in the sequence of an exon (e.g., the
exon flanking the 5' splice site or the exon flanking the 3' splice
site).
[1668] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein, resulting in increased expression of ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA. As used herein, "contacting" or administering
to cells refers to any method of providing an ASO in immediate
proximity with the cells such that the ASO and the cells interact.
A cell that is contacted with an ASO will take up or transport the
ASO into the cell. The method involves contacting a condition or
disease-associated or condition or disease-relevant cell with any
of the ASOs described herein. In some embodiments, the ASO may be
further modified or attached (e.g., covalently attached) to another
molecule to target the ASO to a cell type, enhance contact between
the ASO and the condition or disease-associated or condition or
disease-relevant cell, or enhance uptake of the ASO.
[1669] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[1670] In embodiments, contacting a cell that expresses a ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA RIC pre-mRNA with an ASO that is complementary
to a targeted portion of the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA
transcript results in a measurable increase in the amount of the
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein (e.g., a target protein) encoded
by the pre-mRNA. Methods of measuring or detecting production of a
protein will be evident to one of skill in the art and include any
known method, for example, Western blotting, flow cytometry,
immunofluorescence microscopy, and ELISA.
[1671] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
RIC pre-mRNA transcript results in an increase in the amount of
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein produced by at least 10, 20, 30,
40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or
1000%, compared to the amount of the protein produced by a cell in
the absence of the ASO/absence of treatment. In embodiments, the
total amount of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein produced by the cell
to which the antisense oligomer was contacted is increased about
1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
amount of target protein produced by an control compound. A control
compound can be, for example, an oligonucleotide that is not
complementary to the targeted portion of the RIC pre-mRNA.
[1672] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of a ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
RIC pre-mRNA transcript results in an increase in the amount of
mRNA encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA respectively, including the
mature mRNA encoding the target protein. In some embodiments, the
amount of mRNA encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, or the mature
mRNA encoding the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, is increased
by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300,
350, 400, 450, 500, or 1000%, compared to the amount of the protein
produced by a cell in the absence of the ASO/absence of treatment.
In embodiments, the total amount of the mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein, or the mature mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein produced in the cell to which the antisense
oligomer was contacted is increased about 1.1 to about 10-fold,
about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to
about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold,
about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to
about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold,
about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about
8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3
to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold,
about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about
9-fold, at least about 1.1-fold, at least about 1.5-fold, at least
about 2-fold, at least about 2.5-fold, at least about 3-fold, at
least about 3.5-fold, at least about 4-fold, at least about 5-fold,
or at least about 10-fold compared to the amount of mature RNA
produced in an untreated cell, e.g., an untreated cell or a cell
treated with a control compound. A control compound can be, for
example, an oligonucleotide that is not complementary to the
targeted portion of the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[1673] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA protein in cells, for example, in a subject
having a deficiency in the amount or activity of ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA protein, by increasing the level of mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein, or the mature mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein. In particular, the methods and compositions
as described herein can induce the constitutive splicing of a
retained intron from a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA
transcript encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein, thereby
increasing the level of mRNA encoding ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein, or the mature mRNA encoding ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein and increasing the expression of ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein.
[1674] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[1675] In embodiments, constitutive splicing can correctly remove a
retained intron from a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA, wherein
the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA is transcribed from a
wild-type gene or allele, or a polymorphic gene or allele, that
encodes a fully-functional target protein or functional RNA, and
wherein the gene or allele does not have a mutation that causes
alternative splicing or aberrant splicing of the retained
intron.
[1676] In some embodiments, constitutive splicing of a retained
intron from a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA encoding ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA protein correctly removes a retained intron
from a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA encoding ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA protein, wherein the ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC
pre-mRNA is transcribed from a gene or allele from which the target
gene or functional RNA is produced at a reduced level compared to
production from a wild-type allele, and wherein the gene or allele
does not have a mutation that causes alternative splicing or
aberrant splicing of the retained intron. In these embodiments, the
correct removal of the constitutively spliced retained intron
results in production of target protein or functional RNA that is
functional when compared to an equivalent wild-type protein or
functional RNA.
[1677] In other embodiments, constitutive splicing can correctly
remove a retained intron from a ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC
pre-mRNA, wherein the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA is
transcribed from a gene or allele that encodes a target protein or
functional RNA produced in a form having reduced function compared
to an equivalent wild-type protein or functional RNA, and wherein
the gene or allele does not have a mutation that causes alternative
splicing or aberrant splicing of the retained intron. In these
embodiments, the correct removal of the constitutively spliced
retained intron results in production of partially functional
target protein, or functional RNA that is partially functional when
compared to an equivalent wild-type protein or functional RNA.
[1678] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[1679] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein or the amount of
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA protein by modulating alternative
splicing or aberrant splicing of a pre-mRNA transcribed from the
ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA gene. Modulation of alternative splicing
or aberrant splicing can be measured using any known method for
analyzing the sequence and length of RNA species, e.g., by RT-PCR
and using methods described elsewhere herein and in the literature.
In embodiments, modulation of alternative or aberrant splicing is
determined based on an increase or decrease in the amount of the
spliced species of interest of at least 10% or 1.1-fold. In
embodiments, modulation is determined based on an increase or
decrease at a level that is at least 10% to 100% or 1.1 to 10-fold,
as described herein regarding determining an increase in mRNA
encoding ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA protein in the methods of the
invention.
[1680] In embodiments, the method is a method wherein the ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA RIC pre-mRNA can be produce by partial splicing
of a wild-type ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA pre-mRNA. In embodiments,
the method is a method wherein the ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC
pre-mRNA can be produce by partial splicing of a full-length
wild-type ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA pre-mRNA. In embodiments, the ROM1,
TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8,
CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3,
ALMS1, PER1 or IDUA RIC pre-mRNA can be produce by partial splicing
of a full-length ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4,
MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8,
RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA pre-mRNA. In these
embodiments, a full-length ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA pre-mRNA may
have a polymorphism in a splice site of the retained intron that
does not impair correct splicing of the retained intron as compared
to splicing of the retained intron having the wild-type splice site
sequence.
[1681] In embodiments, the mRNA encoding ROM1, TEAD1, RDH5, NR2E3,
PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN,
RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA
protein is a full-length mature mRNA, or a wild-type mature mRNA.
In these embodiments, a full-length mature mRNA may have a
polymorphism that does not affect the activity of the target
protein or the functional RNA encoded by the mature mRNA, as
compared to the activity of ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX,
FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65,
LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA protein encoded
by the wild-type mature mRNA.
Antisense Oligomers
[1682] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA. As used
herein, the terms "ASO" and "antisense oligomer" are used
interchangeably and refer to an oligomer such as a polynucleotide,
comprising nucleobases, that hybridizes to a target nucleic acid
(e.g., a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA) sequence by
Watson-Crick base pairing or wobble base pairing (G-U). The ASO may
have exact sequence complementary to the target sequence or near
complementarity (e.g., sufficient complementarity to bind the
target sequence and enhancing splicing at a splice site). ASOs are
designed so that they bind (hybridize) to a target nucleic acid
(e.g., a targeted portion of a pre-mRNA transcript) and remain
hybridized under physiological conditions. Typically, if they
hybridize to a site other than the intended (targeted) nucleic acid
sequence, they hybridize to a limited number of sequences that are
not a target nucleic acid (to a few sites other than a target
nucleic acid). Design of an ASO can take into consideration the
occurrence of the nucleic acid sequence of the targeted portion of
the pre-mRNA transcript or a sufficiently similar nucleic acid
sequence in other locations in the genome or cellular pre-mRNA or
transcriptome, such that the likelihood the ASO will bind other
sites and cause "off-target" effects is limited. Any antisense
oligomers known in the art, for example in PCT Application No.
PCT/US2014/054151, published as WO 2015/035091, titled "Reducing
Nonsense-Mediated mRNA Decay," can be used to practice the methods
described herein.
[1683] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[1684] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[1685] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[1686] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and
Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by
reference in their entirety.
[1687] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine,
5,6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[1688] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081
(1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et
al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer
Drug Design 6:539 (1991); Zon et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphorothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[1689] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the invention comprises an ASO having
phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[1690] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention comprises about 5-100% Rp, at least about 5% Rp, at
least about 10% Rp, at least about 15% Rp, at least about 20% Rp,
at least about 25% Rp, at least about 30% Rp, at least about 35%
Rp, at least about 40% Rp, at least about 45% Rp, at least about
50% Rp, at least about 55% Rp, at least about 60% Rp, at least
about 65% Rp, at least about 70% Rp, at least about 75% Rp, at
least about 80% Rp, at least about 85% Rp, at least about 90% Rp,
or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
In embodiments, an ASO used in the methods of the invention
comprises about 10% to about 100% Rp, about 15% to about 100% Rp,
about 20% to about 100% Rp, about 25% to about 100% Rp, about 30%
to about 100% Rp, about 35% to about 100% Rp, about 40% to about
100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp,
about 55% to about 100% Rp, about 60% to about 100% Rp, about 65%
to about 100% Rp, about 70% to about 100% Rp, about 75% to about
100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp,
about 90% to about 100% Rp, or about 95% to about 100% Rp, about
20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about
70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp,
with the remainder Sp.
[1691] In embodiments, an ASO used in the methods of the invention
comprises about 5-100% Sp, at least about 5% Sp, at least about 10%
Sp, at least about 15% Sp, at least about 20% Sp, at least about
25% Sp, at least about 30% Sp, at least about 35% Sp, at least
about 40% Sp, at least about 45% Sp, at least about 50% Sp, at
least about 55% Sp, at least about 60% Sp, at least about 65% Sp,
at least about 70% Sp, at least about 75% Sp, at least about 80%
Sp, at least about 85% Sp, at least about 90% Sp, or at least about
95% Sp, with the remainder Rp, or about 100% Sp. In embodiments, an
ASO used in the methods of the invention, comprises about 10% to
about 100% Sp, about 15% to about 100% Sp, about 20% to about 100%
Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about
35% to about 100% Sp, about 40% to about 100% Sp, about 45% to
about 100% Sp, about 50% to about 100% Sp, about 55% to about 100%
Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about
70% to about 100% Sp, about 75% to about 100% Sp, about 80% to
about 100% Sp, about 85% to about 100% Sp, about 90% to about 100%
Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about
25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about
60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
[1692] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[1693] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[1694] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[1695] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[1696] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[1697] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[1698] In other embodiments, the ASOs are complementary to (and
bind to) a targeted portion of a ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC
pre-mRNA that is downstream (in the 3' direction) of the 5' splice
site of the retained intron in a ROM1, TEAD1, RDH5, NR2E3, PAX6,
CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC
pre-mRNA (e.g., the direction designated by positive numbers
relative to the 5' splice site) (FIG. 1). In some embodiments, the
ASOs are complementary to a targeted portion of the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA RIC pre-mRNA that is within the region +6 to +100
relative to the 5' splice site of the retained intron. In some
embodiments, the ASO is not complementary to nucleotides +1 to +5
relative to the 5' splice site (the first five nucleotides located
downstream of the 5' splice site). In some embodiments, the ASOs
may be complementary to a targeted portion of a ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA RIC pre-mRNA that is within the region between nucleotides +6
and +50 relative to the 5' splice site of the retained intron. In
some aspects, the ASOs are complementary to a targeted portion that
is within the region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6
to +50, +6 to +40, +6 to +30, or +6 to +20 relative to 5' splice
site of the retained intron.
[1699] In some embodiments, the ASOs are complementary to a
targeted region of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA that is
upstream (5' relative) of the 3' splice site of the retained intron
in a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA (e.g., in the direction
designated by negative numbers) (FIG. 1). In some embodiments, the
ASOs are complementary to a targeted portion of the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA RIC pre-mRNA that is within the region -16 to -100
relative to the 3' splice site of the retained intron. In some
embodiments, the ASO is not complementary to nucleotides -1 to -15
relative to the 3' splice site (the first 15 nucleotides located
upstream of the 3' splice site). In some embodiments, the ASOs are
complementary to a targeted portion of the ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA RIC pre-mRNA that is within the region -16 to -50 relative to
the 3' splice site of the retained intron. In some aspects, the
ASOs are complementary to a targeted portion that is within the
region -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50,
-16 to -40, or -16 to -30 relative to 3' splice site of the
retained intron.
[1700] In embodiments, the targeted portion of the ROM1, TEAD1,
RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS,
NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1,
PER1 or IDUA RIC pre-mRNA is within the region +100 relative to the
5' splice site of the retained intron to -100 relative to the 3'
splice site of the retained intron.
[1701] In some embodiments, the ASOs are complementary to a
targeted portion of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA that is
within the exon flanking the 5' splice site (upstream) of the
retained intron (FIG. 1). In some embodiments, the ASOs are
complementary to a targeted portion of the ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA RIC pre-mRNA that is within the region +2e to -4e in the exon
flanking the 5' splice site of the retained intron. In some
embodiments, the ASOs are not complementary to nucleotides -1e to
-3e relative to the 5' splice site of the retained intron. In some
embodiments, the ASOs are complementary to a targeted portion of
the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA that is within the region
-4e to -100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e,
-4e to -50e, -4 to -40e, -4e to -30e, or -4e to -20e relative to
the 5' splice site of the retained intron.
[1702] In some embodiments, the ASOs are complementary to a
targeted portion of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2,
ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT,
RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA that is
within the exon flanking the 3' splice site (downstream) of the
retained intron (FIG. 1). In some embodiments, the ASOs are
complementary to a targeted portion to the ROM1, TEAD1, RDH5,
NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4, MFSD8, CTNS, NXNL1,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 or
IDUA RIC pre-mRNA that is within the region +2e to -4e in the exon
flanking the 3' splice site of the retained intron. In some
embodiments, the ASOs are not complementary to nucleotide +1e
relative to the 3' splice site of the retained intron. In some
embodiments, the ASOs are complementary to a targeted portion of
the ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA that is within the region
+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e,
+2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative to
the 3' splice site of the retained intron. The ASOs may be of any
length suitable for specific binding and effective enhancement of
splicing. In some embodiments, the ASOs consist of 8 to 50
nucleobases. For example, the ASO may be 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some
embodiments, the ASOs consist of more than 50 nucleobases. In some
embodiments, the ASO is from 8 to 50 nucleobases, 8 to 40
nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25
nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50
nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30
nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15
nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35
nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20
nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40
nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25
nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50
nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30
nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15
nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35
nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20
nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35
nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20
nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35
nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20
nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35
nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50
nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to
30 nucleobases in length. In some embodiments, the ASOs are 18
nucleotides in length. In some embodiments, the ASOs are 15
nucleotides in length. In some embodiments, the ASOs are 25
nucleotides in length.
[1703] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[1704] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[1705] In some embodiments, the nucleic acid to be targeted by an
ASO is a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC,
TCF4, MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12,
RGR, CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA expressed in a cell,
such as a eukaryotic cell. In some embodiments, the term "cell" may
refer to a population of cells. In some embodiments, the cell is in
a subject. In some embodiments, the cell is isolated from a
subject. In some embodiments, the cell is ex vivo. In some
embodiments, the cell is a condition or disease-relevant cell or a
cell line. In some embodiments, the cell is in vitro (e.g., in cell
culture).
Pharmaceutical Compositions
[1706] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent
or carrier.
[1707] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose. The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[1708] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[1709] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancers is a surfactant, fatty acid,
bile salt, chelating agent, or non-chelating nonsurfactant.
[1710] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[1711] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[1712] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[1713] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[1714] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[1715] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. The ASOs of the present invention may be
administered to patients parenterally, for example, by intravitreal
injection, subretinal injection, topical application, implantation,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection. In embodiments, delivery is to
the heart or liver. In embodiments, a fetus is treated in utero,
e.g., by administering the ASO composition to the fetus directly or
indirectly (e.g., via the mother).
[1716] The compositions of the present invention may be provided to
muscle cells by any suitable means, including direct administration
(e.g., locally by injection or topical administration at a
treatment site) or systemically (e.g., parenterally or orally),
intranasally, orally, or by intravitreal, subretinal, implantation,
inhalational, enteral, topical, intrauterine, vaginal, sublingual,
rectal, intramuscular, intrapleural, intraventricular,
intraperitoneal, ophthalmic, intravenous, or subcutaneous
means.
Methods of Identifying Additional ASOs that Enhance Splicing
[1717] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of a ROM1, TEAD1, RDH5, NR2E3, PAX6, CRX, FSCN2, ABCA4, MYOC, TCF4,
MFSD8, CTNS, NXNL1, OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR,
CNGA3, ALMS1, PER1 or IDUA RIC pre-mRNA, specifically at the target
intron. ASOs that specifically hybridize to different nucleotides
within the target region of the pre-mRNA may be screened to
identify (determine) ASOs that improve the rate and/or extent of
splicing of the target intron. In some embodiments, the ASO may
block or interfere with the binding site(s) of a splicing
repressor(s)/silencer. Any method known in the art may be used to
identify (determine) an ASO that when hybridized to the target
region of the intron results in the desired effect (e.g., enhanced
splicing, protein or functional RNA production). These methods also
can be used for identifying ASOs that enhance splicing of the
retained intron by binding to a targeted region in an exon flanking
the retained intron, or in a non-retained intron. An example of a
method that may be used is provided below.
[1718] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site.
[1719] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1720] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[1721] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[1722] As described for the ASO walk above, the ASO micro-walk can
be perform by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1723] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. Following administration, the cells, tissues, and/or
organs of the model animals may be assessed to determine the effect
of the ASO treatment by for example evaluating splicing
(efficiency, rate, extent) and protein production by methods known
in the art and described herein. The animal models may also be any
phenotypic or behavioral indication of the disease or disease
severity.
Compositions and Methods for Treatment of Liver Disease
[1724] Liver disease is a debilitating condition that results in an
estimated 60,000 deaths per year in the United States alone. In
many cases, the only hope for those suffering from liver failure is
a liver transplant, though the donor pool is only estimated to be
approximately 4,000. Therefore, the odds of receiving a transplant
is low, and there are few treatments available to ameliorate the
condition for those unable to receive a transplant. Therefore,
there exists a need for compositions and methods for treating liver
diseases.
[1725] Individual introns in primary transcripts of protein-coding
genes having one or more than one intron are spliced from the
primary transcript with different efficiencies. In most cases only
the fully spliced mRNA is exported through nuclear pores for
subsequent translation in the cytoplasm. Unspliced and partially
spliced transcripts are detectable in the nucleus. It is generally
thought that nuclear accumulation of transcripts that are not fully
spliced is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[1726] Substantial levels of partially-spliced transcripts of the
gene, which encodes a protein that is deficient in a subset of
liver diseases, have been discovered in the nucleus of human cells.
These pre-mRNA species comprise at least one retained intron. The
present invention provides compositions and methods for
upregulating splicing of one or more retained introns that are
rate-limiting for the nuclear stages of gene expression to increase
steady-state production of fully-spliced, mature mRNA, and thus,
translated protein levels. These compositions and methods utilize
antisense oligomers (ASOs) that promote constitutive splicing at an
intron splice site of a retained-intron-containing pre-mRNA that
accumulates in the nucleus. Thus, in embodiments, protein is
increased using the methods of the invention to treat a condition
caused by a protein deficiency.
[1727] Liver diseases that can be treated by the invention
described herein are diseases where a subject is deficient in a
gene product, where deficiency in a gene product causes the liver
disease.
[1728] These AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5,
GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1,
HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 and NCOA5 pre-mRNA species comprise at least one
retained intron. The present invention provides compositions and
methods for upregulating splicing of one or more retained AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5
and NCOA5 introns that are rate-limiting for the nuclear stages of
gene expression to increase steady-state production of
fully-spliced, mature mRNA, and thus, translated
aminomethyltransferase, adenosine deaminase, protoporphyrinogen
oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane
synthase, very long chain acyl-CoA dehydrogenase, pyruvate
carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V,
galactose-1-phosphate uridylyltransferase, low density lipoprotein
receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha,
glucokinase, hepatic nuclear factor-1-alpha albumin proximal
factor, protein O-glucosyltransferase 1, phosphatidylinositol
3-kinase regulatory subunit 1, Tribbles-1, transforming growth
factor beta-1, hemochromatosis type 2B, thrombopoietin,
patatin-like phospholipase domain-containing protein 3,
copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5 protein levels. These compositions and methods can utilize
antisense oligomers (ASOs) that promote constitutive splicing at
intron splice sites of a retained-intron-containing AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 pre-mRNA
(RIC pre-mRNA) that accumulates in the nucleus. Thus, in
embodiments, aminomethyltransferase, adenosine deaminase,
protoporphyrinogen oxidase, uroporphyrinogen decarboxylase,
hydroxymethylbilane synthase, very long chain acyl-CoA
dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase,
apolipoprotein A-V, galactose-1-phosphate uridylyltransferase, low
density lipoprotein receptor adaptor protein 1, hepatocyte nuclear
factor 4-alpha, glucokinase, hepatic nuclear factor-1-alpha albumin
proximal factor, protein O-glucosyltransferase 1,
phosphatidylinositol 3-kinase regulatory subunit 1, Tribbles-1,
transforming growth factor beta-1, hemochromatosis type 2B,
thrombopoietin, patatin-like phospholipase domain-containing
protein 3, copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5 protein can be increased using the methods of the invention to
treat a condition caused by aminomethyltransferase, adenosine
deaminase, protoporphyrinogen oxidase, uroporphyrinogen
decarboxylase, hydroxymethylbilane synthase, very long chain
acyl-CoA dehydrogenase, pyruvate carboxylase isovaleryl-CoA
dehydrogenase, apolipoprotein A-V, galactose-1-phosphate
uridylyltransferase, low density lipoprotein receptor adaptor
protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic
nuclear factor-1-alpha albumin proximal factor, protein
O-glucosyltransferase 1, phosphatidylinositol 3-kinase regulatory
subunit 1, Tribbles-1, transforming growth factor beta-1,
hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase
domain-containing protein 3, copper-transporting ATPase 2,
fumarylacetoacetase, argininosuccinate lyase, hereditary
hemochromatosis protein, alstrom syndrome protein 1, 3
beta-hydroxysteroid dehydrogenase type 7, peroxisome proliferator
activated receptor delta, interleukin 6, ceramide synthase 2 or
nuclear receptor coactivator 5 deficiency.
[1729] In some embodiments, disclosed herein are compositions and
methods for upregulating splicing of one or more retained AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5
introns that are rate-limiting for the nuclear stages of gene
expression to increase steady-state production of fully-spliced,
mature mRNA, and thus, translated AMT, ADA, PPOX, UROD, HMBS,
ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1,
HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE,
ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein levels. These
compositions and methods can utilize antisense oligomers (ASOs)
that promote constitutive splicing at intron splice sites of a
retained-intron-containing AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 pre-mRNA (RIC pre-mRNA) that
accumulates in the nucleus. Thus, in embodiments, AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein
can be increased using the methods of the invention to treat a
condition caused by AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD,
APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1,
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 deficiency.
[1730] In other embodiments, the methods of the invention can be
used to increase aminomethyltransferase, adenosine deaminase,
protoporphyrinogen oxidase, uroporphyrinogen decarboxylase,
hydroxymethylbilane synthase, very long chain acyl-CoA
dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase,
apolipoprotein A-V, galactose-1-phosphate uridylyltransferase, low
density lipoprotein receptor adaptor protein 1, hepatocyte nuclear
factor 4-alpha, glucokinase, hepatic nuclear factor-1-alpha albumin
proximal factor, protein O-glucosyltransferase 1,
phosphatidylinositol 3-kinase regulatory subunit 1, Tribbles-1,
transforming growth factor beta-1, hemochromatosis type 2B,
thrombopoietin, patatin-like phospholipase domain-containing
protein 3, copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5 production to treat a condition in a subject in need thereof. In
embodiments, the subject has a condition in which
aminomethyltransferase, adenosine deaminase, protoporphyrinogen
oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane
synthase, very long chain acyl-CoA dehydrogenase, pyruvate
carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V,
galactose-1-phosphate uridylyltransferase, low density lipoprotein
receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha,
glucokinase, hepatic nuclear factor-1-alpha albumin proximal
factor, protein O-glucosyltransferase 1, phosphatidylinositol
3-kinase regulatory subunit 1, Tribbles-1, transforming growth
factor beta-1, hemochromatosis type 2B, thrombopoietin,
patatin-like phospholipase domain-containing protein 3,
copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5 is not necessarily deficient relative to wild-type, but where an
increase in aminomethyltransferase, adenosine deaminase,
protoporphyrinogen oxidase, uroporphyrinogen decarboxylase,
hydroxymethylbilane synthase, very long chain acyl-CoA
dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase,
apolipoprotein A-V, galactose-1-phosphate uridylyltransferase, low
density lipoprotein receptor adaptor protein 1, hepatocyte nuclear
factor 4-alpha, glucokinase, hepatic nuclear factor-1-alpha albumin
proximal factor, protein 0-glucosyltransferase 1,
phosphatidylinositol 3-kinase regulatory subunit 1, Tribbles-1,
transforming growth factor beta-1, hemochromatosis type 2B,
thrombopoietin, patatin-like phospholipase domain-containing
protein 3, copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5 mitigates the condition nonetheless. In embodiments, the
condition can be caused by a AMT, ADA, PPOX, UROD, HMBS, ACADVL,
PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 and NCOA5 haploinsufficiency.
[1731] In embodiments, the described compositions and methods are
used to treat a subject or patient having a liver condition that is
caused by a deficiency in the target protein. In embodiments the
described compositions and methods are used to treat a subject or
patient having a liver condition that is not caused by a deficiency
in the target protein. In embodiments, the subject or patient
having a liver condition can benefit from increased production of
the target protein by supplementing normal production of the target
protein. In related embodiments, the subject or patient having a
liver condition can benefit from increased production of the target
protein by increasing mature mRNA production and/or supplementing
normal production of the target protein. In certain embodiments,
wherein the condition that is not necessarily caused by a
deficiency of the target protein but is nonetheless treated by
increasing production of the target protein using the present
methods, the target protein is TRIB1, TGFB1, HAMP, THPO, PNPLA3,
PPARD, IL6, CERS2, or NCOA5. In embodiments, the target protein
acts on a secondary target to ameliorate or treat the liver
condition in the subject. In embodiments, the secondary target
protein is deficient in the subject. In embodiments, the secondary
target protein is not deficient in the subject.
Glycine Encephalopathy
[1732] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disorder Glycine
Encephalopathy (GCE). The predominant phenotype of GCE is the
neonatal phenotype, which manifests early in life. Symptoms of this
phenotype include lethargy, hypotonia, myoclonic jerks, seizures,
mental retardation, apnea and often death. In other instances, GCE
manifests in childhood, with symptoms including mild mental
retardation, delirium, chorea, and vertical gaze palsy. The late
onset form of GCE results in spastic diplegia and optic atrophy,
but typically does not result in mental retardation or
seizures.
[1733] GCE can manifest as a result of deficiency in the glycine
cleavage system in the liver. Deficiency in the protein
aminomethyltransferase (AMT) has been implicated in the progression
of GCE and studies have linked AMT deficiency and the progression
of GCE. AMT is a component of the glycine cleavage system in the
mitochondria of liver cells. The AMT gene, which codes for the AMT
protein, is a 6 kb gene spanning 9 exons located on 3p21.2.
Mutations in the AMT gene have been shown to cause the clinical
phenotype associated with GCE. In one study, a patient heterozygous
for a G269D mutation. Other studies have examined other missense
mutations in AMT that result in the progression of GCE, thereby
establishing a positive link between AMT deficiency and the
progression of GCE.
Zellweger Syndrome/Heimler Syndrome
[1734] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disorder Zellweger
Syndrome. Zellweger Syndrome is a severe peroxisomal biogenesis
disorder characterized by severe neurologic dysfunction,
craniofacial abnormalities, and liver dysfunction. Patients
afflicted with the classic Zellweger Syndrome phenotype typically
die within the first year of life.
[1735] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disorder Heimler
syndrome. Heimler syndrome is the mildest form the peroxisomal
biogenesis disorders characterized by sensorineural hearing loss,
enamel hypoplasia of the secondary dentition and nail
abnormalities.
Adenosine Deaminase Deficiency
[1736] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disease Adenosine
Deaminase (ADA) deficiency. ADA deficiency generally manifests in
infancy, and is generally fatal, though a small subset of patients
display a late onset form of the disease that is generally milder
than the infantile form. Patients afflicted with the late onset
form of ADA deficiency typically show gradual immunologic
deterioration, which leads to a number of secondary infections.
[1737] Deficiency in the ADA protein results in the clinical
manifestations shown in ADA deficiency. The ADA gene, which is
located at 20q13 and spans 10 exons, codes for the ADA protein.
Mutations in the ADA gene resulting in deficient amounts of ADA
protein have been shown to be responsible for the progression of
ADA deficiency. In one study, a pair of children diagnosed with ADA
deficiency were examined. Both children were found to have
diminished levels of ADA protein, while both of the parents were
found to have intermediate levels of the ADA protein. This finding
supported the recessive pattern of inheritance proposed for the
disease, and provides a positive link between diminished ADA
protein and the clinical manifestations of ADA deficiency.
Porphyria Variegata
[1738] In some embodiments, the invention described herein can be
used to treat the liver disease porphyria variegate (VP). VP is
characterized by cutaneous manifestations, such as increased
photosensitivity, blistering, skin fragility, and postinflammatory
hyperpigmentation. Additional manifestations include abdominal
pain, dark urine, and neuropsychiatric symptoms that characterize
the acute hepatic porphyrias.
[1739] Deficiency in the protoporphyrinogen oxidase (PPOX) protein
results in the clinical manifestations shown in VP. The PPOX gene,
which is located at 1q23.3 and spans 13 exons, codes for the PPOX
protein. Mutations in the PPOX gene resulting in deficient amounts
of PPOX protein have been shown to be responsible for the
progression of VP. While there exists a rare homozygous form of VP,
"classical" VP is characterized by mutations in a single allele of
the PPOX gene, thus proceeding via a haploinsufficiency mechanism.
Several studies have linked heterozygous mutations in PPOX with the
progression of VP, which is a result of diminished levels of PPOX
protein found in patients afflicted with VP.
Porphyria Cutanea Tarda
[1740] In some embodiments, the invention described herein can be
used to treat the autosomal dominant liver disease porphyria
cutanea tarda (PCT). PCT is characterized by light sensitive
dermatitis and excretion of uroporphyrin in urine.
[1741] Deficiency in the uroporphyrinogen decarboxylase (UROD)
protein results in the clinical manifestations shown in PCT. The
UROD gene, which is located at 1p34.1 and spans 10 exons, codes for
the UROD protein. Mutations in the UROD gene resulting in deficient
amounts of UROD protein have been shown to be responsible for the
progression of PCT. In one study, a G381V mutation in the UROD gene
was shown in a patient with the familial version of PCT, which
resulted in diminished levels of the UROD protein. Other studies
have also shown correlation between diminished UROD protein levels
and the progression of PCT.
Acute Intermittent Porphyria
[1742] In some embodiments, the invention described herein can be
used to treat the autosomal dominant liver disease acute
intermittent porphyria (AIP). AIP is characterized by defects in
the biosynthesis of heme. Clinical manifestations of AIP include
abdominal pain, gastrointestinal dysfunction, and neurologic
disturbance that may lead to death.
[1743] Deficiency in the hydroxymethylbilane synthase (HMBS)
protein (nonerythroid, or both erythroid and nonerythroid) results
in the clinical manifestations shown in AIP. The HMBS gene, which
is located at 11q23.3 and spans 15 exons, codes for the HMBS
protein. HMBS also is referred to as porphobilinogen deaminase
(PBGD). Mutations in the HMBS gene resulting in deficient amounts
of HMBS protein have been shown to be responsible for the
progression of AIP. In one study, 19 separate mutations in HMBS
were found in 28 families displaying AIP, further providing a link
between HMBS deficiency and AIP.
Very Long Chain Acyl-CoA Dehydrogenase Deficiency
[1744] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disease, very long
chain acyl-CoA dehydrogenase (VLCAD) deficiency. VLCAD is
characterized by nonketotic hypoglycemia, cardiorespiratory arrest,
hepatomegaly, cardiomegaly, and hypotonia, which are believed to be
manifestations resulting from a defect in mitochondrial fatty acid
oxidation.
[1745] Deficiency in the VLCAD protein results in the clinical
manifestations shown in VLCAD deficiency. The ACADVL gene, which is
located at 17p13.1 and spans 20 exons, codes for the VLCAD protein.
Mutations in the ACADVL gene resulting in deficient amounts of
VLCAD protein have been shown to be responsible for the progression
of VLCAD deficiency. In one study, 2 patients displaying VLCAD
deficiency were found to have a 105 bp deletion in the ACADVL gene.
In another study, 21 different missense mutations were found in 18
children displaying VLCAD deficiency. In aggregate, studies such as
these have shown a positive link between deficiency in VLCAD and
the clinical manifestations seen in patients displaying VLCAD
deficiency.
Pyruvate Carboxylase Deficiency
[1746] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disease pyruvate
carboxylase (PC) deficiency. PC deficiency is categorized into 3
phenotypic subtypes. Patients with type A, which is more prevalent
in North America, display lactic academia and psychomotor
retardation. Patients with type B, which is more severe than type A
and is more prevalent in France and the United Kingdom, display
increased serum lactate, ammonia, citrulline and lysine, and
intracellular redox disturbance with a higher incidence of
mortality. Type C is the more milder form of PC deficiency and is
generally benign.
[1747] Deficiency in the pyruvate carboxylase (PC) protein results
in the clinical manifestations shown in PC deficiency. The PC gene,
which is located at 11q13.2 and spans 19 exons, codes for the PC
protein. The familial inheritance is believed to proceed via an
autosomal recessive mechanism, and the carrier frequency is
estimated to be as high as 1 in 10 in certain families. Mutations
in the PC gene resulting in deficient amounts of PC protein have
been shown to be responsible for the progression of PC deficiency.
In one study, missense mutations in the PC gene in patients
suffering from type A PC deficiency were discovered, thereby
providing a link between a deficiency in PC protein levels and the
clinical manifestations associated with PC deficiency.
Isovaleric Acidemia
[1748] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disease isovaleric
academia (IVA). IVA is categorized into 2 phenotypic subtypes: an
acute and chronic subtype. The acute subtype leads to massive
metabolic acidosis and ultimately death. The chronic subtype
results in periods of attacks of severe ketoacidosis followed by
asymptomatic periods. Clinical manifestations of IVA include
peculiar odor, an aversion to dietary protein and vomiting.
[1749] Deficiency in the isovaleryl-CoA dehydrogenase (IVD) protein
results in the clinical manifestations shown in IVA. The IVD gene,
which is located at 15q13 and spans 12 exons, codes for the IVD
protein. Mutations in the IVD gene resulting in deficient amounts
of IVD protein have been shown to be responsible for the
progression of IVA deficiency. Several studies have looked at a
number of different mutations in IVD that result in IVD deficiency,
and have shown that mutations that result in deficiency in the
amount of IVD protein result in the clinical manifestations seen in
IVA.
Hyperchylomicronemia/Hypertriglyceridemia
[1750] In some embodiments, the invention described herein can be
used to treat the autosomal dominant liver disease
hyperchylomicronemia. Hyperchylomicronemia is characterized by
increased amounts of chylomicrons and very low density lipoprotein
(VLDL) and decreased LDL and high density lipoprotein (HDL) in the
plasma.
[1751] In some embodiments, the invention described herein can be
used to treat the autosomal dominant liver disease
hypertriglyceridemia. Patients afflicted with hypertriglyceridemia
generally have normal levels of cholesterol while displaying
elevated levels of triglycerides. Other than elevated
triglycerides, patients are generally asymptomatic.
[1752] Deficiency in apolipoprotein A-V (APOA5) protein results in
the clinical manifestations shown in both hyperchylomicronemia and
hypertriglyceridemia. The APOA5 gene, which is located at 11q23.3
and spans 4 exons, codes for the APOA5 protein. Mutations in the
APOA5 gene resulting in deficient amounts of APOA5 protein have
been shown to be responsible for the progression of both
hyperchylomicronemia and hypertriglyceridemia. Several studies have
looked at a number of different mutations in APOA5 that result in
both hyperchylomicronemia and hypertriglyceridemia. For example in
one study, a S19W mutation in APOA5 was shown to result in a
deficiency in the amount of APOA5 protein, which manifested as
hyperchylomicronemia in a family of patients.
Galactosemia
[1753] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disease galactosemia.
Galactosemia generally manifests during neonatal development, and
is characterized by jaundice, hepatosplenomegaly, hepatocellular
insufficiency, food intolerance, hypoglycemia, renal tubular
dysfunction, muscle hypotonia, sepsis, and cataracts. Overtime,
patients afflicted with galactosemia experience mental retardation,
verbal dyspraxia, motor abnormalities, and hypergonadotropic
hypogonadism.
[1754] Deficiency in the galactose-1-phosphate uridylyltransferase
(GALT) protein results in the clinical manifestations shown in
galactosemia. The GALT gene, which is located at 15q11.2 and spans
11 exons, codes for the GALT protein. Mutations in the GALT gene
resulting in deficient amounts of GALT protein have been shown to
be responsible for the progression of galactosemia. Several studies
have looked at a number of different mutations in GALT that result
in galactosemia, and have shown that mutations that result in
deficiency in the amount of GALT protein result in the clinical
manifestations seen in galactosemia. In one study, a M142K mutation
was found to decrease the amount of GALT protein to approximately
4% of the normal level, which provides a positive link between
deficiency in the amount of GALT protein and the clinical
progression of galactosemia.
Hypercholesterolemia
[1755] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disease
hypercholesterolemia (ARH). ARH is characterized by severely
elevated plasma low density lipoprotein (LDL) cholesterol, tuberous
and tendon xanthomata and premature atherosclerosis.
[1756] Deficiency in the protein low density lipoprotein receptor
adaptor protein 1 (LDLRAP1) results in the clinical manifestations
shown in ARH. The LDLRAP1 gene, which is located at 1p36.11 and
spans 9 exons, codes for the LDLRAP1 protein. Mutations in the
LDLRAP1 gene resulting in deficient amounts of LDLRAP1 protein have
been shown to be responsible for the progression of ARH. Several
studies have looked at a number of different mutations in LDLRAP1
that result in ARH, and have shown that mutations that result in
deficiency in the amount of LDLRAP1 protein result in the clinical
manifestations seen in ARH. A number of studies examined a
conserved nonsense mutation in the LDLRAP1 gene, which resulted in
substantial decrease in the amount of LDLRAP1 protein, and hence
the clinical manifestations associated with ARH.
Diabetes Mellitus
[1757] In some embodiments, the invention described herein can be
used to treat maturity-onset diabetes of the young type 1 (MODY1).
In other embodiments, the invention described herein can be used to
treat maturity-onset diabetes of the young type 2 (MODY2). In other
embodiments, the invention described herein can be used to treat
maturity-onset diabetes of the young type 3 (MODY3). In other
embodiments, the invention described herein can be used to treat
noninsulin-dependent diabetes mellitus (NIDDM). In other
embodiments, the invention described herein can be used to treat
insulin-dependent diabetes mellitus 1 (IDDM1). In other
embodiments, the invention described herein can be used to treat
insulin-dependent diabetes mellitus 20 (IDDM20). In other
embodiments, the invention described herein can be used to treat
Falconi renotubular syndrome 4 with maturity-onset diabetes of the
young (FRTS4). In other embodiments, the invention described herein
can be used to treat hyperinsulemic hypoglycemia familial 3 (HHF3).
In other embodiments, the invention described herein can be used to
treat permanent neonatal diabetes mellitus (PNDM). Diabetes
mellitus are a group of metabolic diseases characterized by high
blood sugar, with symptoms including frequent urination, increased
thirst, increased hunger, diabetic ketoacidosis, cardiovascular
disease, stroke, chronic kidney failure, foot ulcers and damage to
the eyes.
[1758] While there are a number of factors that can contribute to
the progression of diabetes, deficiency in the protein hepatocyte
nuclear factor 4-alpha (HNF4A) has been shown to correlate to the
incidence of MODY1, NIDDM and FRTS4. The HNF4A gene, which is
located at 20q13.12 and spans 12 exons, codes for the HNF4A
protein. Mutations in the HNF4A gene resulting in deficient amounts
of HNF4A protein have been shown to be responsible for the
progression of MODY1, NIDDM and FRTS4. Several studies have looked
at a number of different mutations in HNF4A that result in MODY1,
NIDDM and FRTS4 and have shown that mutations that result in
deficiency in the amount of HNF4A protein result in the clinical
manifestations seen in diabetes. For example, one study
demonstrated that a nonsense mutation at Q268 was present in a
large population of patients afflicted with MODY1. In another
study, the authors concluded that mutations in HNF4A is associated
with increased birth weight and macrosomia, which eventually
evolves into the hyperinsulinemia seen in patients afflicted with
diabetes. Studies such this and others have positively correlated
the deficiency in the amount of expressed HNF4A protein with the
progression of diabetes.
[1759] Deficiency in the protein glucokinase (GCK) has been shown
to correlate to the incidence of NIDDM, MODY2, HHF3 and PNDM. The
GCK gene, which is located at 7p13 and spans 12 exons, codes for
the GCK protein. Mutations in the GCK gene resulting in deficient
amounts of GCK protein have been shown to be responsible for the
progression of NIDDM, MODY2, HHF3 and PNDM. Several studies have
looked at a number of different mutations in GCK that result in
NIDDM, MODY2, HHF3 and PNDM and have positively correlated the
deficiency in the amount of expressed GCK protein with the
progression of diabetes.
[1760] Deficiency in the protein hepatic nuclear factor-1-alpha
albumin proximal factor (HNF1A) has been shown to correlate to the
incidence of MODY3, IDDM20, IDDM1 and NIDDM. The HNF1A gene, which
is located at 12q24.31 and spans 10 exons, codes for the HNF1A
protein. Mutations in the HNF1A gene resulting in deficient amounts
of HNF1A protein have been shown to be responsible for the
progression of MODY3, IDDM20, IDDM1 and NIDDM. Several studies have
looked at a number of different mutations in HNF1A that result in
MODY3, IDDM20, IDDM1 and NIDDM, and have positively correlated the
deficiency in the amount of expressed HNF1A protein with the
progression of diabetes.
[1761] Deficiency in the protein nuclear receptor coactivator 5
(NCOA5) has been shown to correlate to the incidence of type II
diabetes, glucose intolerance and ultimately liver cancer. The
NCOA5 gene, which is located at 20q13.12 and spans 8 exons, codes
for the NCOA5 protein. Mutations in the NCOA5 gene resulting in
deficient amounts of NCOA5 protein have been shown to be
responsible for the progression of diabetes. Several studies have
looked at a number of different mutations in NCOA5 that result in
diabetes, and have positively correlated the deficiency in the
amount of expressed NCOA5 protein with the progression of
diabetes.
Hepatic Adenoma
[1762] In some embodiments, the invention described herein can be
used to treat the autosomal dominant liver disease hepatic adenoma.
Hepatic adenoma is an uncommon benign liver tumor with a very low
risk of malignant transformation. Patients afflicted with hepatic
adenoma are generally asymptomatic unless the tumor begins to
hemorrhage, which could lead to hypotension, tachycardia and
diaphoresis.
[1763] Deficiency in the HNF1A protein can result in the
progression of hepatic adenoma. Mutations in the HNF1A gene
resulting in deficient amounts of HNF1A protein have been shown to
be responsible for the progression of hepatic adenoma.
Dowling-Degos Disease 4
[1764] In some embodiments, the invention described herein can be
used to treat the autosomal dominant liver disease Dowling-Degos
disease 4 (DDD4). DDD4 is characterized by retricular pigmentation
that presents in adult life, particularly in the folds of the skin.
While the manifestations affect somatic cells, the pigmentation
results from dysfunction of the liver.
[1765] Deficiency in the protein O-glucosyltransferase 1 (POGLUT1)
results in the clinical manifestations shown in DDD4. The POGLUT1
gene, which is located at 3q13.33 and spans 11 exons, codes for the
POGLUT1 protein. Mutations in the POGLUT1 gene resulting in
deficient amounts of POGLUT1 protein have been shown to be
responsible for the progression of DDD4. Several studies have
looked at a number of different mutations in POGLUT1 that result in
DDD4, and have shown that mutations that result in deficiency in
the amount of POGLUT1 protein result in the clinical manifestations
seen in DDD4.
SHORT Syndrome/Immunodeficiency 36/Agammaglobulinemia 7
[1766] In some embodiments, the invention described herein can be
used to treat SHORT syndrome. SHORT syndrome is an acronym for the
clinical conditions that are associated with the condition,
including short stature, hyperextensibility of joints and/or
inguinal hernia, ocular depression, rieger anomaly and teething
delay. Other symptoms characteristic of SHORT syndrome include a
triangular face, small chin with a dimple, loss of fat under the
skin, abnormal position of the ears, hearing loss and delayed
speech.
[1767] In some embodiments, the invention described herein can be
used to treat the autosomal dominant disease immunodeficiency 36
(IMD36). IMD36 is characterized by impaired B-cell function,
hypogammaglobulinemia and recurrent infections.
[1768] In some embodiments, the invention described herein can be
used to treat the autosomal recessive disease agammaglobulinemia 7
(AGM7). AGM7 is characterized by impaired B-cell function,
hypogammaglobulinemia and recurrent infections. AGM7 is an
immunodeficiency disease characterized by low serum antibodies and
low circulating B cells, which results in recurrent infections.
[1769] Deficiency in the protein phosphatidylinositol 3-kinase
regulatory subunit 1 (PIK3R1) results in the clinical
manifestations shown in SHORT syndrome, IMD36 and AGM7. The PIK3R1
gene, which is located at 5q13.1 and spans 16 exons, codes for the
PIK3R1 protein. Mutations in the PIK3R1 gene resulting in deficient
amounts of PIK3R1 protein have been shown to be responsible for the
progression of SHORT syndrome, IMD36 and AGM7. Several studies have
looked at a number of different mutations in PIK3R1 that result in
SHORT syndrome, IMD36 and AGM7. In one study, a R649W mutation in
PIK3R1 was found a population of unrelated individuals diagnosed
with SHORT syndrome. The same mutation was witnessed in an affected
mother and her two sons, which provided evidence of the autosomal
dominant pattern of inheritance of SHORT syndrome.
Lipid Metabolism Dysfunction
[1770] In some embodiments, the invention described herein can be
used to treat the lipid metabolism deficiency caused by deficiency
in the protein Tribbles-1 (TRIB1). Deficiency in the amount of
TRIB1 protein, which is encoded by the TRIB1 gene located on
8q24.13 and spans 3 exons, has been shown to be correlated to an
increased risk of atherosclerosis. Mice lacking TRIB1 were shown to
have diminished adipose tissue mass accompanied by increased
lipolysis, which positively linked diminished levels of TRIB1 with
dysfunction in lipid metabolism.
[1771] In some embodiments, the invention described herein can be
used to treat the lipid metabolism deficiency caused by deficiency
in the protein peroxisome proliferator activated receptor delta
(PPARD). Deficiency in the amount of PPARD protein, which is
encoded by the PPARD gene located on chromosome 6 and spans 8
exons, has been shown to be correlated to increased lipid
metabolism dysfunction.
Liver Inflammation
[1772] In some embodiments, the invention described herein can be
used to treat the liver inflammation caused by deficiency in the
protein transforming growth factor beta-1 (TGFB1). Deficiency in
the amount of TGBF1 protein, which is encoded by the TGBF1 gene
located on 19813.2 and spans 7 exons, has been shown to be
correlated to an increased risk of atherosclerosis. Knockout mice
displaying the TGBF1 (-/-) genotype were shown to develop severe
liver inflammation due to CD4(+) T-cell mediated inflammation. Such
studies provide a positive correlation between deficiency in the
amount of TGBF1 protein and increased incidence of liver
inflammation.
[1773] In some embodiments, the invention described herein can be
used to treat the lipid inflammation caused by deficiency in the
protein interleukin 6 (IL6). Deficiency in the amount of IL6
protein, which is encoded by the IL6 gene located on 7p15.3 and
spans 5 exons, has been shown to be correlated to lipid
inflammation.
[1774] In some embodiments, the invention described herein can be
used to treat the lipid inflammation or steatohepatitis caused by
deficiency in the ceramide synthase 2 (CERS2) protein. Deficiency
in the amount of CERS2 protein, which is encoded by the CERS2 gene
located on 1q21.3 and spans 11 exons, has been shown to be
correlated to steatohepatitis and insulin resistance.
Hemochromatosis Type 2B
[1775] In some embodiments, the invention described herein can be
used to treat the autosomal recessive liver disease hemochromatosis
type 2B (HFE2B). HFE2B (otherwise known as iron overload) is
characterized by joint pain, fatigue and weakness, which ultimately
results in organ damage.
[1776] Deficiency in the protein hepcidin antimicrobial peptide
(HAMP) results in the clinical manifestations shown in HFE2B. The
HAMP gene, which is located at 19q13.12 and spans 3 exons, codes
for the HAMP protein. Mutations in the HAMP gene resulting in
deficient amounts of HAMP protein have been shown to be responsible
for the progression of HFE2B. Nonsense mutations have been reported
at G93 and R56 in patients afflicted with HFE2B, while missense
mutations such as G71D have also been found. These mutations, which
result in diminished amounts of the HAMP protein, were proposed to
be a direct cause of HFE2B, thereby providing a direct correlation
between diminished levels of HAMP and incidence of HFE2B.
Thrombocytopenia
[1777] In some embodiments, the invention described herein can be
used to treat the autosomal dominant disease thrombocytopenia.
Thrombocytopenia is characterized by a decrease in the amount of
thrombocytes in the blood. While many cases of thrombocytopenia are
asymptomatic, some patients experience external bleeding such as
nose bleeds, malaise, fatigue and general weakness.
[1778] Deficiency in the protein thrombopoietin (THPO) has been
shown to be correlated to the incidence of thrombocytopenia. The
THPO gene, which is located at 3q27.1 and spans 6 exons, codes for
the THPO protein. Mutations in the THPO gene resulting in deficient
amounts of THPO protein have been shown to be responsible for the
progression of thrombocytopenia. In one study, a single nucleotide
deletion at 3252 was seen in 3 generations of a family afflicted
with thrombocytopenia. This study correlated the deficiency of THPO
protein levels as a result of the deletion with the incidence of
thrombocytopenia.
Non-Alcoholic Fatty Liver Disease
[1779] In some embodiments, the invention described herein can be
used to treat non-alcoholic fatty liver disease (NAFLD). NAFLD is
characterized by the accumulation of excess triglycerides in the
liver, which can be associated with adverse metabolic consequences
such as insulin resistance and dyslipidemia. Factors that can
influence the progression of NAFLD include obesity, diabetes, and
insulin resistance. In some instances, aggregated fatty deposits in
a liver can promote an inflammatory response, which can progress to
cirrhosis or liver cancer.
[1780] Deficiency in the protein patatin-like phospholipase
domain-containing protein 3 (PNPLA3) has been shown to be
correlated to the incidence of NAFLD. The PNPLA3 gene, which is
located at 22q13 and spans 9 exons, codes for the PNPLA3 protein.
Mutations in the PNPLA3 gene resulting in deficient amounts of
PNPLA3 protein have been shown to be responsible for the
progression of NAFLD. Polymorphisms such as C99G, G115C, I148M,
T216P and K434E have been found in populations manifesting symptoms
of NAFLD. These missense mutations were shown to result in
decreased levels of PNPLA3, which provides a correlation between
the deficiency of the PNPLA3 protein and the progression of
NAFLD.
Wilson Disease
[1781] In some embodiments, the invention described herein can be
used to treat the autosomal recessive disorder Wilson disease.
Wilson disease is characterized by dramatic build-up of
intracellular hepatic copper with subsequent hepatic and neurologic
abnormalities. Wilson disease may present itself in a patient with
tiredness, increased bleeding tendency or confusion (due to hepatic
encephalopathy) and portal hypertension.
[1782] Deficiency in the protein copper-transporting ATPase 2
(ATP7B) has been shown to be correlated to the incidence of Wilson
disease. The ATP7B gene, which is located at 13q14.3 and spans 21
exons, codes for the ATP7B protein. Mutations in the ATP7B gene
resulting in deficient amounts of ATP7B protein have been shown to
be responsible for the progression of Wilson disease. Mutations in
ATP7B such as L492S, Y532H, G591D, R616Q and G626A have been found
in populations manifesting symptoms of Wilson disease. These
missense mutations were shown to result in decreased levels of
ATP7B, which provides a correlation between the deficiency of the
ATP7B protein and the progression of Wilson disease.
Tyrosinemia
[1783] In some embodiments, the invention described herein can be
used to treat tyrosinemia. Tyrosinemia is characterized by
progressive liver disease and a secondary renal tubular dysfunction
leading to hypophosphatemic rickets. Onset varies from infancy to
adolescence. In the most acute form patients present with severe
liver failure within weeks after birth, whereas rickets may be the
major symptom in chronic tyrosinemia. Untreated, patients die from
cirrhosis or hepatocellular carcinoma at a young age
[1784] Deficiency in the protein fumarylacetoacetase (FAH) has been
shown to be correlated to the incidence of tyrosinemia. The FAH
gene, which is located at 15q25.1 and spans 14 exons, codes for the
FAH protein. Mutations in the FAH gene resulting in deficient
amounts of FAH protein have been shown to be responsible for the
progression of FAH. Polymorphisms such as N16I, A134D, C193R, D233V
and Q279R have been found in populations manifesting symptoms of
FAH. These missense mutations were shown to result in decreased
levels of FAH, which provides a correlation between the deficiency
of the tyrosinemia protein and the progression of FAH.
Argininosuccinate Lyase Deficiency
[1785] In some embodiments, the invention described herein can be
used to treat the autosomal recessive disorder argininosuccinate
lyase deficiency. Argininosuccinate lyase deficiency is
characterized by mental and physical retardation, liver
enlargement, skin lesions, dry and brittle hair showing
trichorrhexis nodosa microscopically and fluorescing red,
convulsions, and episodic unconsciousness.
[1786] Deficiency in the protein argininosuccinate lyase (ASL) has
been shown to be correlated to the incidence of argininosuccinate
lyase deficiency. The ASL gene, which is located at 7q11.21 and
spans 17 exons, codes for the ASL protein. Mutations in the ASL
gene resulting in deficient amounts of ASL protein have been shown
to be responsible for the progression of argininosuccinate lyase
deficiency. Polymorphisms such as R113Q, V178M, R182Q, R236W, Q286R
and R456W have been found in populations manifesting symptoms of
argininosuccinate lyase deficiency. These missense mutations were
shown to result in decreased levels of ASL, which provides a
correlation between the deficiency of the ASL protein and the
progression of argininosuccinate lyase deficiency.
Hemochromatosis Type 1
[1787] In some embodiments, the invention described herein can be
used to treat the autosomal recessive disorder hemochromatosis type
1. Hemochromatosis type 1 is characterized by iron overload. Excess
iron is deposited in a variety of organs leading to their failure,
and resulting in serious illnesses including cirrhosis, hepatomas,
diabetes, cardiomyopathy, arthritis, and hypogonadotropic
hypogonadism. Severe effects of the disease usually do not appear
until after decades of progressive iron loading.
[1788] Deficiency in the hereditary hemochromatosis protein has
been shown to be correlated to the incidence of hemochromatosis
type 1. The HFE gene, which is located at 6p22.2 and spans 6 exons,
codes for the hereditary hemochromatosis protein. Mutations in the
ASL gene resulting in deficient amounts of hereditary
hemochromatosis protein have been shown to be responsible for the
progression of hemochromatosis type 1. Polymorphisms such as S65C,
Q127H, A176V, C282Y, Q283P and V295A have been found in populations
manifesting symptoms of hemochromatosis type 1. These missense
mutations were shown to result in decreased levels of hereditary
hemochromatosis protein, which provides a correlation between the
deficiency of the hereditary hemochromatosis protein and the
progression of hemochromatosis type 1.
Alstrom Syndrome
[1789] In some embodiments, the invention described herein can be
used to treat the autosomal recessive disorder Alstrom syndrome.
Alstrom syndrome is characterized by progressive cone-rod retinal
dystrophy, neurosensory hearing loss, early childhood obesity and
diabetes mellitus type 2. Dilated cardiomyopathy, acanthosis
nigricans, male hypogonadism, hypothyroidism, developmental delay
and hepatic dysfunction can also be associated with the
syndrome.
[1790] Deficiency in the protein alstrom syndrome protein 1 has
been shown to be correlated to the incidence of Alstrom syndrome.
The ALMS1 gene, which is located at 2p13.1 and spans 23 exons,
codes for the alstrom syndrome protein. Mutations in the ALMS1 gene
resulting in deficient amounts of alstrom syndrome protein 1 have
been shown to be responsible for the progression of Alstrom
syndrome. Polymorphisms such as V671G, G1412A, I1875V, S2111R,
D2672H and K3434E have been found in populations manifesting
symptoms of Alstrom syndrome. These missense mutations were shown
to result in decreased levels of alstrom syndrome protein 1, which
provides a correlation between the deficiency of the alstrom
syndrome protein 1 and the progression of Alstrom syndrome.
Congenital Bile Acid Synthesis Defect 1
[1791] In some embodiments, the invention described herein can be
used to treat the autosomal recessive disorder congenital bile acid
synthesis defect 1 (CBAS1). CBAS1 is a primary defect in bile
synthesis leading to progressive liver disease. Clinical features
include neonatal jaundice, severe intrahepatic cholestasis,
cirrhosis.
[1792] Deficiency in the protein 3 beta-hydroxysteroid
dehydrogenase type 7 (3BHS7) has been shown to be correlated to the
incidence of CBAS1. The HSD3B7 gene, which is located at 16p11.2
and spans 6 exons, codes for the 3BHS7 protein. Mutations in the
HSD3B7 gene resulting in deficient amounts of 3BHS7 protein have
been shown to be responsible for the progression of CBAS1.
Polymorphisms such as G19S, E147K, T250A and L347P have been found
in populations manifesting symptoms of CBAS1. These missense
mutations were shown to result in decreased levels of 3BHS7, which
provides a correlation between the deficiency of the 3BHS7 protein
and the progression of CBAS1.
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[1793] In embodiments, the methods of the present invention can
exploit the presence of retained-intron-containing pre-mRNA (RIC
pre-mRNA) transcribed from the AMT, ADA, PPOX, UROD, HMBS, ACADVL,
PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 gene and encoding
aminomethyltransferase, adenosine deaminase, protoporphyrinogen
oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane
synthase, very long chain acyl-CoA dehydrogenase, pyruvate
carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V,
galactose-1-phosphate uridylyltransferase, low density lipoprotein
receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha,
glucokinase, hepatic nuclear factor-1-alpha albumin proximal
factor, protein O-glucosyltransferase 1, phosphatidylinositol
3-kinase regulatory subunit 1, Tribbles-1, transforming growth
factor beta-1, hemochromatosis type 2B, thrombopoietin,
patatin-like phospholipase domain-containing protein 3,
copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5 protein, in the cell nucleus. Splicing of AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA species
to produce mature, fully-spliced, AMT, ADA, PPOX, UROD, HMBS,
ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1,
HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE,
ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA, can be induced
using ASOs that stimulate splicing out of the retained introns. The
resulting mature AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD,
APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1,
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 mRNA can be exported to the cytoplasm and
translated, thereby increasing the amount of
aminomethyltransferase, adenosine deaminase, protoporphyrinogen
oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane
synthase, very long chain acyl-CoA dehydrogenase, pyruvate
carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V,
galactose-1-phosphate uridylyltransferase, low density lipoprotein
receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha,
glucokinase, hepatic nuclear factor-1-alpha albumin proximal
factor, protein O-glucosyltransferase 1, phosphatidylinositol
3-kinase regulatory subunit 1, Tribbles-1, transforming growth
factor beta-1, hemochromatosis type 2B, thrombopoietin,
patatin-like phospholipase domain-containing protein 3,
copper-transporting ATPase 2, fumarylacetoacetase,
argininosuccinate lyase, hereditary hemochromatosis protein,
alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase
type 7, peroxisome proliferator activated receptor delta,
interleukin 6, ceramide synthase 2 or nuclear receptor coactivator
5 protein in the patient's cells and alleviating symptoms of the
CNS disease or conditions caused by deficiency in each protein.
This method, described further below, is known as Targeted
Augmentation of Nuclear Gene Output (TANGO).
[1794] AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP,
THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7,
CERS2 or NCOA5 gene can be analyzed for intron-retention events. In
some cases, AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5,
GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1,
HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 gene can be analyzed for intron-retention
events. RNA sequencing (RNAseq), can be visualized in the UCSC
genome browser, and can show AMT, ADA, PPOX, UROD, HMBS, ACADVL,
PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 transcripts expressed in human
liver cells and localized in either the cytoplasmic or nuclear
fraction. In some embodiments, the retained-intron containing
pre-mRNA transcripts are retained in the nucleus and are not
exported out to the cytoplasm
[1795] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 65%, about 5% to about 60%, about 5%
to about 55%, about 5% to about 50%, about 5% to about 45%, about
5% to about 40%, about 5% to about 35%, about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 10% to about 100%, about 10% to about 95%, about 10% to
about 90%, about 10% to about 85%, about 10% to about 80%, about
10% to about 75%, about 10% to about 70%, about 10% to about 65%,
about 10% to about 60%, about 10% to about 65%, about 10% to about
60%, about 10% to about 55%, about 10% to about 50%, about 10% to
about 45%, about 10% to about 40%, about 10% to about 35%, about
10% to about 30%, about 10% to about 25%, about 10% to about 20%,
about 15% to about 100%, about 15% to about 95%, about 15% to about
90%, about 15% to about 85%, about 15% to about 80%, about 15% to
about 75%, about 15% to about 70%, about 15% to about 65%, about
15% to about 60%, about 15% to about 65%, about 15% to about 60%,
about 15% to about 55%, about 15% to about 50%, about 15% to about
45%, about 15% to about 40%, about 15% to about 35%, about 15% to
about 30%, about 15% to about 25%, about 20% to about 100%, about
20% to about 95%, about 20% to about 90%, about 20% to about 85%,
about 20% to about 80%, about 20% to about 75%, about 20% to about
70%, about 20% to about 65%, about 20% to about 60%, about 20% to
about 65%, about 20% to about 60%, about 20% to about 55%, about
20% to about 50%, about 20% to about 45%, about 20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about
100%, about 25% to about 95%, about 25% to about 90%, about 25% to
about 85%, about 25% to about 80%, about 25% to about 75%, about
25% to about 70%, about 25% to about 65%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 60%, about 25% to about
55%, about 25% to about 50%, about 25% to about 45%, about 25% to
about 40%, or about 25% to about 35%, retention. In embodiments,
other ASOs useful for this purpose are identified, using, e.g.,
methods described herein.
[1796] In embodiments, the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 intron numbering corresponds to
the one or more mRNA sequences at shown in Table 10 or Table 11. In
embodiments, the targeted portion of the AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA is in
one or more introns as shown in Table 10 or Table 11. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of one or more retained introns as
shown in Table 10 or Table 11 and subsequently increases AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5
protein production. It is understood that the intron numbering may
change in reference to a different AMT, ADA, PPOX, UROD, HMBS,
ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1,
HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE,
ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 isoform sequence. One of
skill in the art can determine the corresponding intron number in
any isoform based on an intron sequence provided herein or using
the number provided in reference to the one or more mRNA sequence
at shown in Table 10 or Table 11. One of skill in the art also can
determine the sequences of flanking exons in any AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 isoform
for targeting using the methods of the invention, based on an
intron sequence provided herein or using the intron number provided
in reference to the one or more mRNA sequence at shown in Table 10
or Table 11.
[1797] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 genomic sequence. In some
embodiments, the ASOs disclosed herein target a AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA sequence.
[1798] In some embodiments, the ASO targets a sequence of a RIC
pre-mRNA transcript encoded by a AMT, ADA, PPOX, UROD, HMBS,
ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1,
HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE,
ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 genomic sequence comprising a retained intron as shown in
Table 10 or Table 11. In some embodiments, the ASO targets a RIC
pre-mRNA encoded by SEQ ID NOs: 61504-61534.
[1799] In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 61535-61633. In some embodiments, the ASO
targets a RIC pre-mRNA transcript of SEQ ID NO: 61535-61633
comprising a retained intron as shown in Table 10 or Table 11. In
some embodiments, the ASOs target SEQ ID NO: 139852-140004. In some
embodiments, the ASO comprises a sequence of any one of SEQ ID NOs:
61634-139851.
[1800] In some embodiments, the ASO targets an exon sequence
upstream of a retained intron as shown in Table 10 or Table 11 of a
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table
10 or Table 11. In some embodiments, the ASO targets an exon
sequence upstream (or 5') from the 5' splice site of a retained
intron as shown in Table 10 or Table 11 of a AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
comprising a retained intron as shown in Table 10 or Table 11. In
some embodiments, the ASO targets an exon sequence about 4 to about
1000 nucleotides upstream (or 5') from the 5' splice site of a AMT,
ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table
10 or Table 11.
[1801] In some embodiments, the ASO targets an intron sequence
upstream of a retained intron as shown in Table 10 or Table 11 of a
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table
10 or Table 11. In some embodiments, the ASO targets an intron
sequence downstream (or 3') from the 5' splice site of a retained
intron as shown in Table 10 or Table 11 of a AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
comprising a retained intron as shown in Table 10 or Table 11. In
some embodiments, the ASO targets an exon sequence about 6 to about
500 nucleotides downstream (or 3') from the 5' splice site of a
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table
10 or Table 11.
[1802] In some embodiments, the ASO targets an exon sequence
downstream of a retained intron as shown in Table 10 or Table 11 of
a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP,
THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7,
CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown
in Table 10 or Table 11. In some embodiments, the ASO targets an
exon sequence downstream (or 3') from the 3' splice site of a
retained intron as shown in Table 10 or Table 11 of a AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA comprising a retained intron as shown in Table 10 or Table
11. In some embodiments, the ASO targets an exon sequence about 2
to about 1000 nucleotides downstream (or 3') from the 3' splice
site of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP,
THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7,
CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown
in Table 10 or Table 11.
Protein Expression
[1803] In embodiments, the methods described herein are used to
increase the production of a functional protein. As used herein,
the term "functional" refers to the amount of activity or function
of a protein that is necessary to eliminate any one or more
symptoms of a treated condition. In embodiments, the methods are
used to increase the production of a partially functional protein.
As used herein, the term "partially functional" refers to any
amount of activity or function of the protein that is less than the
amount of activity or function that is necessary to eliminate or
prevent any one or more symptoms of a disease or condition. In some
embodiments, a partially functional protein or RNA will have at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 75%, at least 80%, 85%, at
least 90%, or at least 95% less activity relative to the fully
functional protein or RNA.
[1804] In embodiments, the method is a method of increasing the
expression of the protein by cells of a subject having a RIC
pre-mRNA encoding the protein, wherein the subject has a condition
described herein caused by a deficient amount of activity of a
protein described herein. In some embodiments, the deficient amount
of the protein is caused by haploinsufficiency of the protein. In
such an embodiment, the subject has a first allele encoding a
functional protein, and a second allele from which the protein is
not produced. In another such embodiment, the subject has a first
allele encoding a functional protein, and a second allele encoding
a nonfunctional protein. In another such embodiment, the subject
has a first allele encoding a functional protein, and a second
allele encoding a partially functional protein. In any of these
embodiments, the antisense oligomer binds to a targeted portion of
the RIC pre-mRNA transcribed from the first allele (encoding
functional protein), thereby inducing constitutive splicing of the
retained intron from the RIC pre-mRNA, and causing an increase in
the level of mature mRNA encoding functional protein, and an
increase in the expression of the protein in the cells of the
subject.
[1805] In embodiments, the subject has a first allele encoding a
functional protein, and a second allele encoding a partially
functional protein, and the antisense oligomer binds to a targeted
portion of the RIC pre-mRNA transcribed from the first allele or a
targeted portion of the RIC pre-mRNA transcribed from the second
allele (encoding partially functional protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding the
protein, and an increase in the expression of functional or
partially functional protein in the cells of the subject.
[1806] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of a protein
described herein in cells of a subject having a RIC pre-mRNA
encoding the protein, wherein the subject has a deficiency in the
amount or function of the protein.
[1807] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease or condition is targeted
by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the
disease is targeted by the ASOs. For example, a disease that is the
result of a mutation or deficiency of a first protein in a
particular pathway may be ameliorated by targeting a RIC pre-mRNA
that encodes a second protein, thereby increasing production of the
second protein. In some embodiments, the function of the second
protein is able to compensate for the mutation or deficiency of the
first protein (which is causative of the disease or condition).
[1808] In embodiments, the subject has:
[1809] a) a first mutant allele from which [1810] i) the protein is
produced at a reduced level compared to production from a wild-type
allele, [1811] ii) the protein is produced in a form having reduced
function compared to an equivalent wild-type protein, or [1812]
iii) the protein or functional RNA is not produced; and
[1813] b) a second mutant allele from which [1814] i) the protein
is produced at a reduced level compared to production from a
wild-type allele, [1815] ii) the protein is produced in a form
having reduced function compared to an equivalent wild-type
protein, or [1816] iii) the protein is not produced, and wherein
the RIC pre-mRNA is transcribed from the first allele and/or the
second allele. In these embodiments, the ASO binds to a targeted
portion of the RIC pre-mRNA transcribed from the first allele or
the second allele, thereby inducing constitutive splicing of the
retained intron from the RIC pre-mRNA, and causing an increase in
the level of mRNA encoding a protein and an increase in the
expression of the target protein or functional RNA in the cells of
the subject. In these embodiments, the target protein or functional
RNA having an increase in expression level resulting from the
constitutive splicing of the retained intron from the RIC pre-mRNA
is either in a form having reduced function compared to the
equivalent wild-type protein (partially-functional), or having full
function compared to the equivalent wild-type protein
(fully-functional).
[1817] In embodiments, the level of mRNA encoding a protein
described herein is increased 1.1 to 10-fold, when compared to the
amount of mRNA encoding the protein that is produced in a control
cell, e.g., one that is not treated with the antisense oligomer or
one that is treated with an antisense oligomer that does not bind
to the targeted portion of the RIC pre-mRNA.
[1818] In embodiments, the condition caused by a deficient amount
or activity of a protein is not a condition caused by alternative
or aberrant splicing of the retained intron to which the ASO is
targeted. In embodiments, the condition caused by a deficient
amount or activity of the protein is not a condition caused by
alternative or aberrant splicing of any retained intron in a RIC
pre-mRNA encoding the protein. In embodiments, alternative or
aberrant splicing may occur in a pre-mRNA transcribed from the
gene, however the compositions and methods of the invention do not
prevent or correct this alternative or aberrant splicing.
[1819] In embodiments, a subject treated using the methods of the
invention expresses a partially functional protein from one allele,
wherein the partially functional protein is caused by a frameshift
mutation, a nonsense mutation, a missense mutation, or a partial
gene deletion. In embodiments, a subject treated using the methods
of the invention expresses a nonfunctional protein from one allele,
wherein the nonfunctional protein is caused by a frameshift
mutation, a nonsense mutation, a missense mutation, a partial gene
deletion, in one allele. In embodiments, a subject treated using
the methods of the invention has a whole gene deletion, in one
allele.
Use of TANGO for Increasing Protein Expression
[1820] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) is used in the methods of the invention
to increase expression of a protein. In these embodiments, a
retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding a
protein is present in the nucleus of a cell. Cells having a RIC
pre-mRNA that comprises a retained intron, an exon flanking the 5'
splice site, and an exon flanking the 3' splice site, encoding the
protein, are contacted with antisense oligomers (ASOs) that are
complementary to a targeted portion of the RIC pre-mRNA.
Hybridization of the ASOs to the targeted portion of the RIC
pre-mRNA results in enhanced splicing at the splice site (5' splice
site or 3' splice site) of the retained intron and subsequently
increases target protein production.
[1821] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[1822] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a RIC pre-mRNA encoding the protein.
[1823] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g. in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features (e.g.
in the case of an antisense oligomer, the presence of additional,
unrecited nucleobases).
[1824] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g. nucleobase sequence) as well as those which do not materially
affect the character or function of the claimed invention. As used
herein, the term "consisting" is used to indicate the presence of
the recited feature (e.g. nucleobase sequence) alone (so that in
the case of an antisense oligomer consisting of a specified
nucleobase sequence, the presence of additional, unrecited
nucleobases is excluded).
[1825] In embodiments, the targeted region is in a retained intron
that is the second most abundant intron in a RIC pre-mRNA encoding
a protein described herein. For example, the second most abundant
retained intron may be targeted rather than the most abundant
retained intron due to the uniqueness of the nucleotide sequence of
the second most abundant retained intron, ease of ASO design to
target a particular nucleotide sequence, and/or amount of increase
in protein production resulting from targeting the intron with an
ASO. In embodiments, the retained intron is the second most
abundant intron in a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein in a cell, wherein the
population of RIC pre-mRNAs comprises two or more retained introns.
In embodiments, an antisense oligomer targeted to the second most
abundant intron in the population of RIC pre-mRNAs encoding the
target protein induces splicing out of two or more retained introns
in the population, including the retained intron to which the
antisense oligomer is targeted or binds. In embodiments,
fully-spliced (mature) RNA encoding the target protein is thereby
produced.
[1826] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[1827] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[1828] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for a
gene in the published reference genome deposited in the NCBI
repository of biological and scientific information (operated by
National Center for Biotechnology Information, National Library of
Medicine, 8600 Rockville Pike, Bethesda, Md. USA 20894). Also used
herein, a nucleotide position denoted with an "e" indicates the
nucleotide is present in the sequence of an exon (e.g., the exon
flanking the 5' splice site or the exon flanking the 3' splice
site).
[1829] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding a protein
described herein, resulting in increased expression of the protein.
As used herein, "contacting" or administering to cells refers to
any method of providing an ASO in immediate proximity with the
cells such that the ASO and the cells interact. A cell that is
contacted with an ASO will take up or transport the ASO into the
cell. The method involves contacting a condition or
disease-associated or condition or disease-relevant cell with an
ASO. In some embodiments, the ASO may be further modified or
attached (e.g., covalently attached) to another molecule to target
the ASO to a cell type, enhance contact between the ASO and the
condition or disease-associated or condition or disease-relevant
cell, or enhance uptake of the ASO.
[1830] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[1831] In embodiments, contacting a cell that expresses a RIC
pre-mRNA with an ASO that is complementary to a targeted portion of
the RIC pre-mRNA transcript results in a measurable increase in the
amount of a protein (e.g., a target protein) encoded by the
pre-mRNA. Methods of measuring or detecting production of a protein
will be evident to one of skill in the art and include any known
method, for example, Western blotting, flow cytometry,
immunofluorescence microscopy, and ELISA.
[1832] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of an RIC pre-mRNA transcript
results in an increase in the amount of a protein produced by at
least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350,
400, 450, 500, or 1000%, compared to the amount of the protein
produced by a cell in the absence of the ASO/absence of treatment.
In embodiments, the total amount of a protein produced by the cell
to which the antisense oligomer was contacted is increased about
1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
amount of target protein produced by an control compound. A control
compound can be, for example, an oligonucleotide that is not
complementary to the targeted portion of the RIC pre-mRNA.
[1833] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of an RIC pre-mRNA transcript
results in an increase in the amount of mRNA encoding a protein,
including the mature mRNA encoding the target protein. In some
embodiments, the amount of mRNA encoding a protein, or the mature
mRNA encoding the protein, is increased by at least 10, 20, 30, 40,
50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%,
compared to the amount of the protein produced by a cell in the
absence of the ASO/absence of treatment. In embodiments, the total
amount of the mRNA encoding a protein, or the mature mRNA encoding
a protein produced in the cell to which the antisense oligomer was
contacted is increased about 1.1 to about 10-fold, about 1.5 to
about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold,
about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to
about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold,
about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to
about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold,
about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about
7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4
to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold,
at least about 1.1-fold, at least about 1.5-fold, at least about
2-fold, at least about 2.5-fold, at least about 3-fold, at least
about 3.5-fold, at least about 4-fold, at least about 5-fold, or at
least about 10-fold compared to the amount of mature RNA produced
in an untreated cell, e.g., an untreated cell or a cell treated
with a control compound. A control compound can be, for example, an
oligonucleotide that is not complementary to the targeted portion
of the RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[1834] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of a
protein in cells, for example, in a subject having a condition
described herein caused by a deficiency in the amount or activity
of a protein described herein, by increasing the level of mRNA
encoding the protein, or the mature mRNA encoding the protein. In
particular, the methods and compositions as described herein induce
the constitutive splicing of a retained intron from an RIC pre-mRNA
transcript encoding the protein, thereby increasing the level of
mRNA encoding the protein, or the mature mRNA encoding the protein
and increasing the expression of the protein.
[1835] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[1836] In embodiments, constitutive splicing correctly removes a
retained intron from an RIC pre-mRNA, wherein the RIC pre-mRNA is
transcribed from a wild-type gene or allele, or a polymorphic gene
or allele, that encodes a fully-functional target protein or
functional RNA, and wherein the gene or allele does not have a
mutation that causes alternative splicing or aberrant splicing of
the retained intron.
[1837] In some embodiments, constitutive splicing of a retained
intron from an RIC pre-mRNA encoding a protein correctly removes a
retained intron from an RIC pre-mRNA encoding the protein, wherein
the RIC pre-mRNA is transcribed from a gene or allele from which
the target gene or functional RNA is produced at a reduced level
compared to production from a wild-type allele, and wherein the
gene or allele does not have a mutation that causes alternative
splicing or aberrant splicing of the retained intron. In these
embodiments, the correct removal of the constitutively spliced
retained intron results in production of target protein or
functional RNA that is functional when compared to an equivalent
wild-type protein or functional RNA.
[1838] In other embodiments, constitutive splicing correctly
removes a retained intron from an RIC pre-mRNA, wherein the RIC
pre-mRNA is transcribed from a gene or allele that encodes a target
protein or functional RNA produced in a form having reduced
function compared to an equivalent wild-type protein or functional
RNA, and wherein the gene or allele does not have a mutation that
causes alternative splicing or aberrant splicing of the retained
intron. In these embodiments, the correct removal of the
constitutively spliced retained intron results in production of
partially functional target protein, or functional RNA that is
partially functional when compared to an equivalent wild-type
protein or functional RNA.
[1839] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[1840] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding a protein or the amount of a protein by modulating
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene. Modulation of alternative splicing or aberrant
splicing can be measured using any known method for analyzing the
sequence and length of RNA species, e.g., by RT-PCR and using
methods described elsewhere herein and in the literature. In
embodiments, modulation of alternative or aberrant splicing is
determined based on an increase or decrease in the amount of the
spliced species of interest of at least 10% or 1.1-fold. In
embodiments, modulation is determined based on an increase or
decrease at a level that is at least 10% to 100% or 1.1 to 10-fold,
as described herein regarding determining an increase in mRNA
encoding the protein in the methods of the invention.
[1841] In embodiments, the method is a method wherein the RIC
pre-mRNA was produced by partial splicing of a wild-type pre-mRNA.
In embodiments, the method is a method wherein the RIC pre-mRNA was
produced by partial splicing of a full-length wild-type pre-mRNA.
In embodiments, the RIC pre-mRNA was produced by partial splicing
of a full-length pre-mRNA. In these embodiments, a full-length
pre-mRNA may have a polymorphism in a splice site of the retained
intron that does not impair correct splicing of the retained intron
as compared to splicing of the retained intron having the wild-type
splice site sequence.
[1842] In embodiments, the mRNA encoding a protein is a full-length
mature mRNA, or a wild-type mature mRNA. In these embodiments, a
full-length mature mRNA may have a polymorphism that does not
affect the activity of the target protein or the functional RNA
encoded by the mature mRNA, as compared to the activity of the
protein encoded by the wild-type mature mRNA.
Antisense Oligomers
[1843] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of an RIC pre-mRNA. As used herein, the terms
"ASO" and "antisense oligomer" are used interchangeably and refer
to an oligomer such as a polynucleotide, comprising nucleobases,
that hybridizes to a target nucleic acid (e.g., an RIC pre-mRNA)
sequence by Watson-Crick base pairing or wobble base pairing (G-U).
The ASO may have exact sequence complementary to the target
sequence or near complementarity (e.g., sufficient complementarity
to bind the target sequence and enhancing splicing at a splice
site). ASOs are designed so that they bind (hybridize) to a target
nucleic acid (e.g., a targeted portion of a pre-mRNA transcript)
and remain hybridized under physiological conditions. Typically, if
they hybridize to a site other than the intended (targeted) nucleic
acid sequence, they hybridize to a limited number of sequences that
are not a target nucleic acid (to a few sites other than a target
nucleic acid). Design of an ASO can take into consideration the
occurrence of the nucleic acid sequence of the targeted portion of
the pre-mRNA transcript or a sufficiently similar nucleic acid
sequence in other locations in the genome or cellular pre-mRNA or
transcriptome, such that the likelihood the ASO will bind other
sites and cause "off-target" effects is limited. Any antisense
oligomers known in the art, for example in PCT Application No.
PCT/US2014/054151, published as WO 2015/035091, titled "Reducing
Nonsense-Mediated mRNA Decay," can be used to practice the methods
described herein.
[1844] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[1845] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[1846] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[1847] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Pat. Pub. No. 2012/0190728, and Dias and Stein,
Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by
reference in their entirety.
[1848] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine,
5,6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[1849] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081
(1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et
al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer
Drug Design 6:539 (1991); Zon et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphorothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[1850] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the invention comprises an ASO having
phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[1851] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention comprises about 5-100% Rp, at least about 5% Rp, at
least about 10% Rp, at least about 15% Rp, at least about 20% Rp,
at least about 25% Rp, at least about 30% Rp, at least about 35%
Rp, at least about 40% Rp, at least about 45% Rp, at least about
50% Rp, at least about 55% Rp, at least about 60% Rp, at least
about 65% Rp, at least about 70% Rp, at least about 75% Rp, at
least about 80% Rp, at least about 85% Rp, at least about 90% Rp,
or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
In embodiments, an ASO used in the methods of the invention
comprises about 10% to about 100% Rp, about 15% to about 100% Rp,
about 20% to about 100% Rp, about 25% to about 100% Rp, about 30%
to about 100% Rp, about 35% to about 100% Rp, about 40% to about
100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp,
about 55% to about 100% Rp, about 60% to about 100% Rp, about 65%
to about 100% Rp, about 70% to about 100% Rp, about 75% to about
100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp,
about 90% to about 100% Rp, or about 95% to about 100% Rp, about
20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about
70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp,
with the remainder Sp.
[1852] In embodiments, an ASO used in the methods of the invention
comprises about 5-100% Sp, at least about 5% Sp, at least about 10%
Sp, at least about 15% Sp, at least about 20% Sp, at least about
25% Sp, at least about 30% Sp, at least about 35% Sp, at least
about 40% Sp, at least about 45% Sp, at least about 50% Sp, at
least about 55% Sp, at least about 60% Sp, at least about 65% Sp,
at least about 70% Sp, at least about 75% Sp, at least about 80%
Sp, at least about 85% Sp, at least about 90% Sp, or at least about
95% Sp, with the remainder Rp, or about 100% Sp. In embodiments, an
ASO used in the methods of the invention comprises about 10% to
about 100% Sp, about 15% to about 100% Sp, about 20% to about 100%
Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about
35% to about 100% Sp, about 40% to about 100% Sp, about 45% to
about 100% Sp, about 50% to about 100% Sp, about 55% to about 100%
Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about
70% to about 100% Sp, about 75% to about 100% Sp, about 80% to
about 100% Sp, about 85% to about 100% Sp, about 90% to about 100%
Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about
25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about
60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
[1853] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[1854] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[1855] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[1856] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[1857] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[1858] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[1859] In other embodiments, the ASOs are complementary to (and
bind to) a targeted portion of an AMT, ADA, PPOX, UROD, HMBS,
ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1,
HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE,
ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is
downstream (in the 3' direction) of the 5' splice site of the
retained intron in an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD,
APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1,
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 RIC pre-mRNA (e.g., the direction designated
by positive numbers relative to the 5' splice site) (FIG. 1). In
some embodiments, the ASOs are complementary to a targeted portion
of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP,
THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7,
CERS2 or NCOA5 RIC pre-mRNA that is within the region +6 to +100
relative to the 5' splice site of the retained intron. In some
embodiments, the ASO is not complementary to nucleotides +1 to +5
relative to the 5' splice site (the first five nucleotides located
downstream of the 5' splice site). In some embodiments, the ASOs
may be complementary to a targeted portion of an AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA that is within the region between nucleotides +6 and +50
relative to the 5' splice site of the retained intron. In some
aspects, the ASOs are complementary to a targeted portion of the
AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1,
HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO,
PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or
NCOA5 RIC pre-mRNA that is within the region +6 to +500, +6 to
+400, +6 to +300, +6 to +200, or +6 to +90, +6 to +80, +6 to +70,
+6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20 relative
to 5' splice site of the retained intron.
[1860] In some embodiments, the ASOs are complementary to a
targeted region of an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD,
APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1,
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is upstream (5' relative)
of the 3' splice site of the retained intron in an AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA (e.g., in the direction designated by negative numbers)
(FIG. 1). In some embodiments, the ASOs are complementary to a
targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the
region -16 to -500, -16 to -400, -16 to -300, -6 to -200, or -16 to
-100 relative to the 3' splice site of the retained intron. In some
embodiments, the ASO is not complementary to nucleotides -1 to -15
relative to the 3' splice site (the first 15 nucleotides located
upstream of the 3' splice site). In some embodiments, the ASOs are
complementary to a targeted portion of the AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is
within the region -16 to -50 relative to the 3' splice site of the
retained intron. In some aspects, the ASOs are complementary to a
targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the
region -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50,
-16 to -40, or -16 to -30 relative to 3' splice site of the
retained intron.
[1861] In embodiments, the targeted portion of the AMT, ADA, PPOX,
UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK,
POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B,
FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA is within the region +100 relative to the 5' splice site
of the retained intron to -100 relative to the 3' splice site of
the retained intron.
[1862] In some embodiments, the ASOs are complementary to a
targeted portion of an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD,
APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1,
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the exon
flanking the 5' splice site (upstream) of the retained intron (FIG.
1). In some embodiments, the ASOs are complementary to a targeted
portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5,
GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1,
HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region +2e
to -4e in the exon flanking the 5' splice site of the retained
intron. In some embodiments, the ASOs are not complementary to
nucleotides -1e to -3e relative to the 5' splice site of the
retained intron. In some embodiments, the ASOs are complementary to
a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the
region -4e to -100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to
-60e, -4e to -50e, -4 to -40e, -4e to -30e, or -4e to -20e relative
to the 5' splice site of the retained intron.
[1863] In some embodiments, the ASOs are complementary to a
targeted portion of an RIC pre-mRNA that is within the exon
flanking the 3' splice site (downstream) of the retained intron
(FIG. 1). In some embodiments, the ASOs are complementary to a
targeted portion to the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A,
TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the
region +2e to -4e in the exon flanking the 3' splice site of the
retained intron. In some embodiments, the ASOs are not
complementary to nucleotide +1e relative to the 3' splice site of
the retained intron. In some embodiments, the ASOs are
complementary to a targeted portion of the AMT, ADA, PPOX, UROD,
HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1,
PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is
within the region +2e to +100e, +2e to +90e, +2e to +80e, +2e to
+70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2 to
+20e relative to the 3' splice site of the retained intron. The
ASOs may be of any length suitable for specific binding and
effective enhancement of splicing. In some embodiments, the ASOs
consist of 8 to 50 nucleobases. For example, the ASO may be 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in
length. In some embodiments, the ASOs consist of more than 50
nucleobases. In some embodiments, the ASO is from 8 to 50
nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30
nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15
nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35
nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20
nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25
nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50
nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15
nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35
nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20
nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40
nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25
nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40
nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25
nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40
nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25
nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40
nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25
nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35
nucleobases, or 25 to 30 nucleobases in length. In some
embodiments, the ASOs are 30 nucleotides in length. In some
embodiments, the ASOs are 29 nucleotides in length. In some
embodiments, the ASOs are 28 nucleotides in length. In some
embodiments, the ASOs are 27 nucleotides in length. In some
embodiments, the ASOs are 26 nucleotides in length. In some
embodiments, the ASOs are 25 nucleotides in length. In some
embodiments, the ASOs are 24 nucleotides in length. In some
embodiments, the ASOs are 23 nucleotides in length. In some
embodiments, the ASOs are 22 nucleotides in length. In some
embodiments, the ASOs are 21 nucleotides in length. In some
embodiments, the ASOs are 20 nucleotides in length. In some
embodiments, the ASOs are 19 nucleotides in length. In some
embodiments, the ASOs are 18 nucleotides in length. In some
embodiments, the ASOs are 17 nucleotides in length. In some
embodiments, the ASOs are 16 nucleotides in length. In some
embodiments, the ASOs are 15 nucleotides in length. In some
embodiments, the ASOs are 14 nucleotides in length. In some
embodiments, the ASOs are 13 nucleotides in length. In some
embodiments, the ASOs are 12 nucleotides in length. In some
embodiments, the ASOs are 11 nucleotides in length. In some
embodiments, the ASOs are 10 nucleotides in length.
[1864] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[1865] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[1866] In some embodiments, the nucleic acid to be targeted by an
ASO is an RIC pre-mRNA expressed in a cell, such as a eukaryotic
cell. In some embodiments, the term "cell" may refer to a
population of cells. In some embodiments, the cell is in a subject.
In some embodiments, the cell is isolated from a subject. In some
embodiments, the cell is ex vivo. In some embodiments, the cell is
a condition or disease-relevant cell or a cell line. In some
embodiments, the cell is in vitro (e.g., in cell culture).
Pharmaceutical Compositions
[1867] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent
or carrier.
[1868] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose. The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[1869] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[1870] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancers is a surfactant, fatty acid,
bile salt, chelating agent, or non-chelating nonsurfactant.
[1871] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[1872] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[1873] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[1874] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[1875] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[1876] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. Multiple tissues and organs can be affected in a
condition described herein, with the liver being the most
significantly affected tissue. The ASOs of the present invention
may be administered to patients parenterally, for example, by
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection. In embodiments, delivery is to
the heart or liver. In embodiments, a fetus is treated in utero,
e.g., by administering the ASO composition to the fetus directly or
indirectly (e.g., via the mother).
Methods of Identifying Additional ASOs that Enhance Splicing
[1877] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of an RIC pre-mRNA, specifically at the target intron. ASOs that
specifically hybridize to different nucleotides within the target
region of the pre-mRNA may be screened to identify (determine) ASOs
that improve the rate and/or extent of splicing of the target
intron. In some embodiments, the ASO may block or interfere with
the binding site(s) of a splicing repressor(s)/silencer. Any method
known in the art may be used to identify (determine) an ASO that
when hybridized to the target region of the intron results in the
desired effect (e.g., enhanced splicing, protein or functional RNA
production). These methods also can be used for identifying ASOs
that enhance splicing of the retained intron by binding to a
targeted region in an exon flanking the retained intron, or in a
non-retained intron. An example of a method that may be used is
provided below.
[1878] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site.
[1879] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1880] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[1881] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[1882] As described for the ASO walk above, the ASO micro-walk is
performed by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[1883] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. Following administration, the cells, tissues, and/or
organs of the model animals may be assessed to determine the effect
of the ASO treatment by for example evaluating splicing
(efficiency, rate, extent) and protein production by methods known
in the art and described herein. The animal models may also be any
phenotypic or behavioral indication of the disease or disease
severity.
Compositions and Methods for Treatment of Kidney Diseases
[1884] Kidney failure is a debilitating condition that is estimated
to affect more than 600,000 in the United States alone. In many
cases, renal dialysis is able to prolong life, with an estimated
400,000 patients receiving renal dialysis according to a 2009
study. Still, the only hope for many afflicted with kidney failure
is a kidney transplant, though the donor pool is only sufficient to
treat a small portion of those in need. Therefore, the odds of
receiving a transplant is low, and there are few treatments
available to ameliorate the condition for those unable to receive a
transplant. Therefore, there exists a need for compositions and
methods for treating kidney diseases.
[1885] Individual introns in primary transcripts of protein-coding
genes having more than one intron are spliced from the primary
transcript with different efficiencies. In most cases only the
fully spliced mRNA is exported through nuclear pores for subsequent
translation in the cytoplasm. Unspliced and partially spliced
transcripts are detectable in the nucleus. It is generally thought
that nuclear accumulation of transcripts that are not fully spliced
is a mechanism to prevent the accumulation of potentially
deleterious mRNAs in the cytoplasm that may be translated to
protein. For some genes, splicing of the least efficient intron is
a rate-limiting post-transcriptional step in gene expression, prior
to translation in the cytoplasm.
[1886] Substantial levels of partially-spliced transcripts of the
gene, which encodes a protein that is deficient in a subset of
kidney diseases, have been discovered in the nucleus of human
cells. These pre-mRNA species comprise at least one retained
intron. The present invention provides compositions and methods for
upregulating splicing of one or more retained introns that are
rate-limiting for the nuclear stages of gene expression to increase
steady-state production of fully-spliced, mature mRNA, and thus,
translated protein levels. These compositions and methods utilize
antisense oligomers (ASOs) that promote constitutive splicing at an
intron splice site of a retained-intron-containing pre-mRNA that
accumulates in the nucleus. Thus, in embodiments, protein is
increased using the methods of the invention to treat a condition
caused by a protein deficiency.
[1887] Kidney diseases that can be treated by the invention
described herein are diseases where a subject is deficient in a
gene product, where deficiency in a gene product causes the kidney
disease.
[1888] These CTNS, PAX2, CYP24A1 and PPARD pre-mRNA species
comprise at least one retained intron. The present invention
provides compositions and methods for upregulating splicing of one
or more retained CTNS, PAX2, CYP24A1 or PPARD introns that are
rate-limiting for the nuclear stages of gene expression to increase
steady-state production of fully-spliced, mature mRNA, and thus,
translated CTNS, PAX2, CYP24A1 or PPARD protein levels.
[1889] These compositions and methods can utilize antisense
oligomers (ASOs) that promote constitutive splicing at intron
splice sites of a retained-intron-containing CTNS, PAX2, CYP24A1 or
PPARD pre-mRNA (RIC pre-mRNA) that accumulates in the nucleus.
Thus, in embodiments, CTNS, PAX2, CYP24A1 or PPARD protein can be
increased using the methods of the invention to treat a condition
caused by CTNS, PAX2, CYP24A1 or PPARD deficiency.
[1890] In other embodiments, the methods of the invention can be
used to increase CTNS, PAX2, CYP24A1 or PPARD production to treat a
condition in a subject in need thereof. In embodiments, the subject
has a condition in which CTNS, PAX2, CYP24A1 or PPARD is not
necessarily deficient relative to wild-type, but where an increase
in CTNS, PAX2, CYP24A1 or PPARD mitigates the condition
nonetheless. In embodiments, the condition can be caused by a CTNS,
PAX2, CYP24A1 or PPARD haploinsufficiency.
Nephropathic Cystinosis/Late-Onset Cystinosis
[1891] In some embodiments, the invention described herein can be
used to treat the autosomal recessive kidney disorder infantile
nephropathic cystinosis. Infantile nephropathic cystinosis is
characterized by the accumulation of cysteine in lysosomes.
Clinical manifestations of infantile nephropathic cystinosis
include low blood sugar and electrolytes, excessive excretion of
proteins in the urine, slow body growth, weak bones,
hypothyroidism, blindness, muscle weakness, pulmonary dysfunction
and kidney failure. Typically infantile nephropathic cystinosis is
diagnosed in infancy.
[1892] In some embodiments, the invention described herein can be
used to treat the autosomal recessive kidney disorder late-onset
cystinosis. Late onset cystinosis has a similar clinical
manifestation as the infantile form, though the late onset form
typically manifests in either late adolescence or early
adulthood.
[1893] Deficiency in the protein cystinosin (CTNS) results in the
clinical manifestations shown in both infantile nephropathic
cystinosis and late-onset cystinosis. The CTNS gene, which is
located at 17q13.2 and spans 12 exons, codes for the CTNS protein.
Mutations in the CTNS gene resulting in deficient amounts of CTNS
protein have been shown to be responsible for the progression of
both infantile nephropathic cystinosis and late-onset cystinosis.
In one study, a G169D missense mutation was discovered in patients
with infantile nephropathic cystinosis. This missense mutation
resulted in diminished levels of CTNS, which provided a positive
link between deficiency in CTNS and the progression of infantile
nephropathic cystinosis. Focal Segmental Glomerulosclerosis
7/Papillorenal Syndrome
[1894] In some embodiments, the invention described herein can be
used to treat the autosomal dominant kidney disorder focal
segmental glomerulosclerosis 7 (FSGS7). FSGS7 is one of the leading
causes of kidney failure in adults. FSGS7 is characterized by
edema, hypoalbuminemia, hyperlipidemia and hypertension, which
ultimately results in kidney failure.
[1895] In some embodiments, the invention described herein can be
used to treat the autosomal dominant kidney disorder Papillorenal
syndrome (PAPRS). PAPRS is characterized by hypoplastic kidneys,
hypodysplasia, multicystic dysplastic kidney, oligomeganephronia,
renal insufficiency and vesicoureteral reflux.
[1896] Deficiency in the protein paired box gene 2 (PAX2) results
in the clinical manifestations shown in both FSGS7 and PAPRS. The
PAX2 gene, which is located at 10q24.31 and spans 12 exons, codes
for the PAX2 protein. Mutations in the PAX2 gene resulting in
deficient amounts of PAX2 protein have been shown to be responsible
for the progression of both FSGS7 and PAPRS. In one study, a G76S
missense mutation was discovered in 5 generations of a family
afflicted with PAPRS. This missense mutation resulted in diminished
levels of PAX2 protein, which provided a positive link between
deficiency in PAX2 protein and the progression of PAPRS.
Infantile Hypercalcemia
[1897] In some embodiments, the invention described herein can be
used to treat the autosomal recessive kidney disease infantile
hypercalcemia. Infantile hypercalcemia is characterized by elevated
calcium levels in the blood, which can result in renal stones, bone
pain, abdominal pain, nausea, vomiting, polyuria and psychiatric
conditions such as depression, anxiety, cognitive dysfunction,
insomnia and coma.
[1898] Deficiency in the protein cytochrome P450 family 24,
subfamily A, polypeptide 1 (CYP24A1) results in the clinical
manifestations shown in infantile hypercalcemia. The CYP24A1 gene,
which is located at 20q13.2 and spans 2 exons, codes for the
CYP24A1 protein. Mutations in the CYP24A1 gene resulting in
deficient amounts of CYP24A1 protein have been shown to be
responsible for the progression of infantile hypercalcemia. In one
study, a number of children diagnosed with infantile hypercalcemia
were examined. Children displaying an R396W or an E322K missense
mutation were shown to have complete ablation of CYP24A1 activity,
which children displaying an L409S mutation retained small, but
measurable levels. This finding provides a positive link between
diminished CYP24A1 protein and the clinical manifestations of
infantile hypercalcemia.
Lipid Metabolism Dysfunction and Chronic Kidney Disease
[1899] In some embodiments, the invention described herein can be
used to treat the lipid metabolism deficiency, chronic kidney
disease (CKD), end-stage renal disease (ESRD) or cardiovascular
disease (CVD) caused by deficiency in the protein peroxisome
proliferator activated receptor delta (PPARD). Deficiency in the
amount of PPARD protein, which is encoded by the PPARD gene located
on chromosome 6 and spans 8 exons, has been shown to be correlated
to increased lipid metabolism dysfunction.
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[1900] In embodiments, the methods of the present invention exploit
the presence of retained-intron-containing pre-mRNA (RIC pre-mRNA)
transcribed from a gene and encoding a protein that is found to be
deficient in a disease described herein, in the cell nucleus.
Splicing of the identified RIC pre-mRNA species to produce mature,
fully-spliced, mRNA, is induced using ASOs that stimulate splicing
out of the retained introns. The resulting mature mRNA can be
exported to the cytoplasm and translated, thereby increasing the
amount of protein in the patient's cells and alleviating symptoms
of a disease or condition described herein. In embodiments, the
methods of the present invention can exploit the presence of
retained-intron-containing pre-mRNA (RIC pre-mRNA) transcribed from
the CTNS, PAX2, CYP24A1 or PPARD gene and encoding CTNS, PAX2,
CYP24A1 or PPARD protein, in the cell nucleus. Splicing of CTNS,
PAX2, CYP24A1 or PPARD RIC pre-mRNA species to produce mature,
fully-spliced, CTNS, PAX2, CYP24A1 or PPARD mRNA, can be induced
using ASOs that stimulate splicing out of the retained introns. The
resulting mature CTNS, PAX2, CYP24A1 or PPARD mRNA can be exported
to the cytoplasm and translated, thereby increasing the amount of
CTNS, PAX2, CYP24A1 or PPARD protein in the patient's cells and
alleviating symptoms of the CNS disease or conditions caused by
deficiency in CTNS, PAX2, CYP24A1 or PPARD. This method, described
further below, is known as Targeted Augmentation of Nuclear Gene
Output (TANGO).
[1901] In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
retention. In embodiments, a retained intron is an intron that is
identified as a retained intron based on a determination of about
5% to about 100%, about 5% to about 95%, about 5% to about 90%,
about 5% to about 85%, about 5% to about 80%, about 5% to about
75%, about 5% to about 70%, about 5% to about 65%, about 5% to
about 60%, about 5% to about 65%, about 5% to about 60%, about 5%
to about 55%, about 5% to about 50%, about 5% to about 45%, about
5% to about 40%, about 5% to about 35%, about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 10% to about 100%, about 10% to about 95%, about 10% to
about 90%, about 10% to about 85%, about 10% to about 80%, about
10% to about 75%, about 10% to about 70%, about 10% to about 65%,
about 10% to about 60%, about 10% to about 65%, about 10% to about
60%, about 10% to about 55%, about 10% to about 50%, about 10% to
about 45%, about 10% to about 40%, about 10% to about 35%, about
10% to about 30%, about 10% to about 25%, about 10% to about 20%,
about 15% to about 100%, about 15% to about 95%, about 15% to about
90%, about 15% to about 85%, about 15% to about 80%, about 15% to
about 75%, about 15% to about 70%, about 15% to about 65%, about
15% to about 60%, about 15% to about 65%, about 15% to about 60%,
about 15% to about 55%, about 15% to about 50%, about 15% to about
45%, about 15% to about 40%, about 15% to about 35%, about 15% to
about 30%, about 15% to about 25%, about 20% to about 100%, about
20% to about 95%, about 20% to about 90%, about 20% to about 85%,
about 20% to about 80%, about 20% to about 75%, about 20% to about
70%, about 20% to about 65%, about 20% to about 60%, about 20% to
about 65%, about 20% to about 60%, about 20% to about 55%, about
20% to about 50%, about 20% to about 45%, about 20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about
100%, about 25% to about 95%, about 25% to about 90%, about 25% to
about 85%, about 25% to about 80%, about 25% to about 75%, about
25% to about 70%, about 25% to about 65%, about 25% to about 60%,
about 25% to about 65%, about 25% to about 60%, about 25% to about
55%, about 25% to about 50%, about 25% to about 45%, about 25% to
about 40%, or about 25% to about 35%, retention. In embodiments,
other ASOs useful for this purpose are identified, using, e.g.,
methods described herein.
[1902] In embodiments, the CTNS intron numbering corresponds to the
mRNA sequence at NM_001031681 or NM_001031681. In embodiments, the
targeted portion of the CTNS RIC pre-mRNA is in intron 9 and/or 10.
In embodiments, the targeted portion of the CTNS RIC pre-mRNA is in
intron 10. In embodiments, the percent retained intron can be 18%.
In embodiments, the targeted portion of the CTNS RIC pre-mRNA is in
intron 9. In embodiments, the percent retained intron can be 10%.
In embodiments, hybridization of an ASO to the targeted portion of
the RIC pre-mRNA results in enhanced splicing at the splice site
(5' splice site or 3' splice site) of retained intron 9 and/or 10
and subsequently increases CTNS protein production. It is
understood that the intron numbering may change in reference to a
different CTNS isoform sequence. One of skill in the art can
determine the corresponding intron number in any isoform based on
an intron sequence provided herein or using the number provided in
reference to the mRNA sequence at NM_001031681 or NM_001031681. One
of skill in the art also can determine the sequences of flanking
exons in any CTNS isoform for targeting using the methods of the
invention, based on an intron sequence provided herein or using the
intron number provided in reference to the mRNA sequence at
NM_001031681 or NM_001031681.
[1903] In embodiments, the PAX2 intron numbering corresponds to the
mRNA sequence at NM_001304569, NM_000278, NM_003990, NM_003988,
NM_003987 or NM_003989. In embodiments, the targeted portion of the
PAX2 RIC pre-mRNA is in intron 1 or 2. In embodiments,
hybridization of an ASO to the targeted portion of the RIC pre-mRNA
results in enhanced splicing at the splice site (5' splice site or
3' splice site) of retained intron 1 or 2 and subsequently
increases PAX2 protein production. It is understood that the intron
numbering may change in reference to a different PAX2 isoform
sequence. One of skill in the art can determine the corresponding
intron number in any isoform based on an intron sequence provided
herein or using the number provided in reference to the mRNA
sequence at NM_001304569, NM_000278, NM_003990, NM_003988,
NM_003987 or NM_003989. One of skill in the art also can determine
the sequences of flanking exons in any PAX2 isoform for targeting
using the methods of the invention, based on an intron sequence
provided herein or using the intron number provided in reference to
the mRNA sequence at NM_001304569, NM_000278, NM_003990, NM_003988,
NM_003987 or NM_003989.
[1904] In embodiments, the CYP24A1 intron numbering corresponds to
the mRNA sequence at NM_000782 or NM_001128915. In embodiments, the
targeted portion of the CYP24A1 RIC pre-mRNA is in intron 10 and/or
9 or 11. In embodiments, the targeted portion of the CYP24A1 RIC
pre-mRNA is in intron 9. In embodiments, the targeted portion of
the CYP24A1 RIC pre-mRNA is in intron 11. In embodiments, the
percent retained intron can be 23%. In embodiments, the targeted
portion of the CYP24A1 RIC pre-mRNA is in intron 10. In
embodiments, the percent retained intron can be 50%. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of retained intron 10 and/or 9 or 11
and subsequently increases CYP24A1 protein production. It is
understood that the intron numbering may change in reference to a
different CYP24A1 isoform sequence. One of skill in the art can
determine the corresponding intron number in any isoform based on
an intron sequence provided herein or using the number provided in
reference to the mRNA sequence at NM_000782 or NM_001128915. One of
skill in the art also can determine the sequences of flanking exons
in any CYP24A1 isoform for targeting using the methods of the
invention, based on an intron sequence provided herein or using the
intron number provided in reference to the mRNA sequence at
NM_000782 or NM_001128915.
[1905] In embodiments, the PPARD intron numbering corresponds to
the mRNA sequence at NM_006238, NM_177435, NM_001171818,
NM_001171819 or NM_001171820. In embodiments, the targeted portion
of the PPARD RIC pre-mRNA is in intron 3 or 4 or 5 or 6 or 7 and/or
2 or 3 or 4 or 5 or 6 or 7 or 8. In embodiments, the targeted
portion of the PPARD RIC pre-mRNA is in intron 2. In embodiments,
the targeted portion of the PPARD RIC pre-mRNA is in intron 3. In
embodiments, the targeted portion of the PPARD RIC pre-mRNA is in
intron 4. In embodiments, the targeted portion of the PPARD RIC
pre-mRNA is in intron 5. In embodiments, the targeted portion of
the PPARD RIC pre-mRNA is in intron 6. In embodiments, the targeted
portion of the PPARD RIC pre-mRNA is in intron 7. In embodiments,
the targeted portion of the PPARD RIC pre-mRNA is in intron 8. In
embodiments, hybridization of an ASO to the targeted portion of the
RIC pre-mRNA results in enhanced splicing at the splice site (5'
splice site or 3' splice site) of a retained intron subsequently
increases PPARD protein production. It is understood that the
intron numbering may change in reference to a different PPARD
isoform sequence. One of skill in the art can determine the
corresponding intron number in any isoform based on an intron
sequence provided herein or using the number provided in reference
to the mRNA sequence at NM_006238, NM_177435, NM_001171818,
NM_001171819 or NM_001171820. One of skill in the art also can
determine the sequences of flanking exons in any PPARD isoform for
targeting using the methods of the invention, based on an intron
sequence provided herein or using the intron number provided in
reference to the mRNA sequence at NM_006238, NM_177435,
NM_001171818, NM_001171819 or NM_001171820.
[1906] In some embodiments, the ASOs disclosed herein target a RIC
pre-mRNA transcribed from a CTNS, PAX2, CYP24A1 or PPARD genomic
sequence. In some embodiments, the ASOs disclosed herein target a
CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA sequence.
[1907] In some embodiments, the ASO targets a sequence of a RIC
pre-mRNA transcript encoded by a CTNS genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a
CTNS genomic sequence comprising retained intron 9 and/or 10. In
some embodiments, the ASO targets a RIC pre-mRNA encoded by SEQ ID
NO: 140007. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of SEQ ID NO: 140020 or 140021. In some embodiments, the
ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 140020 or
140021 comprising a retained intron 9 and/or 10. In some
embodiments, the ASOs target SEQ ID NO: 150752 and/or 150738. In
some embodiments, the ASO comprises a sequence of any one of SEQ ID
NOs: 140024-146956.
[1908] In some embodiments, the ASO targets a sequence of a RIC
pre-mRNA transcript encoded by a PAX2 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a
PAX2 genomic sequence comprising retained intron 1 or 2. In some
embodiments, the ASO targets a RIC pre-mRNA transcript encoded by
SEQ ID NO: 140006. In some embodiments, the ASO targets a RIC
pre-mRNA transcript of one or more of SEQ ID NO: 140014-140019. In
some embodiments, the ASO targets a RIC pre-mRNA transcript of one
or more of SEQ ID NO: 140014-140019 comprising a retained intron 1
or 2. In some embodiments, the ASOs target SEQ ID NO: 150744 and/or
150742. In some embodiments, the ASO comprises a sequence of any
one of SEQ ID NOs: 146957-148627.
[1909] In some embodiments, the ASO targets a sequence of a RIC
pre-mRNA transcript encoded by a CYP24A1 genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a
CYP24A1 genomic sequence comprising retained intron 10 and/or 9 or
11. In some embodiments, the ASO targets a RIC pre-mRNA transcript
encoded by SEQ ID NO: 140008. In some embodiments, the ASO targets
a RIC pre-mRNA transcript of SEQ ID NO: 18 or 19. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID
NO: 140022 or 140023 comprising a retained intron 10 and/or 9 or
11. In some embodiments, the ASOs target SEQ ID NOs: 150752,
150738, 150750, 150749 and/or 150745. In some embodiments, the ASO
comprises a sequence of any one of SEQ ID NOs: 149524-150737.
[1910] In some embodiments, the ASO targets a sequence of a RIC
pre-mRNA transcript encoded by a PPARD genomic sequence. In some
embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a
PPARD genomic sequence comprising retained intron 3 or 4 or 5 or 6
or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8. In some embodiments,
the ASO targets a RIC pre-mRNA transcript encoded by SEQ ID NO:
140005. In some embodiments, the ASO targets a RIC pre-mRNA
transcript of one or more of SEQ ID NO: 140009-140013. In some
embodiments, the ASO targets a RIC pre-mRNA transcript of one or
more of SEQ ID NO: 140009-140013 comprising a retained intron 3 or
4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8. In some
embodiments, the ASOs target SEQ ID NO 150740, 150751, 150743,
150747, 150746, 150741, 150739, and/or 150748. In some embodiments,
the ASO comprises a sequence of any one of SEQ ID NOs:
140024-146956.
[1911] In some embodiments, the ASO targets exon 9 and/or 10 of a
CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10. In some
embodiments, the ASO targets an exon 9 and/or 10 sequence upstream
(or 5') from the 5' splice site of a CTNS RIC pre-mRNA comprising a
retained intron 9 and/or 10.
[1912] In some embodiments, the ASO targets intron 9 and/or 10 in a
CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10. In some
embodiments, the ASO targets an intron 9 and/or 10 sequence
downstream (or 3') from the 5' splice site of a CTNS RIC pre-mRNA
comprising a retained intron 9 and/or 10.
[1913] In some embodiments, the ASO targets an intron 9 and/or 10
sequence upstream (or 5') from the 3' splice site of a CTNS RIC
pre-mRNA comprising a retained intron 9 and/or 10.
[1914] In some embodiments, the ASO targets exon 10 and/or 11 in a
CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10. In some
embodiments, the ASO targets an exon 10 and/or 11 sequence
downstream (or 3') from the 3' splice site of a CTNS RIC pre-mRNA
comprising a retained intron 9 and/or 10.
[1915] In some embodiments, the ASO targets exon 1 or 2 of a PAX2
RIC pre-mRNA comprising a retained intron 1 or 2. In some
embodiments, the ASO targets an exon 1 or 2 sequence upstream (or
5') from the 5' splice site of a PAX2 RIC pre-mRNA comprising a
retained intron 1 or 2.
[1916] In some embodiments, the ASO targets intron 1 or 2 in a PAX2
RIC pre-mRNA comprising a retained intron 1 or 2. In some
embodiments, the ASO targets an intron 1 or 2 sequence downstream
(or 3') from the 5' splice site of a PAX2 RIC pre-mRNA comprising a
retained intron 1 or 2.
[1917] In some embodiments, the ASO targets an intron 1 or 2
sequence upstream (or 5') from the 3' splice site of a PAX2 RIC
pre-mRNA comprising a retained intron 1 or 2.
[1918] In some embodiments, the ASO targets exon 2 or 3 in a PAX2
RIC pre-mRNA comprising a retained intron 1 or 2. In some
embodiments, the ASO targets an exon 2 or 3 sequence downstream (or
3') from the 3' splice site of a PAX2 RIC pre-mRNA comprising a
retained intron 1 or 2.
[1919] In some embodiments, the ASO targets exon 10 and/or 9 or 11
of a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9
or 11. In some embodiments, the ASO targets an exon 10 and/or 9 or
11 sequence upstream (or 5') from the 5' splice site of a CYP24A1
RIC pre-mRNA comprising a retained intron 10 and/or 9 or 11.
[1920] In some embodiments, the ASO targets intron 10 and/or 9 or
11 in a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or
9 or 11. In some embodiments, the ASO targets an intron 10 and/or 9
or 11 sequence downstream (or 3') from the 5' splice site of a
CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9 or
11.
[1921] In some embodiments, the ASO targets an intron 10 and/or 9
or 11 sequence upstream (or 5') from the 3' splice site of a
CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9 or
11.
[1922] In some embodiments, the ASO targets exon 11 and/or 10 or 12
in a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9
or 11. In some embodiments, the ASO targets an exon 11 and/or 10 or
12 sequence downstream (or 3') from the 3' splice site of a CYP24A1
RIC pre-mRNA comprising a retained intron 10 and/or 9 or 11.
[1923] In some embodiments, the ASO targets exon 3 or 4 or 5 or 6
or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 of a PPARD RIC pre-mRNA
comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or
4 or 5 or 6 or 7 or 8. In some embodiments, the ASO targets an exon
3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8
sequence upstream (or 5') from the 5' splice site of a PPARD RIC
pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or
2 or 3 or 4 or 5 or 6 or 7 or 8.
[1924] In some embodiments, the ASO targets intron 3 or 4 or 5 or 6
or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 in a PPARD RIC pre-mRNA
comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or
4 or 5 or 6 or 7 or 8. In some embodiments, the ASO targets an
intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8
sequence downstream (or 3') from the 5' splice site of a PPARD RIC
pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or
2 or 3 or 4 or 5 or 6 or 7 or 8.
[1925] In some embodiments, the ASO targets an intron 3 or 4 or 5
or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 sequence upstream
(or 5') from the 3' splice site of a PPARD RIC pre-mRNA comprising
a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or
6 or 7 or 8.
[1926] In some embodiments, the ASO targets exon 4 or 5 or 6 or 7
or 8 and/or 3 or 4 or 5 or 6 or 7 or 8 or 9 in a PPARD RIC pre-mRNA
comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or
4 or 5 or 6 or 7 or 8. In some embodiments, the ASO targets an exon
4 or 5 or 6 or 7 or 8 and/or 3 or 4 or 5 or 6 or 7 or 8 or 9
sequence downstream (or 3') from the 3' splice site of a PPARD RIC
pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or
2 or 3 or 4 or 5 or 6 or 7 or 8.
Protein Expression
[1927] In embodiments, the methods described herein are used to
increase the production of a functional protein. As used herein,
the term "functional" refers to the amount of activity or function
of a protein that is necessary to eliminate any one or more
symptoms of a treated condition. In embodiments, the methods are
used to increase the production of a partially functional protein.
As used herein, the term "partially functional" refers to any
amount of activity or function of the protein that is less than the
amount of activity or function that is necessary to eliminate or
prevent any one or more symptoms of a disease or condition. In some
embodiments, a partially functional protein or RNA will have at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 75%, at least 80%, 85%, at
least 90%, or at least 95% less activity relative to the fully
functional protein or RNA.
[1928] In embodiments, the method is a method of increasing the
expression of the protein by cells of a subject having a RIC
pre-mRNA encoding the protein, wherein the subject has a condition
described herein caused by a deficient amount of activity of a
protein described herein. In some embodiments, the deficient amount
of the protein is caused by haploinsufficiency of the protein. In
such an embodiment, the subject has a first allele encoding a
functional protein, and a second allele from which the protein is
not produced. In another such embodiment, the subject has a first
allele encoding a functional protein, and a second allele encoding
a nonfunctional protein. In another such embodiment, the subject
has a first allele encoding a functional protein, and a second
allele encoding a partially functional protein. In any of these
embodiments, the antisense oligomer binds to a targeted portion of
the RIC pre-mRNA transcribed from the first allele (encoding
functional protein), thereby inducing constitutive splicing of the
retained intron from the RIC pre-mRNA, and causing an increase in
the level of mature mRNA encoding functional protein, and an
increase in the expression of the protein in the cells of the
subject.
[1929] In embodiments, the subject has a first allele encoding a
functional protein, and a second allele encoding a partially
functional protein, and the antisense oligomer binds to a targeted
portion of the RIC pre-mRNA transcribed from the first allele or a
targeted portion of the RIC pre-mRNA transcribed from the second
allele (encoding partially functional protein), thereby inducing
constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in the level of mature mRNA encoding the
protein, and an increase in the expression of functional or
partially functional protein in the cells of the subject.
[1930] In related embodiments, the method is a method of using an
ASO to increase the expression of a protein or functional RNA. In
embodiments, an ASO is used to increase the expression of a protein
described herein in cells of a subject having a RIC pre-mRNA
encoding the protein, wherein the subject has a deficiency in the
amount or function of the protein.
[1931] In embodiments, the RIC pre-mRNA transcript that encodes the
protein that is causative of the disease or condition is targeted
by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the
disease is targeted by the ASOs. For example, a disease that is the
result of a mutation or deficiency of a first protein in a
particular pathway may be ameliorated by targeting a RIC pre-mRNA
that encodes a second protein, thereby increasing production of the
second protein. In some embodiments, the function of the second
protein is able to compensate for the mutation or deficiency of the
first protein (which is causative of the disease or condition).
[1932] In embodiments, the subject has:
[1933] a. a first mutant allele from which [1934] i) the protein is
produced at a reduced level compared to production from a wild-type
allele, [1935] ii) the protein is produced in a form having reduced
function compared to an equivalent wild-type protein, or [1936]
iii) the protein or functional RNA is not produced; and
[1937] b. a second mutant allele from which [1938] i) the protein
is produced at a reduced level compared to production from a
wild-type allele, [1939] ii) the protein is produced in a form
having reduced function compared to an equivalent wild-type
protein, or [1940] iii) the protein is not produced, and wherein
the RIC pre-mRNA is transcribed from the first allele and/or the
second allele. In these embodiments, the ASO binds to a targeted
portion of the RIC pre-mRNA transcribed from the first allele or
the second allele, thereby inducing constitutive splicing of the
retained intron from the RIC pre-mRNA, and causing an increase in
the level of mRNA encoding a protein and an increase in the
expression of the target protein or functional RNA in the cells of
the subject. In these embodiments, the target protein or functional
RNA having an increase in expression level resulting from the
constitutive splicing of the retained intron from the RIC pre-mRNA
is either in a form having reduced function compared to the
equivalent wild-type protein (partially-functional), or having full
function compared to the equivalent wild-type protein
(fully-functional).
[1941] In embodiments, the level of mRNA encoding a protein
described herein is increased 1.1 to 10-fold, when compared to the
amount of mRNA encoding the protein that is produced in a control
cell, e.g., one that is not treated with the antisense oligomer or
one that is treated with an antisense oligomer that does not bind
to the targeted portion of the RIC pre-mRNA.
[1942] In embodiments, the condition caused by a deficient amount
or activity of a protein is not a condition caused by alternative
or aberrant splicing of the retained intron to which the ASO is
targeted. In embodiments, the condition caused by a deficient
amount or activity of the protein is not a condition caused by
alternative or aberrant splicing of any retained intron in a RIC
pre-mRNA encoding the protein. In embodiments, alternative or
aberrant splicing may occur in a pre-mRNA transcribed from the
gene, however the compositions and methods of the invention do not
prevent or correct this alternative or aberrant splicing.
[1943] In embodiments, a subject treated using the methods of the
invention expresses a partially functional protein from one allele,
wherein the partially functional protein is caused by a frameshift
mutation, a nonsense mutation, a missense mutation, or a partial
gene deletion. In embodiments, a subject treated using the methods
of the invention expresses a nonfunctional protein from one allele,
wherein the nonfunctional protein is caused by a frameshift
mutation, a nonsense mutation, a missense mutation, a partial gene
deletion, in one allele. In embodiments, a subject treated using
the methods of the invention has a whole gene deletion, in one
allele.
Use of TANGO for Increasing Protein Expression
[1944] As described above, in embodiments, Targeted Augmentation of
Nuclear Gene Output (TANGO) is used in the methods of the invention
to increase expression of a protein. In these embodiments, a
retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding a
protein is present in the nucleus of a cell. Cells having a RIC
pre-mRNA that comprises a retained intron, an exon flanking the 5'
splice site, and an exon flanking the 3' splice site, encoding the
protein, are contacted with antisense oligomers (ASOs) that are
complementary to a targeted portion of the RIC pre-mRNA.
Hybridization of the ASOs to the targeted portion of the RIC
pre-mRNA results in enhanced splicing at the splice site (5' splice
site or 3' splice site) of the retained intron and subsequently
increases target protein production.
[1945] The terms "pre-mRNA," and "pre-mRNA transcript" may be used
interchangeably and refer to any pre-mRNA species that contains at
least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts
comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In
embodiments, pre-mRNA or pre-mRNA transcripts comprise both a
5'-7-methylguanosine cap and a poly-A tail. In some embodiments,
the pre-mRNA transcript does not comprise a 5'-7-methylguanosine
cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive
messenger RNA (mRNA) molecule if it is not translated into a
protein (or transported into the cytoplasm from the nucleus).
[1946] As used herein, a "retained-intron-containing pre-mRNA"
("RIC pre-mRNA") is a pre-mRNA transcript that contains at least
one retained intron. The RIC pre-mRNA contains a retained intron,
an exon flanking the 5' splice site of the retained intron, an exon
flanking the 3' splice site of the retained intron, and encodes the
target protein. An "RIC pre-mRNA encoding a target protein" is
understood to encode the target protein when fully spliced. A
"retained intron" is any intron that is present in a pre-mRNA
transcript when one or more other introns, such as an adjacent
intron, encoded by the same gene have been spliced out of the same
pre-mRNA transcript. In some embodiments, the retained intron is
the most abundant intron in RIC pre-mRNA encoding the target
protein. In embodiments, the retained intron is the most abundant
intron in a population of RIC pre-mRNAs transcribed from the gene
encoding the target protein in a cell, wherein the population of
RIC pre-mRNAs comprises two or more retained introns. In
embodiments, an antisense oligomer targeted to the most abundant
intron in the population of RIC pre-mRNAs encoding the target
protein induces splicing out of two or more retained introns in the
population, including the retained intron to which the antisense
oligomer is targeted or binds. In embodiments, a mature mRNA
encoding the target protein is thereby produced. The terms "mature
mRNA," and "fully-spliced mRNA," are used interchangeably herein to
describe a fully processed mRNA encoding a target protein (e.g.,
mRNA that is exported from the nucleus into the cytoplasm and
translated into target protein) or a fully processed functional
RNA. The term "productive mRNA," also can be used to describe a
fully processed mRNA encoding a target protein. In embodiments, the
targeted region is in a retained intron that is the most abundant
intron in a RIC pre-mRNA encoding the protein.
[1947] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited feature (e.g. in the case of an antisense
oligomer, a defined nucleobase sequence) but not the exclusion of
any other features. Thus, as used herein, the term "comprising" is
inclusive and does not exclude additional, unrecited features (e.g.
in the case of an antisense oligomer, the presence of additional,
unrecited nucleobases).
[1948] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of" The phrase "consisting
essentially of" is used herein to require the specified feature(s)
(e.g. nucleobase sequence) as well as those which do not materially
affect the character or function of the claimed invention. As used
herein, the term "consisting" is used to indicate the presence of
the recited feature (e.g. nucleobase sequence) alone (so that in
the case of an antisense oligomer consisting of a specified
nucleobase sequence, the presence of additional, unrecited
nucleobases is excluded).
[1949] In embodiments, the targeted region is in a retained intron
that is the second most abundant intron in a RIC pre-mRNA encoding
a protein described herein. For example, the second most abundant
retained intron may be targeted rather than the most abundant
retained intron due to the uniqueness of the nucleotide sequence of
the second most abundant retained intron, ease of ASO design to
target a particular nucleotide sequence, and/or amount of increase
in protein production resulting from targeting the intron with an
ASO. In embodiments, the retained intron is the second most
abundant intron in a population of RIC pre-mRNAs transcribed from
the gene encoding the target protein in a cell, wherein the
population of RIC pre-mRNAs comprises two or more retained introns.
In embodiments, an antisense oligomer targeted to the second most
abundant intron in the population of RIC pre-mRNAs encoding the
target protein induces splicing out of two or more retained introns
in the population, including the retained intron to which the
antisense oligomer is targeted or binds. In embodiments,
fully-spliced (mature) RNA encoding the target protein is thereby
produced.
[1950] In embodiments, an ASO is complementary to a targeted region
that is within a non-retained intron in a RIC pre-mRNA. In
embodiments, the targeted portion of the RIC pre-mRNA is within:
the region +6 to +100 relative to the 5' splice site of the
non-retained intron; or the region -16 to -100 relative to the 3'
splice site of the non-retained intron. In embodiments, the
targeted portion of the RIC pre-mRNA is within the region +100
relative to the 5' splice site of the non-retained intron to -100
relative to the 3' splice site of the non-retained intron. As used
to identify the location of a region or sequence, "within" is
understood to include the residues at the positions recited. For
example, a region +6 to +100 includes the residues at positions +6
and +100. In embodiments, fully-spliced (mature) RNA encoding the
target protein is thereby produced.
[1951] In embodiments, the retained intron of the RIC pre-mRNA is
an inefficiently spliced intron. As used herein, "inefficiently
spliced" may refer to a relatively low frequency of splicing at a
splice site adjacent to the retained intron (5' splice site or 3'
splice site) as compared to the frequency of splicing at another
splice site in the RIC pre-mRNA. The term "inefficiently spliced"
may also refer to the relative rate or kinetics of splicing at a
splice site, in which an "inefficiently spliced" intron may be
spliced or removed at a slower rate as compared to another intron
in a RIC pre-mRNA.
[1952] In embodiments, the 9-nucleotide sequence at -3e to -1e of
the exon flanking the 5' splice site and +1 to +6 of the retained
intron is identical to the corresponding wild-type sequence. In
embodiments, the 16 nucleotide sequence at -15 to -1 of the
retained intron and +1e of the exon flanking the 3' splice site is
identical to the corresponding wild-type sequence. As used herein,
the "wild-type sequence" refers to the nucleotide sequence for a
gene in the published reference genome deposited in the NCBI
repository of biological and scientific information (operated by
National Center for Biotechnology Information, National Library of
Medicine, 8600 Rockville Pike, Bethesda, Md. USA 20894). Also used
herein, a nucleotide position denoted with an "e" indicates the
nucleotide is present in the sequence of an exon (e.g., the exon
flanking the 5' splice site or the exon flanking the 3' splice
site).
[1953] The methods involve contacting cells with an ASO that is
complementary to a portion of a pre-mRNA encoding a protein
described herein, resulting in increased expression of the protein.
As used herein, "contacting" or administering to cells refers to
any method of providing an ASO in immediate proximity with the
cells such that the ASO and the cells interact. A cell that is
contacted with an ASO will take up or transport the ASO into the
cell. The method involves contacting a condition or
disease-associated or condition or disease-relevant cell with an
ASO. In some embodiments, the ASO may be further modified or
attached (e.g., covalently attached) to another molecule to target
the ASO to a cell type, enhance contact between the ASO and the
condition or disease-associated or condition or disease-relevant
cell, or enhance uptake of the ASO.
[1954] As used herein, the term "increasing protein production" or
"increasing expression of a target protein" means enhancing the
amount of protein that is translated from an mRNA in a cell. A
"target protein" may be any protein for which increased
expression/production is desired.
[1955] In embodiments, contacting a cell that expresses a RIC
pre-mRNA with an ASO that is complementary to a targeted portion of
the RIC pre-mRNA transcript results in a measurable increase in the
amount of a protein (e.g., a target protein) encoded by the
pre-mRNA. Methods of measuring or detecting production of a protein
will be evident to one of skill in the art and include any known
method, for example, Western blotting, flow cytometry,
immunofluorescence microscopy, and ELISA.
[1956] In embodiments, contacting cells with an ASO that is
complementary to a targeted portion of an RIC pre-mRNA transcript
results in an increase in the amount of a protein produced by at
least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350,
400, 450, 500, or 1000%, compared to the amount of the protein
produced by a cell in the absence of the ASO/absence of treatment.
In embodiments, the total amount of a protein produced by the cell
to which the antisense oligomer was contacted is increased about
1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about
10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about
1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about
7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about
2 to about 5-fold, about 2 to about 6-fold, about 2 to about
7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3
to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about
8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least
about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at
least about 3-fold, at least about 3.5-fold, at least about 4-fold,
at least about 5-fold, or at least about 10-fold, compared to the
amount of target protein produced by an control compound. A control
compound can be, for example, an oligonucleotide that is not
complementary to the targeted portion of the RIC pre-mRNA.
[1957] In some embodiments, contacting cells with an ASO that is
complementary to a targeted portion of an RIC pre-mRNA transcript
results in an increase in the amount of mRNA encoding a protein,
including the mature mRNA encoding the target protein. In some
embodiments, the amount of mRNA encoding a protein, or the mature
mRNA encoding the protein, is increased by at least 10, 20, 30, 40,
50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%,
compared to the amount of the protein produced by a cell in the
absence of the ASO/absence of treatment. In embodiments, the total
amount of the mRNA encoding a protein, or the mature mRNA encoding
a protein produced in the cell to which the antisense oligomer was
contacted is increased about 1.1 to about 10-fold, about 1.5 to
about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold,
about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to
about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold,
about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to
about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold,
about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about
7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4
to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold,
at least about 1.1-fold, at least about 1.5-fold, at least about
2-fold, at least about 2.5-fold, at least about 3-fold, at least
about 3.5-fold, at least about 4-fold, at least about 5-fold, or at
least about 10-fold compared to the amount of mature RNA produced
in an untreated cell, e.g., an untreated cell or a cell treated
with a control compound. A control compound can be, for example, an
oligonucleotide that is not complementary to the targeted portion
of the RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC Pre-mRNA
[1958] The methods and antisense oligonucleotide compositions
provided herein are useful for increasing the expression of a
protein in cells, for example, in a subject having a condition
described herein caused by a deficiency in the amount or activity
of a protein described herein, by increasing the level of mRNA
encoding the protein, or the mature mRNA encoding the protein. In
particular, the methods and compositions as described herein induce
the constitutive splicing of a retained intron from an RIC pre-mRNA
transcript encoding the protein, thereby increasing the level of
mRNA encoding the protein, or the mature mRNA encoding the protein
and increasing the expression of the protein.
[1959] Constitutive splicing of a retained intron from a RIC
pre-mRNA correctly removes the retained intron from the RIC
pre-mRNA, wherein the retained intron has wild-type splice
sequences. Constitutive splicing, as used herein, does not
encompass splicing of a retained intron from a RIC pre-mRNA
transcribed from a gene or allele having a mutation that causes
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene or allele. For example, constitutive splicing of a
retained intron, as induced using the methods and antisense
oligonucleotides provided herein, does not correct aberrant
splicing in or influence alternative splicing of a pre-mRNA to
result in an increased expression of a target protein or functional
RNA.
[1960] In embodiments, constitutive splicing correctly removes a
retained intron from an RIC pre-mRNA, wherein the RIC pre-mRNA is
transcribed from a wild-type gene or allele, or a polymorphic gene
or allele, that encodes a fully-functional target protein or
functional RNA, and wherein the gene or allele does not have a
mutation that causes alternative splicing or aberrant splicing of
the retained intron.
[1961] In some embodiments, constitutive splicing of a retained
intron from an RIC pre-mRNA encoding a protein correctly removes a
retained intron from an RIC pre-mRNA encoding the protein, wherein
the RIC pre-mRNA is transcribed from a gene or allele from which
the target gene or functional RNA is produced at a reduced level
compared to production from a wild-type allele, and wherein the
gene or allele does not have a mutation that causes alternative
splicing or aberrant splicing of the retained intron. In these
embodiments, the correct removal of the constitutively spliced
retained intron results in production of target protein or
functional RNA that is functional when compared to an equivalent
wild-type protein or functional RNA.
[1962] In other embodiments, constitutive splicing correctly
removes a retained intron from an RIC pre-mRNA, wherein the RIC
pre-mRNA is transcribed from a gene or allele that encodes a target
protein or functional RNA produced in a form having reduced
function compared to an equivalent wild-type protein or functional
RNA, and wherein the gene or allele does not have a mutation that
causes alternative splicing or aberrant splicing of the retained
intron. In these embodiments, the correct removal of the
constitutively spliced retained intron results in production of
partially functional target protein, or functional RNA that is
partially functional when compared to an equivalent wild-type
protein or functional RNA.
[1963] "Correct removal" of the retained intron by constitutive
splicing refers to removal of the entire intron, without removal of
any part of an exon.
[1964] In embodiments, an antisense oligomer as described herein or
used in any method described herein does not increase the amount of
mRNA encoding a protein or the amount of a protein by modulating
alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the gene. Modulation of alternative splicing or aberrant
splicing can be measured using any known method for analyzing the
sequence and length of RNA species, e.g., by RT-PCR and using
methods described elsewhere herein and in the literature. In
embodiments, modulation of alternative or aberrant splicing is
determined based on an increase or decrease in the amount of the
spliced species of interest of at least 10% or 1.1-fold. In
embodiments, modulation is determined based on an increase or
decrease at a level that is at least 10% to 100% or 1.1 to 10-fold,
as described herein regarding determining an increase in mRNA
encoding the protein in the methods of the invention.
[1965] In embodiments, the method is a method wherein the RIC
pre-mRNA was produced by partial splicing of a wild-type pre-mRNA.
In embodiments, the method is a method wherein the RIC pre-mRNA was
produced by partial splicing of a full-length wild-type pre-mRNA.
In embodiments, the RIC pre-mRNA was produced by partial splicing
of a full-length pre-mRNA. In these embodiments, a full-length
pre-mRNA may have a polymorphism in a splice site of the retained
intron that does not impair correct splicing of the retained intron
as compared to splicing of the retained intron having the wild-type
splice site sequence.
[1966] In embodiments, the mRNA encoding a protein is a full-length
mature mRNA, or a wild-type mature mRNA. In these embodiments, a
full-length mature mRNA may have a polymorphism that does not
affect the activity of the target protein or the functional RNA
encoded by the mature mRNA, as compared to the activity of the
protein encoded by the wild-type mature mRNA.
Antisense Oligomers
[1967] One aspect of the present disclosure is a composition
comprising antisense oligomers that enhances splicing by binding to
a targeted portion of an RIC pre-mRNA. As used herein, the terms
"ASO" and "antisense oligomer" are used interchangeably and refer
to an oligomer such as a polynucleotide, comprising nucleobases,
that hybridizes to a target nucleic acid (e.g., an RIC pre-mRNA)
sequence by Watson-Crick base pairing or wobble base pairing (G-U).
The ASO may have exact sequence complementary to the target
sequence or near complementarity (e.g., sufficient complementarity
to bind the target sequence and enhancing splicing at a splice
site). ASOs are designed so that they bind (hybridize) to a target
nucleic acid (e.g., a targeted portion of a pre-mRNA transcript)
and remain hybridized under physiological conditions. Typically, if
they hybridize to a site other than the intended (targeted) nucleic
acid sequence, they hybridize to a limited number of sequences that
are not a target nucleic acid (to a few sites other than a target
nucleic acid). Design of an ASO can take into consideration the
occurrence of the nucleic acid sequence of the targeted portion of
the pre-mRNA transcript or a sufficiently similar nucleic acid
sequence in other locations in the genome or cellular pre-mRNA or
transcriptome, such that the likelihood the ASO will bind other
sites and cause "off-target" effects is limited. Any antisense
oligomers known in the art, for example in PCT Application No.
PCT/US2014/054151, published as WO 2015/035091, titled "Reducing
Nonsense-Mediated mRNA Decay," can be used to practice the methods
described herein.
[1968] In some embodiments, ASOs "specifically hybridize" to or are
"specific" to a target nucleic acid or a targeted portion of a RIC
pre-mRNA. Typically such hybridization occurs with a Tm
substantially greater than 37.degree. C., preferably at least
50.degree. C., and typically between 60.degree. C. to approximately
90.degree. C. Such hybridization preferably corresponds to
stringent hybridization conditions. At a given ionic strength and
pH, the Tm is the temperature at which 50% of a target sequence
hybridizes to a complementary oligonucleotide.
[1969] Oligomers, such as oligonucleotides, are "complementary" to
one another when hybridization occurs in an antiparallel
configuration between two single-stranded polynucleotides. A
double-stranded polynucleotide can be "complementary" to another
polynucleotide, if hybridization can occur between one of the
strands of the first polynucleotide and the second. Complementarity
(the degree to which one polynucleotide is complementary with
another) is quantifiable in terms of the proportion (e.g., the
percentage) of bases in opposing strands that are expected to form
hydrogen bonds with each other, according to generally accepted
base-pairing rules. The sequence of an antisense oligomer (ASO)
need not be 100% complementary to that of its target nucleic acid
to hybridize. In certain embodiments, ASOs can comprise at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence complementarity to a target region within the target
nucleic acid sequence to which they are targeted. For example, an
ASO in which 18 of 20 nucleobases of the oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered together or interspersed with complementary nucleobases
and need not be contiguous to each other or to complementary
nucleobases. Percent complementarity of an ASO with a region of a
target nucleic acid can be determined routinely using BLAST
programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[1970] An ASO need not hybridize to all nucleobases in a target
sequence and the nucleobases to which it does hybridize may be
contiguous or noncontiguous. ASOs may hybridize over one or more
segments of a pre-mRNA transcript, such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure or hairpin structure may be formed). In
certain embodiments, an ASO hybridizes to noncontiguous nucleobases
in a target pre-mRNA transcript. For example, an ASO can hybridize
to nucleobases in a pre-mRNA transcript that are separated by one
or more nucleobase(s) to which the ASO does not hybridize.
[1971] The ASOs described herein comprise nucleobases that are
complementary to nucleobases present in a target portion of a RIC
pre-mRNA. The term ASO embodies oligonucleotides and any other
oligomeric molecule that comprises nucleobases capable of
hybridizing to a complementary nucleobase on a target mRNA but does
not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
The ASOs may comprise naturally-occurring nucleotides, nucleotide
analogs, modified nucleotides, or any combination of two or three
of the preceding. The term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" includes nucleotides with modified or
substituted sugar groups and/or having a modified backbone. In some
embodiments, all of the nucleotides of the ASO are modified
nucleotides. Chemical modifications of ASOs or components of ASOs
that are compatible with the methods and compositions described
herein will be evident to one of skill in the art and can be found,
for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No.
5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and
Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by
reference in their entirety.
[1972] The nucleobase of an ASO may be any naturally occurring,
unmodified nucleobase such as adenine, guanine, cytosine, thymine
and uracil, or any synthetic or modified nucleobase that is
sufficiently similar to an unmodified nucleobase such that it is
capable of hydrogen bonding with a nucleobase present on a target
pre-mRNA. Examples of modified nucleobases include, without
limitation, hypoxanthine, xanthine, 7-methylguanine,
5,6-dihydrouracil, 5-methylcytosine, and
5-hydroxymethoylcytosine.
[1973] The ASOs described herein also comprise a backbone structure
that connects the components of an oligomer. The term "backbone
structure" and "oligomer linkages" may be used interchangeably and
refer to the connection between monomers of the ASO. In naturally
occurring oligonucleotides, the backbone comprises a 3'-5'
phosphodiester linkage connecting sugar moieties of the oligomer.
The backbone structure or oligomer linkages of the ASOs described
herein may include (but are not limited to) phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the
like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081
(1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et
al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer
Drug Design 6:539 (1991); Zon et al., Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed.,
Oxford University Press, Oxford England (1991)); Stec et al., U.S.
Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543
(1990). In some embodiments, the backbone structure of the ASO does
not contain phosphorous but rather contains peptide bonds, for
example in a peptide nucleic acid (PNA), or linking groups
including carbamate, amides, and linear and cyclic hydrocarbon
groups. In some embodiments, the backbone modification is a
phosphorothioate linkage. In some embodiments, the backbone
modification is a phosphoramidate linkage.
[1974] In embodiments, the stereochemistry at each of the
phosphorus internucleotide linkages of the ASO backbone is random.
In embodiments, the stereochemistry at each of the phosphorus
internucleotide linkages of the ASO backbone is controlled and is
not random. For example, U.S. Pat. App. Pub. No. 2014/0194610,
"Methods for the Synthesis of Functionalized Nucleic Acids,"
incorporated herein by reference, describes methods for
independently selecting the handedness of chirality at each
phosphorous atom in a nucleic acid oligomer. In embodiments, an ASO
used in the methods of the invention comprises an ASO having
phosphorus internucleotide linkages that are not random. In
embodiments, a composition used in the methods of the invention
comprises a pure diastereomeric ASO. In embodiments, a composition
used in the methods of the invention comprises an ASO that has
diastereomeric purity of at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, about 100%, about 90% to about 100%, about
91% to about 100%, about 92% to about 100%, about 93% to about
100%, about 94% to about 100%, about 95% to about 100%, about 96%
to about 100%, about 97% to about 100%, about 98% to about 100%, or
about 99% to about 100%.
[1975] In embodiments, the ASO has a nonrandom mixture of Rp and Sp
configurations at its phosphorus internucleotide linkages. For
example, it has been suggested that a mix of Rp and Sp is required
in antisense oligonucleotides to achieve a balance between good
activity and nuclease stability (Wan, et al., 2014, "Synthesis,
biophysical properties and biological activity of second generation
antisense oligonucleotides containing chiral phosphorothioate
linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated
herein by reference). In embodiments, an ASO used in the methods of
the invention comprises about 5-100% Rp, at least about 5% Rp, at
least about 10% Rp, at least about 15% Rp, at least about 20% Rp,
at least about 25% Rp, at least about 30% Rp, at least about 35%
Rp, at least about 40% Rp, at least about 45% Rp, at least about
50% Rp, at least about 55% Rp, at least about 60% Rp, at least
about 65% Rp, at least about 70% Rp, at least about 75% Rp, at
least about 80% Rp, at least about 85% Rp, at least about 90% Rp,
or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
In embodiments, an ASO used in the methods of the invention
comprises about 10% to about 100% Rp, about 15% to about 100% Rp,
about 20% to about 100% Rp, about 25% to about 100% Rp, about 30%
to about 100% Rp, about 35% to about 100% Rp, about 40% to about
100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp,
about 55% to about 100% Rp, about 60% to about 100% Rp, about 65%
to about 100% Rp, about 70% to about 100% Rp, about 75% to about
100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp,
about 90% to about 100% Rp, or about 95% to about 100% Rp, about
20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about
70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp,
with the remainder Sp.
[1976] In embodiments, an ASO used in the methods of the invention
comprises about 5-100% Sp, at least about 5% Sp, at least about 10%
Sp, at least about 15% Sp, at least about 20% Sp, at least about
25% Sp, at least about 30% Sp, at least about 35% Sp, at least
about 40% Sp, at least about 45% Sp, at least about 50% Sp, at
least about 55% Sp, at least about 60% Sp, at least about 65% Sp,
at least about 70% Sp, at least about 75% Sp, at least about 80%
Sp, at least about 85% Sp, at least about 90% Sp, or at least about
95% Sp, with the remainder Rp, or about 100% Sp. In embodiments, an
ASO used in the methods of the invention comprises about 10% to
about 100% Sp, about 15% to about 100% Sp, about 20% to about 100%
Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about
35% to about 100% Sp, about 40% to about 100% Sp, about 45% to
about 100% Sp, about 50% to about 100% Sp, about 55% to about 100%
Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about
70% to about 100% Sp, about 75% to about 100% Sp, about 80% to
about 100% Sp, about 85% to about 100% Sp, about 90% to about 100%
Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about
25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about
60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
[1977] Any of the ASOs described herein may contain a sugar moiety
that comprises ribose or deoxyribose, as present in naturally
occurring nucleotides, or a modified sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified
sugar moieties include 2' substitutions such as 2'-O-methyl
(2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;
N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy,
2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium
ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the sugar moiety modification is selected from
2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety
modification is an extra bridge bond, such as in a locked nucleic
acid (LNA). In some embodiments the sugar analog contains a
morpholine ring, such as phosphorodiamidate morpholino (PMO). In
some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl modification. In some embodiments, the sugar
moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE)
modifications. In some embodiments, the sugar moiety comprises cEt
2', 4' constrained 2'-O ethyl BNA modifications. In some
embodiments, the sugar moiety comprises tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises
ethylene nucleic acid (ENA) modifications. In some embodiments, the
sugar moiety comprises MCE modifications. Modifications are known
in the art and described in the literature, e.g., by Jarver, et
al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping
and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[1978] In some examples, each monomer of the ASO is modified in the
same way, for example each linkage of the backbone of the ASO
comprises a phosphorothioate linkage or each ribose sugar moiety
comprises a 2'O-methyl modification. Such modifications that are
present on each of the monomer components of an ASO are referred to
as "uniform modifications." In some examples, a combination of
different modifications may be desired, for example, an ASO may
comprise a combination of phosphorodiamidate linkages and sugar
moieties comprising morpholine rings (morpholinos). Combinations of
different modifications to an ASO are referred to as "mixed
modifications" or "mixed chemistries."
[1979] In some embodiments, the ASO comprises one or more backbone
modification. In some embodiments, the ASO comprises one or more
sugar moiety modification. In some embodiments, the ASO comprises
one or more backbone modification and one or more sugar moiety
modification. In some embodiments, the ASO comprises 2'MOE
modifications and a phosphorothioate backbone. In some embodiments,
the ASO comprises a phosphorodiamidate morpholino (PMO). In some
embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of
the ASOs or any component of an ASO (e.g., a nucleobase, sugar
moiety, backbone) described herein may be modified in order to
achieve desired properties or activities of the ASO or reduce
undesired properties or activities of the ASO. For example, an ASO
or one or more component of any ASO may be modified to enhance
binding affinity to a target sequence on a pre-mRNA transcript;
reduce binding to any non-target sequence; reduce degradation by
cellular nucleases (i.e., RNase H); improve uptake of the ASO into
a cell and/or into the nucleus of a cell; alter the
pharmacokinetics or pharmacodynamics of the ASO; and modulate the
half-life of the ASO.
[1980] In some embodiments, the ASOs are comprised of
2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
ASOs comprised of such nucleotides are especially well-suited to
the methods disclosed herein; oligomers having such modifications
have been shown to have significantly enhanced resistance to
nuclease degradation and increased bioavailability, making them
suitable, for example, for oral delivery in some embodiments
described herein. See e.g., Geary et al., J Pharmacol Exp Ther.
2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[1981] Methods of synthesizing ASOs will be known to one of skill
in the art. Alternatively or in addition, ASOs may be obtained from
a commercial source.
[1982] Unless specified otherwise, the left-hand end of
single-stranded nucleic acid (e.g., pre-mRNA transcript,
oligonucleotide, ASO, etc.) sequences is the 5' end and the
left-hand direction of single or double-stranded nucleic acid
sequences is referred to as the 5' direction. Similarly, the
right-hand end or direction of a nucleic acid sequence (single or
double stranded) is the 3' end or direction. Generally, a region or
sequence that is 5' to a reference point in a nucleic acid is
referred to as "upstream," and a region or sequence that is 3' to a
reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the
initiation or start codon is located, while the 3' end or direction
is where the termination codon is located. In some aspects,
nucleotides that are upstream of a reference point in a nucleic
acid may be designated by a negative number, while nucleotides that
are downstream of a reference point may be designated by a positive
number. For example, a reference point (e.g., an exon-exon junction
in mRNA) may be designated as the "zero" site, and a nucleotide
that is directly adjacent and upstream of the reference point is
designated "minus one," e.g., "-1," while a nucleotide that is
directly adjacent and downstream of the reference point is
designated "plus one," e.g., "+1."
[1983] In other embodiments, the ASOs are complementary to (and
bind to) a targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA that is downstream (in the 3' direction) of the 5' splice
site of the retained intron in a CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA (e.g., the direction designated by positive numbers
relative to the 5' splice site) (FIG. 1). In some embodiments, the
ASOs are complementary to a targeted portion of the CTNS, PAX2,
CYP24A1 or PPARD RIC pre-mRNA that is within the region 1 to +4000,
1 to +3500, 1 to +3000, 1 to +2500, 1 to +2000, 1 to +1500, 1 to
+1000, 1 to +500, +2 to +4000, +2 to +3500, +2 to +3000, +2 to
+2500, +2 to +2000, +2 to +1500, +2 to +1000, +2 to +500, +2 to
+400, +2 to +300, +2 to +200, +6 to +4000, +6 to +3500, +6 to
+3000, +6 to +2500, +6 to +2000, +6 to +1500, +6 to +1000, +6 to
+500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 relative to
the 5' splice site of the retained intron. In some embodiments, the
ASO is not complementary to nucleotides +1 to +5 relative to the 5'
splice site (the first five nucleotides located downstream of the
5' splice site). In some embodiments, the ASOs may be complementary
to a targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA that is within the region between nucleotides +6 and +50
relative to the 5' splice site of the retained intron. In some
aspects, the ASOs are complementary to a targeted portion of the
CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region
+6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40,
+6 to +30, or +6 to +20 relative to 5' splice site of the retained
intron.
[1984] In some embodiments, the ASOs are complementary to a
targeted region of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that
is upstream (5' relative) of the 3' splice site of the retained
intron in an RIC pre-mRNA (e.g., in the direction designated by
negative numbers) (FIG. 1). In some embodiments, the ASOs are
complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or
PPARD RIC pre-mRNA that is within the region -16 to -4000, -16 to
-3000, -16 to -2000, -16 to -1000, -16 to -500, -16 to -400, -16 to
-300, -6 to -200, or -16 to -100 relative to the 3' splice site of
the retained intron. In some embodiments, the ASO is not
complementary to nucleotides -1 to -15 relative to the 3' splice
site (the first 15 nucleotides located upstream of the 3' splice
site). In some embodiments, the ASOs are complementary to a
targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA
that is within the region -16 to -50 relative to the 3' splice site
of the retained intron. In some aspects, the ASOs are complementary
to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC
pre-mRNA that is within the region -16 to -90, -16 to -80, -16 to
-70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30 relative to
3' splice site of the retained intron.
[1985] In embodiments, the targeted portion of the RIC pre-mRNA is
within the region +100 relative to the 5' splice site of the
retained intron to -100 relative to the 3' splice site of the
retained intron.
[1986] In some embodiments, the ASOs are complementary to a
targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA
that is within the exon flanking the 5' splice site (upstream) of
the retained intron (FIG. 1). In some embodiments, the ASOs are
complementary to a targeted portion of the RIC pre-mRNA that is
within the region +2e to -4e in the exon flanking the 5' splice
site of the retained intron. In some embodiments, the ASOs are not
complementary to nucleotides -1e to -3e relative to the 5' splice
site of the retained intron. In some embodiments, the ASOs are
complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or
PPARD RIC pre-mRNA that is within the region -4e to -100e, -4e to
-90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to
-40e, -4e to -30e, or -4e to -20e relative to the 5' splice site of
the retained intron.
[1987] In some embodiments, the ASOs are complementary to a
targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA
that is within the exon flanking the 3' splice site (downstream) of
the retained intron (FIG. 1). In some embodiments, the ASOs are
complementary to a targeted portion to the CTNS, PAX2, CYP24A1 or
PPARD RIC pre-mRNA that is within the region +2e to -4e in the exon
flanking the 3' splice site of the retained intron. In some
embodiments, the ASOs are not complementary to nucleotide +1e
relative to the 3' splice site of the retained intron. In some
embodiments, the ASOs are complementary to a targeted portion of
the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the
region +2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to
+60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative
to the 3' splice site of the retained intron. The ASOs may be of
any length suitable for specific binding and effective enhancement
of splicing. In some embodiments, the ASOs consist of 8 to 50
nucleobases. For example, the ASO may be 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some
embodiments, the ASOs consist of more than 50 nucleobases. In some
embodiments, the ASO is from 8 to 50 nucleobases, 8 to 40
nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25
nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50
nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30
nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15
nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35
nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20
nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40
nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25
nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50
nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30
nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15
nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35
nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20
nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35
nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20
nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35
nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20
nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35
nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50
nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to
30 nucleobases in length. In some embodiments, the ASOs are 30
nucleotides in length. In some embodiments, the ASOs are 29
nucleotides in length. In some embodiments, the ASOs are 28
nucleotides in length. In some embodiments, the ASOs are 27
nucleotides in length. In some embodiments, the ASOs are 26
nucleotides in length. In some embodiments, the ASOs are 25
nucleotides in length. In some embodiments, the ASOs are 24
nucleotides in length. In some embodiments, the ASOs are 23
nucleotides in length. In some embodiments, the ASOs are 22
nucleotides in length. In some embodiments, the ASOs are 21
nucleotides in length. In some embodiments, the ASOs are 20
nucleotides in length. In some embodiments, the ASOs are 19
nucleotides in length. In some embodiments, the ASOs are 18
nucleotides in length. In some embodiments, the ASOs are 17
nucleotides in length. In some embodiments, the ASOs are 16
nucleotides in length. In some embodiments, the ASOs are 15
nucleotides in length. In some embodiments, the ASOs are 14
nucleotides in length. In some embodiments, the ASOs are 13
nucleotides in length. In some embodiments, the ASOs are 12
nucleotides in length. In some embodiments, the ASOs are 11
nucleotides in length. In some embodiments, the ASOs are 10
nucleotides in length.
[1988] In some embodiments, two or more ASOs with different
chemistries but complementary to the same targeted portion of the
RIC pre-mRNA are used. In some embodiments, two or more ASOs that
are complementary to different targeted portions of the RIC
pre-mRNA are used.
[1989] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
[1990] In some embodiments, the nucleic acid to be targeted by an
ASO is an RIC pre-mRNA expressed in a cell, such as a eukaryotic
cell. In some embodiments, the term "cell" may refer to a
population of cells. In some embodiments, the cell is in a subject.
In some embodiments, the cell is isolated from a subject. In some
embodiments, the cell is ex vivo. In some embodiments, the cell is
a condition or disease-relevant cell or a cell line. In some
embodiments, the cell is in vitro (e.g., in cell culture).
Pharmaceutical Compositions
[1991] Pharmaceutical compositions or formulations comprising the
antisense oligonucleotide of the described compositions and for use
in any of the described methods can be prepared according to
conventional techniques well known in the pharmaceutical industry
and described in the published literature. In embodiments, a
pharmaceutical composition or formulation for treating a subject
comprises an effective amount of any antisense oligomer as
described above, or a pharmaceutically acceptable salt, solvate,
hydrate or ester thereof, and a pharmaceutically acceptable
diluent. The antisense oligomer of a pharmaceutical formulation may
further comprise a pharmaceutically acceptable excipient, diluent
or carrier.
[1992] Pharmaceutically acceptable salts are suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response, etc., and are commensurate
with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et
al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated
herein by reference for this purpose. The salts can be prepared in
situ during the final isolation and purification of the compounds,
or separately by reacting the free base function with a suitable
organic acid. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
documented methodologies such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
[1993] In embodiments, the compositions are formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. In embodiments, the compositions are formulated as
suspensions in aqueous, non-aqueous or mixed media. Aqueous
suspensions may further contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers. In embodiments, a pharmaceutical
formulation or composition of the present invention includes, but
is not limited to, a solution, emulsion, microemulsion, foam or
liposome-containing formulation (e.g., cationic or noncationic
liposomes).
[1994] The pharmaceutical composition or formulation of the present
invention may comprise one or more penetration enhancer, carrier,
excipients or other active or inactive ingredients as appropriate
and well known to those of skill in the art or described in the
published literature. In embodiments, liposomes also include
sterically stabilized liposomes, e.g., liposomes comprising one or
more specialized lipids. These specialized lipids result in
liposomes with enhanced circulation lifetimes. In embodiments, a
sterically stabilized liposome comprises one or more glycolipids or
is derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. In embodiments, a surfactant is
included in the pharmaceutical formulation or compositions. The use
of surfactants in drug products, formulations and emulsions is well
known in the art. In embodiments, the present invention employs a
penetration enhancer to effect the efficient delivery of the
antisense oligonucleotide, e.g., to aid diffusion across cell
membranes and/or enhance the permeability of a lipophilic drug. In
embodiments, the penetration enhancers is a surfactant, fatty acid,
bile salt, chelating agent, or non-chelating nonsurfactant.
[1995] In embodiments, the pharmaceutical formulation comprises
multiple antisense oligonucleotides. In embodiments, the antisense
oligonucleotide is administered in combination with another drug or
therapeutic agent. In embodiments, the antisense oligonucleotide is
administered with one or more agents capable of promoting
penetration of the subject antisense oligonucleotide across the
blood-brain barrier by any method known in the art. For example,
delivery of agents by administration of an adenovirus vector to
motor neurons in muscle tissue is described in U.S. Pat. No.
6,632,427, "Adenoviral-vector-mediated gene transfer into medullary
motor neurons," incorporated herein by reference. Delivery of
vectors directly to the brain, e.g., the striatum, the thalamus,
the hippocampus, or the substantia nigra, is described, e.g., in
U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of
foreign genes into cells of the central nervous system particularly
in brain," incorporated herein by reference.
[1996] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that provide desirable pharmaceutical or
pharmacodynamic properties. In embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to
promote penetration or transport across the blood-brain barrier,
e.g., an antibody to the transferrin receptor. In embodiments, the
antisense oligonucleotide is linked with a viral vector, e.g., to
render the antisense compound more effective or increase transport
across the blood-brain barrier. In embodiments, osmotic blood brain
barrier disruption is assisted by infusion of sugars, e.g., meso
erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose,
D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+)
raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol,
D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-)
lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g.,
glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine, leucine, methionine,
phenylalanine, proline, serine, threonine, tyrosine, valine, and
taurine. Methods and materials for enhancing blood brain barrier
penetration are described, e.g., in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain
barrier," U.S. Pat. No. 6,294,520, "Material for passage through
the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral
delivery systems," each incorporated herein by reference.
[1997] In embodiments, the antisense oligonucleotides of the
invention are chemically linked to one or more moieties or
conjugates, e.g., a targeting moiety or other conjugate that
enhances the activity or cellular uptake of the oligonucleotide.
Such moieties include, but are not limited to, a lipid moiety,
e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic
chain, e.g., dodecandiol or undecyl residues, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid.
Oligonucleotides comprising lipophilic moieties, and preparation
methods have been described in the published literature. In
embodiments, the antisense oligonucleotide is conjugated with a
moiety including, but not limited to, an abasic nucleotide, a
polyether, a polyamine, a polyamide, a peptides, a carbohydrate,
e.g., N-acetylgalactosamine (GalNAc), N--Ac-Glucosamine (GluNAc),
or mannose (e.g., mannose-6-phosphate), a lipid, or a
polyhydrocarbon compound. Conjugates can be linked to one or more
of any nucleotides comprising the antisense oligonucleotide at any
of several positions on the sugar, base or phosphate group, as
understood in the art and described in the literature, e.g., using
a linker. Linkers can include a bivalent or trivalent branched
linker. In embodiments, the conjugate is attached to the 3' end of
the antisense oligonucleotide. Methods of preparing oligonucleotide
conjugates are described, e.g., in U.S. Pat. No. 8,450,467,
"Carbohydrate conjugates as delivery agents for oligonucleotides,"
incorporated by reference herein.
Treatment of Subjects
[1998] Any of the compositions provided herein may be administered
to an individual. "Individual" may be used interchangeably with
"subject" or "patient." An individual may be a mammal, for example
a human or animal such as a non-human primate, a rodent, a rabbit,
a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a
pig, or a sheep. In embodiments, the individual is a human. In
embodiments, the individual is a fetus, an embryo, or a child. In
other embodiments, the individual may be another eukaryotic
organism, such as a plant. In some embodiments, the compositions
provided herein are administered to a cell ex vivo.
[1999] In some embodiments, the compositions provided herein are
administered to an individual as a method of treating a disease or
disorder. In some embodiments, the individual has a genetic
disease, such as any of the diseases described herein. In some
embodiments, the individual is at risk of having the disease, such
as any of the diseases described herein. In some embodiments, the
individual is at increased risk of having a disease or disorder
caused by insufficient amount of a protein or insufficient activity
of a protein. If an individual is "at an increased risk" of having
a disease or disorder caused insufficient amount of a protein or
insufficient activity of a protein, the method involves
preventative or prophylactic treatment. For example, an individual
may be at an increased risk of having such a disease or disorder
because of family history of the disease. Typically, individuals at
an increased risk of having such a disease or disorder benefit from
prophylactic treatment (e.g., by preventing or delaying the onset
or progression of the disease or disorder).
[2000] Suitable routes for administration of ASOs of the present
invention may vary depending on cell type to which delivery of the
ASOs is desired. Multiple tissues and organs can be affected in a
condition described herein, with the kidney being the most
significantly affected tissue. The ASOs of the present invention
may be administered to patients parenterally, for example, by
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or intravenous injection. In embodiments, delivery is to
the kidney. In embodiments, a fetus is treated in utero, e.g., by
administering the ASO composition to the fetus directly or
indirectly (e.g., via the mother).
Methods of Identifying Additional ASOs that Enhance Splicing
[2001] Also within the scope of the present invention are methods
for identifying (determining) additional ASOs that enhance splicing
of an RIC pre-mRNA, specifically at the target intron. ASOs that
specifically hybridize to different nucleotides within the target
region of the pre-mRNA may be screened to identify (determine) ASOs
that improve the rate and/or extent of splicing of the target
intron. In some embodiments, the ASO may block or interfere with
the binding site(s) of a splicing repressor(s)/silencer. Any method
known in the art may be used to identify (determine) an ASO that
when hybridized to the target region of the intron results in the
desired effect (e.g., enhanced splicing, protein or functional RNA
production). These methods also can be used for identifying ASOs
that enhance splicing of the retained intron by binding to a
targeted region in an exon flanking the retained intron, or in a
non-retained intron. An example of a method that may be used is
provided below.
[2002] A round of screening, referred to as an ASO "walk" may be
performed using ASOs that have been designed to hybridize to a
target region of a pre-mRNA. For example, the ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100
nucleotides upstream of the 5' splice site of the retained intron
(e.g., a portion of sequence of the exon located upstream of the
target/retained intron) to approximately 100 nucleotides downstream
of the 5' splice site of the target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the
retained intron to approximately 100 nucleotides downstream of the
3' splice site of the target/retained intron (e.g., a portion of
sequence of the exon located downstream of the target/retained
intron). For example, a first ASO of 15 nucleotides in length may
be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of the target/retained intron. A
second ASO is designed to specifically hybridize to nucleotides +11
to +25 relative to the 5' splice site of the target/retained
intron. ASOs are designed as such spanning the target region of the
pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g.,
every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled
from 100 nucleotides downstream of the 5' splice site, to 100
nucleotides upstream of the 3' splice site.
[2003] One or more ASOs, or a control ASO (an ASO with a scrambled
sequence, sequence that is not expected to hybridize to the target
region) are delivered, for example by transfection, into a
disease-relevant cell line that expresses the target pre-mRNA
(e.g., the RIC pre-mRNA described elsewhere herein). The
splicing-inducing effects of each of the ASOs may be assessed by
any method known in the art, for example by reverse transcriptase
(RT)-PCR using primers that span the splice junction, as described
herein (see "Identification of intron-retention events"). A
reduction or absence of the RT-PCR product produced using the
primers spanning the splice junction in ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of
the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[2004] A second round of screening, referred to as an ASO
"micro-walk" may be performed using ASOs that have been designed to
hybridize to a target region of a pre-mRNA. The ASOs used in the
ASO micro-walk are tiled every 1 nucleotide to further refine the
nucleotide acid sequence of the pre-mRNA that when hybridized with
an ASO results in enhanced splicing.
[2005] Regions defined by ASOs that promote splicing of the target
intron are explored in greater detail by means of an ASO
"micro-walk", involving ASOs spaced in 1-nt steps, as well as
longer ASOs, typically 18-25 nt.
[2006] As described for the ASO walk above, the ASO micro-walk is
performed by delivering one or more ASOs, or a control ASO (an ASO
with a scrambled sequence, sequence that is not expected to
hybridize to the target region), for example by transfection, into
a disease-relevant cell line that expresses the target pre-mRNA.
The splicing-inducing effects of each of the ASOs may be assessed
by any method known in the art, for example by reverse
transcriptase (RT)-PCR using primers that span the splice junction,
as described herein (see "Identification of intron-retention
events"). A reduction or absence of the RT-PCR product produced
using the primers spanning the splice junction in ASO-treated cells
as compared to in control ASO-treated cells indicates that splicing
of the target intron has been enhanced. In some embodiments, the
splicing efficiency, the ratio of spliced to unspliced pre-mRNA,
the rate of splicing, or the extent of splicing may be improved
using the ASOs described herein. The amount of protein or
functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine whether each ASO achieved the desired effect
(e.g., enhanced protein production). Any method known in the art
for assessing and/or quantifying protein production, such as
Western blotting, flow cytometry, immunofluorescence microscopy,
and ELISA, can be used.
[2007] ASOs that when hybridized to a region of a pre-mRNA result
in enhanced splicing and increased protein production may be tested
in vivo using animal models, for example transgenic mouse models in
which the full-length human gene has been knocked-in or in
humanized mouse models of disease. Suitable routes for
administration of ASOs may vary depending on the disease and/or the
cell types to which delivery of the ASOs is desired. ASOs may be
administered, for example, by intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous
injection. Following administration, the cells, tissues, and/or
organs of the model animals may be assessed to determine the effect
of the ASO treatment by for example evaluating splicing
(efficiency, rate, extent) and protein production by methods known
in the art and described herein. The animal models may also be any
phenotypic or behavioral indication of the disease or disease
severity.
[2008] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
[2009] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
EXAMPLES
[2010] The present invention will be more specifically illustrated
by the following Examples. However, it should be understood that
the present invention is not limited by these examples in any
manner.
Example 1: Identification of Intron Retention Events in JAG1
Transcripts by RNAseq Using Next Generation Sequencing
[2011] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the JAG1 gene to identify intron-retention events. For
this purpose, polyA+ RNA from nuclear and cytoplasmic fractions of
THLE-3 (human liver epithelial) cells was isolated cDNA libraries
constructed using Illumina's TruSeq Stranded mRNA library Prep Kit.
The libraries were pair-end sequenced resulting in 100-nucleotide
reads that were mapped to the human genome (February 2009,
GRCh37/hg19 assembly). The sequencing results for JAG1 are shown in
FIG. 3. Briefly, FIG. 3 shows the mapped reads visualized using the
UCSC genome browser (operated by the UCSC Genome Informatics Group
(Center for Biomolecular Science & Engineering, University of
California, Santa Cruz, 1156 High Street, Santa Cruz, Calif. 95064)
and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome
Browser database: 2015 update," Nucleic Acids Research 43, Database
Issue, doi: 10.1093/nar/gku1177) and the coverage and number of
reads can be inferred by the peak signals. The height of the peaks
indicates the level of expression given by the density of the reads
in a particular region. A schematic representation of JAG1 (drawn
to scale) is provided by the UCSC genome browser (below the read
signals) so that peaks can be matched to JAG1 exonic and intronic
regions. Based on this display, we identified three introns (13,
18, and 23, indicated by arrows) that have high read density in the
nuclear fraction of THLE-3 cells, but have very low to no reads in
the cytoplasmic fraction of these cells (as shown for intron 13 in
the bottom diagram of FIG. 3, for intron 18 in the bottom diagram
of FIG. 7, and for intron 23 in the bottom diagram of FIG. 11).
This indicates that these introns are retained and that the
intron-13, intron-18, and intron-23 containing transcripts remain
in the nucleus, and suggests that these retained JAG1 RIC pre-mRNAs
are non-productive, as they are not exported out to the
cytoplasm.
Example 2: Design of ASO-Walk Targeting Intron 13 of JAG1
[2012] An ASO walk was designed to target intron 13 using the
method described herein (FIG. 4; Table 2). A region immediately
downstream of the intron 13 5' splice site spanning nucleotides +6
to +69 and a region immediately upstream of intron 13 3' splice
site spanning nucleotides -16 to -68 of the intron were targeted
with 2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals (with the exception of 1 ASO, JAG1-IVS13+52) (FIG. 4;
Table 2).
Example 3: Improved Splicing Efficiency Via ASO-Targeting of JAG1
Intron 13 Increases Transcript Levels
[2013] To determine whether we can achieve an increase in JAG1
expression by improving splicing efficiency of JAG1 intron 13 using
ASOs we used the method described herein (FIG. 5). To this end,
ARPE-19 cells were mock-transfected, or transfected with each of
the targeting ASOs described in FIG. 4 and Table 2, or a
non-targeting SMN-ASO control, independently, using RNAiMAX (RiM)
(Invitrogen) delivery reagents. Experiments were performed using 60
nM ASOs (as indicated in FIG. 5) for 48 hrs. Radioactive RT-PCR
results show that several targeting ASOs (+6, SEQ ID NO: 135; +11,
SEQ ID NO: 136; +21, SEQ ID NO: 138; +26, SEQ ID NO: 139; +31, SEQ
ID NO: 140; +36, SEQ ID NO: 141; +52; -16, SEQ ID NO: 301; -36, SEQ
ID NO: 297; -41, SEQ ID NO: 296; -46, SEQ ID NO: 295; and -51 SEQ
ID NO: 294) increase JAG1 transcript level compared to the
mock-transfected or non-targeting ASO (FIG. 5). Intensities of the
bands corresponding to the JAG1 PCR products from
targeting-ASO-transfected cells were normalized to Beta actin and
plotted relative to the normalized JAG1 PCR product from
mock-treated cells. Results of this analysis indicate that several
targeting ASOs increase JAG1 transcript level 2-6 fold (FIG. 5).
These results were confirmed by RT-qPCR using primers elsewhere in
the JAG1 transcript, showing the same trend of JAG1 upregulation
evidenced by the fold-change plots in FIG. 6 (top, normalized to
Beta actin, bottom normalized to RPL32). Altogether, these results
confirm that improving the splicing efficiency of a rate limiting
intron in the JAG1 gene using ASOs leads to an increase in gene
expression.
Example 4: Design of an ASO-Walk Targeting Intron 18 of JAG1
[2014] An ASO walk was designed to target intron 18 using the
method described herein (FIG. 8, Table 2). A region immediately
downstream of intron 18 5' splice site spanning nucleotides +15 to
+67 and a region immediately upstream of intron 18 3' splice site
spanning nucleotides -16 to -68 of the intron were targeted with
2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals (FIG. 8, Table 2).
Example 5: Improved Splicing Efficiency Via ASO-Targeting of JAG1
Intron 18 Increases Transcript Levels
[2015] To determine whether an increase in JAG1 expression could be
achieved by improving splicing efficiency of JAG1 intron 18 using
ASOs, the method described herein (FIG. 9) was used. To this end,
ARPE-19 cells were mock-transfected, transfected with each of the
targeting ASOs described in FIG. 8 and Table 2, or transfected with
a non-targeting SMN-ASO control, independently, using RNAiMAX (RiM)
(Invitrogen) delivery reagents. Experiments were performed using 60
nM ASOs (as indicated in FIG. 5) for 48 hrs. Radioactive RT-PCR
results show that several targeting ASOs (+15; +20; and -26, SEQ ID
NO: 107) increase JAG1 transcript level compared to the
mock-transfected or non-targeting ASO (FIG. 9). Intensities of the
bands corresponding to the JAG1 PCR products from
targeting-ASO-transfected cells were normalized to Beta actin and
plotted relative to the normalized JAG1 PCR product from
mock-treated cells. Results of this analysis indicate that three
targeting ASOs increase JAG1 transcript level at least 1.5 fold
(FIG. 9). These results were confirmed by RT-qPCR using primers
elsewhere in the JAG1 transcript, showing the same trend of JAG1
upregulation evidenced by the fold-change plots in FIG. 10 (top,
normalized to Beta actin, bottom normalized to RPL32). Altogether,
these results confirm that improving the splicing efficiency of a
rate limiting intron in the JAG1 gene using ASOs leads to an
increase in gene expression.
[2016] A second method can be used to determine whether an increase
in JAG1 target gene intron splicing efficiency can be achieved with
ASOs. In brief, ARPE-19 cells, a human retinal epithelium cell line
(American Type Culture Collection (ATCC), USA), or Huh-7, a human
hepatoma cell line (NIBIOHN, Japan), or SK-N-AS, a human
neuroblastoma cell line (ATCC) are mock-transfected, or transfected
with the targeting ASOs described in Tables 1 and 2. Cells are
transfected using Lipofectamine RNAiMax transfection reagent
(Thermo Fisher) according to vendor's specifications. ASOs are
plated in 96-well tissue culture plates and combined with RNAiMax
diluted in Opti-MEM. Cells are detached using trypsin and
resuspended in full medium, and approximately 25,000 cells are
added the ASO-transfection mixture. Transfection experiments are
carried out in triplicate plate replicates. Final ASO concentration
is 80 nM. Media is changed 6h post-transfection, and cells
harvested at 24h, using the Cells-to-Ct lysis reagent, supplemented
with DNAse (Thermo Fisher), according to vendor's specifications.
cDNA is generated with Cells-to-Ct RT reagents (Thermo Fisher)
according to vendor's specifications. To quantify the amount of
splicing at the intron of interest, quantitative PCR is carried out
using Taqman assays with probes spanning the corresponding
exon-exon junction (Thermo Fisher). Taqman assays are carried out
according to vendor's specifications, on a QuantStudio 7 Flex
Real-Time PCR system (Thermo Fisher). Target gene assay values are
normalized to RPL32 (deltaCt) and plate-matched mock transfected
samples (delta-delta Ct), generating fold-change over mock
quantitation (2{circumflex over ( )}-(delta-deltaCt).
Example 6: Design of ASO-Walk Targeting Intron 23 of JAG1
[2017] An ASO walk was designed to target intron 23 using the
method described herein (FIG. 12, Table 2). A region immediately
downstream of intron 23 5' splice site spanning nucleotides +6 to
+58 and a region immediately upstream of intron 23 3' splice site
spanning nucleotides -16 to -68 of the intron were targeted with
2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals (FIG. 12; Table 2).
TABLE-US-00004 TABLE 2 ASOs targeting the JAG1 Gene SEQ ID NO ASO
Name Sequence 5' to 3' Intron 135 JAG-IVS13+6 aggucccgggagaaggga 13
136 JAG-IVS13+11 cagccaggucccgggaga 13 137 JAG-IVS13+16
ggagacagccaggucccg 13 138 JAG-IVS13+21 agucuggagacagccagg 13 139
JAG-IVS13+26 gagcaagucuggagacag 13 140 JAG-IVS13+31
aaaaggagcaagucugga 13 141 JAG-IVS13+36 gggacaaaaggagcaagu 13 150753
JAG-IVS13+52 gcagugguaguaaguggg 13 301 JAG-IVS13-16
gggaucagaucacagcca 13 300 JAG-IVS13-21 cagaucacagccaugcac 13 299
JAG-IVS13-26 cacagccaugcacccaca 13 298 JAG-IVS13-31
ccaugcacccacagaugc 13 297 JAG-IVS13-36 cacccacagaugcggcau 13 296
JAG-IVS13-41 acagaugcggcauuccua 13 295 JAG-IVS13-46
ugcggcauuccuaagcca 13 294 JAG-IVS13-51 cauuccuaagccaagggc 13 150754
JAG-IVS18+15 ccugggagaguucaaggg 18 150755 JAG-IVS18+20
acagcccugggagaguuc 18 150756 JAG-IVS18+25 cuccgacagcccugggag 18
150757 JAG-IVS18+30 aucugcuccgacagcccu 18 150758 JAG-IVS18+35
ucaggaucugcuccgaca 18 150759 JAG-IVS18+40 gggugucaggaucugcuc 18
150760 JAG-IVS18+45 cccaagggugucaggauc 18 150761 JAG-IVS18+50
aaguccccaaggguguca 18 109 JAG-IVS18-16 aacuuaauagugaggacu 18 108
JAG-IVS18-21 aauagugaggacuucaac 18 107 JAG-IVS18-26
ugaggacuucaacaggga 18 106 JAG-IVS18-31 acuucaacagggaaagcg 18 105
JAG-IVS18-36 aacagggaaagcggucuu 18 104 JAG-IVS18-41
ggaaagcggucuuagccc 18 103 JAG-IVS18-46 gcggucuuagcccuaagu 18 102
JAG-IVS18-51 cuuagcccuaaguaaaac 18 369 JAG-IVS23+6
aaaaaacaaagguuguua 23 370 JAG-IVS23+11 caaaaaaaaaacaaaggu 23 371
JAG-IVS23+16 ccauucaaaaaaaaaaca 23 372 JAG-IVS23+21
auccaccauucaaaaaaa 23 373 JAG-IVS23+26 cagacauccaccauucaa 23 374
JAG-IVS23+31 gcaagcagacauccacca 23 375 JAG-IVS23+36
agcaagcaagcagacauc 23 376 JAG-IVS23+41 uuuaaagcaagcaagcag 23 414
JAG-IVS23-16 aaaacccacacacgugua 23 413 JAG-IVS23-21
ccacacacguguaagauu 23 412 JAG-IVS23-26 cacguguaagauugagag 23 411
JAG-IVS23-31 guaagauugagaggaaga 23 410 JAG-IVS23-36
auugagaggaagaacaaa 23 409 JAG-IVS23-41 gaggaagaacaaaaggcu 23 408
JAG-IVS23-46 agaacaaaaggcugagau 23 407 JAG-IVS23-51
aaaaggcugagauguucu 23
Example 7: Identification of Intron Retention Events in TSC2
Transcripts by RNAseq Using Next Generation Sequencing
[2018] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the TSC2 gene to identify intron-retention events. For
this purpose, polyA+ RNA from nuclear and cytoplasmic fractions of
HCN (human cortical neurons), renal epithelial cells, bronchial
epithelial cells, and THLE-3 (human liver epithelial) cells was
isolated and cDNA libraries constructed using Illumina's TruSeq
Stranded mRNA library Prep Kit. The libraries were pair-end
sequenced resulting in 100-nucleotide reads that were mapped to the
human genome (February 2009, GRCh37/hg19 assembly). The sequencing
results for TSC2 are shown in FIG. 13. Briefly, FIG. 13 shows the
mapped reads visualized using the UCSC genome browser (operated by
the UCSC Genome Informatics Group (Center for Biomolecular Science
& Engineering, University of California, Santa Cruz, 1156 High
Street, Santa Cruz, Calif. 95064) and described by, e.g.,
Rosenbloom, et al., 2015, "The UCSC Genome Browser database: 2015
update," Nucleic Acids Research 43, Database Issue, doi:
10.1093/nar/gku1177) and the coverage and number of reads can be
inferred by the peak signals. The height of the peaks indicates the
level of expression given by the density of the reads in a
particular region. A schematic representation of TSC2 (drawn to
scale) is provided by the UCSC genome browser (below the read
signals) so that peaks can be matched to TSC2 exonic and intronic
regions. Based on this display, we identified five introns (4, 25,
26, 31, and 32, indicated by arrows) that have high read density in
the nuclear fraction of HCN, but have very low to no reads in the
cytoplasmic fraction of these cells (as shown for intron 4 in the
bottom diagram of FIG. 13, for introns 25 and 26 in the bottom
diagram of FIG. 15, and for introns 31 and 32 in the bottom diagram
of FIG. 17). This indicates that these introns are retained and
that the intron-4, intron-25, intron-26, intron-31, and intron-32
containing transcripts remain in the nucleus, and suggests that
these retained TSC2 RIC pre-mRNAs are non-productive, as they are
not exported out to the cytoplasm.
Example 8: Design of ASO-Walk Targeting Intron 4 of TSC2
[2019] An ASO walk was designed to target intron 4 using the method
described herein (FIG. 14; Table 4). A region immediately
downstream of the 5' splice site of intron 4 spanning nucleotides
+6 to +58 and a region immediately upstream of the 3' splice site
of intron 4 spanning nucleotides -16 to -68 of the intron were
targeted with 2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by
5-nucleotide intervals.
Example 9: Design of ASO-Walk Targeting Intron 25 and 26 of
TSC2
[2020] An ASO walk was designed to target introns 25 and 26 using
the method described herein (FIG. 16; Table 4). A region
immediately downstream of the intron 25 5' splice site spanning
nucleotides +6 to +58 and a region immediately upstream of intron
26 3' splice site spanning nucleotides -16 to -68 of the intron
were targeted with 2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by
5-nucleotide intervals. The splice site intronic regions flanking
alternative exon 26 are not targeted to avoid affecting the
inclusion level of exon 26.
Example 10: Design of ASO-Walk Targeting Intron 31 and 32 of
TSC2
[2021] An ASO walk was designed to target introns 31 and 32 using
the method described herein (FIG. 18; Table 4). A region
immediately downstream of the 5' splice site of intron 31 spanning
nucleotides +6 to +58 and a region immediately upstream 3' splice
site of intron 32 spanning nucleotides -18 to -68 were targeted
with 2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals (with the exception of 2 ASOs, TSC2-IVS32-33 and
TSC2-IVS32-51). The splice site intronic regions flanking
alternative exon 32 are not targeted to avoid affecting the
inclusion level of exon 32.
Example 11: Improved Splicing Efficiency Via ASO-Targeting of TSC2
Intron 4, 25, 26, 31 or 32 Increases Transcript Levels
[2022] To determine whether an increase in TSC2 expression could be
achieved by improving splicing efficiency of TSC2 intron 4, 25, 26,
31 or 32 using ASOs, RT-PCR products were evaluated using
radioactive RT-PCR and RT-qPCR. ARPE-19 cells, a human retinal
epithelium cell line (American Type Culture Collection (ATCC),
USA), or Huh-7, a human hepatoma cell line (NIBIOHN, Japan), or
SK-N-AS, a human neuroblastoma cell line (ATCC) were
mock-transfected, or transfected with the targeting ASOs described
in FIG. 20, FIG. 22, FIG. 25 and Tables 1-2. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher), listed in Tables 1-2. Taqman assays were carried
out according to vendor's specifications, on a QuantStudio 7 Flex
Real-Time PCR system (Thermo Fisher). Target gene assay values were
normalized to RPL32 (deltaCt) and plate-matched mock transfected
samples (delta-delta Ct), generating fold-change over mock
quantitation (2{circumflex over ( )}-(delta-deltaCt). Average
fold-change over mock of the three plate replicates was plotted
(FIG. 20, FIG. 22 and FIG. 25). In FIG. 20, FIG. 22 and FIG. 25,
several ASOs were identified to increase the target gene
expression, indicating an increase in splicing at the respective
target intron.
TABLE-US-00005 TABLE 4 ASOs targeting the TSC2 Gene SEQ ID NO ASO
Name Sequence 5' to 3' Intron 689, 1760, 2343, 3210, 4452
TSC2-IVS4+6 cccagcgucgcccugggc 4 690, 1761, 2344, 3211, 4453
TSC2-IVS4+11 cccaucccagcgucgccc 4 691, 1762, 2345, 3212, 4454
TSC2-IVS4+16 cgucacccaucccagcgu 4 692, 1763, 2346, 3213, 4455
TSC2-IVS4+21 ccugacgucacccauccc 4 693, 1764, 2347, 3214, 4456
TSC2-IVS4+26 ggcagccugacgucaccc 4 694, 1765, 2348, 3215, 4457
TSC2-IVS4+31 cagugggcagccugacgu 4 695, 1766, 2349, 3216, 4458
TSC2-IVS4+36 acagucagugggcagccu 4 696, 1767, 2350, 3217, 4459
TSC2-IVS4+41 acaggacagucagugggc 4 840, 1911, 2494, 3361, 4603, 5276
TSC2-IVS4-16 acaggaucagcagagccu 4 839, 1910, 2493, 3360, 4602, 5275
TSC2-IVS4-21 aucagcagagccugccag 4 838, 1909, 2492, 3359, 4601, 5274
TSC2-IVS4-26 cagagccugccagcgucg 4 837, 1908, 2491, 3358, 4600, 5273
TSC2-IVS4-31 ccugccagcgucgcccac 4 836, 1907, 2490, 3357, 4599, 5272
TSC2-IVS4-36 cagcgucgcccacacggc 4 835, 1906, 2489, 3356, 4598, 5271
TSC2-IVS4-41 ucgcccacacggcugcca 4 834, 1905, 2488, 3355, 4597, 5270
TSC2-IVS4-46 cacacggcugccaagaag 4 833, 1904, 2487, 3354, 4596, 5269
TSC2-IVS4-51 ggcugccaagaagucccu 4 460, 1316, 2114, 2766, 4001,
4223, TSC2-IVS25+6 aggcacacccccgcaggc 25 4875 461, 1317, 2115,
2767, 4002, 4224, TSC2-IVS25+11 acuccaggcacacccccg 25 4876 462,
1318, 2116, 2768, 4003, 4225, TSC2-IVS25+16 caccgacuccaggcacac 25
4877 463, 1319, 2117, 2769, 4004, 4226, TSC2-IVS25+21
ccccacaccgacuccagg 25 4878 464, 1320, 2118, 2770, 4005, 4227,
TSC2-IVS25+26 ccccaccccacaccgacu 25 4879 465, 1321, 2119, 2771,
4006, 4228, TSC2-IVS25+31 uccuuccccaccccacac 25 4880 466, 1322,
2120, 2772, 4007, 4229, TSC2-IVS25+36 ccauguccuuccccaccc 25 4881
467, 1323, 2121, 2773, 4008, 4230, TSC2-IVS25+41 cagccccauguccuuccc
25 4882 639, 1710, 3160, 5054 TSC2-IVS26-16 gugaccagggucagggug 26
638, 1709, 3159, 5053 TSC2-IVS26-21 cagggucagggugccagg 26 637,
1708, 3158, 5052 TSC2-IVS26-26 ucagggugccagguaggg 26 636, 1707,
3157, 5051 TSC2-IVS26-31 gugccagguagggcgggc 26 635, 1706, 3156,
5050 TSC2-IVS26-36 agguagggcgggccgagc 26 634, 1705, 3155, 5049
TSC2-IVS26-41 gggcgggccgagccaccu 26 633, 1704, 3154, 5048
TSC2-IVS26-46 ggccgagccaccuaucac 26 632, 1703, 3153, 5047
TSC2-IVS26-51 agccaccuaucaccaagg 26 899, 1970, 2553, 3420, 3653,
4662, TSC2-IVS31+6 ccaaggcccgccaugcca 31 5335 900, 1971, 2554,
3421, 3654, 4663, TSC2-IVS31+11 ccgugccaaggcccgcca 31 5336 901,
1972, 2555, 3422, 3655, 4664, TSC2-IVS31+16 agagcccgugccaaggcc 31
5337 902, 1973, 2556, 3423, 3656, 4665, TSC2-IVS31+21
ggagcagagcccgugcca 31 5338 903, 1974, 2557, 3424, 3657, 4666,
TSC2-IVS31+26 cagugggagcagagcccg 31 5339 904, 1975, 2558, 3425,
3658, 4667, TSC2-IVS31+31 caggccagugggagcaga 31 5340 905, 1976,
2559, 3426, 3659, 4668, TSC2-IVS31+36 agcaccaggccaguggga 31 5341
906, 1977, 2560, 3427, 3660, 4669, TSC2-IVS31+41 ccgggagcaccaggccag
31 5342 150762 TSC2-IVS32-18 gggcugcuggauguggg 32 150763
TSC2-IVS32-33 gggcugggccaggcccug 32 150764 TSC2-IVS32-38
gggccaggcccuggcccu 32 150765 TSC2-IVS32-43 aggcccuggcccugacgu 32
150766 TSC2-IVS32-48 cuggcccugacguggccu 32 1078, 1282, 2732, 3599,
3967, 4841, TSC2-IVS32-51 gcccugacguggccuccg 32 5514
Example 12: Identification of Intron Retention Events in PKD2
Transcripts by RNAseq Using Next Generation Sequencing
[2023] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the PKD2 gene to identify intron-retention events. For
this purpose, polyA+ RNA from nuclear and cytoplasmic fractions of
renal epithelial cells was isolated and cDNA libraries constructed
using Illumina's TruSeq Stranded mRNA library Prep Kit. The
libraries were pair-end sequenced resulting in 100-nucleotide reads
that were mapped to the human genome (February 2009, GRCh37/hg19
assembly). The sequencing results for PKD2 are shown in FIG. 27.
Briefly, FIG. 27 shows the mapped reads visualized using the UCSC
genome browser (operated by the UCSC Genome Informatics Group
(Center for Biomolecular Science & Engineering, University of
California, Santa Cruz, 1156 High Street, Santa Cruz, Calif. 95064)
and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome
Browser database: 2015 update," Nucleic Acids Research 43, Database
Issue, doi: 10.1093/nar/gku1177) and the coverage and number of
reads can be inferred by the peak signals. The height of the peaks
indicates the level of expression given by the density of the reads
in a particular region. A schematic representation of PKD2 (drawn
to scale) is provided by the UCSC genome browser (below the read
signals) so that peaks can be matched to PKD2 exonic and intronic
regions. Based on this display, we identified one intron (intron 5,
as indicated) that has high read density in the nuclear fraction of
HCN, but very low to no reads in the cytoplasmic fraction of these
cells (as shown for intron 5 in the bottom diagram of FIG. 27).
This indicates that intron 5 is retained and that the intron-5
containing transcripts remain in the nucleus, suggesting that this
retained PKD2 RIC pre-mRNAs is non-productive, as it is not
exported out to the cytoplasm.
Example 13: Design of ASO-Walk Targeting Intron 5 of PKD2
[2024] An ASO walk was designed to target intron 5 using the method
described herein (FIG. 28; Table 5, SEQ ID NOS: 5547-5824). A
region immediately upstream and downstream of the 5' splice site of
intron 5, spanning nucleotides +497 to -204e, and a region
immediately upstream and downstream of the 3' splice site of intron
5, spanning nucleotides -496 to +212e were utilized to design ASOs
to target retained intron 5 PKD2 RIC pre-mRNAs. Table 5 lists
exemplary ASOs that were designed and their target sequences. From
this design, 2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by
5-nucleotide intervals can be produced and utilized to target PKD2
RIC pre-mRNAs to increase PC-2 protein production.
TABLE-US-00006 TABLE 5 List of ASOs targeting the PKD2 gene Target
Gene Pre-mRNA Retained Sequence SEQ ID NO: SEQ ID NO: ASOs Intron
SEQ ID NO: PKD2 PKD2:NM_000297 SEQ ID Intron 5 SEQ ID NO: SEQ ID
NO: SEQ ID NO: 5546 NOs: 5825 5545 5547-5824
Example 14: Improved Splicing Efficiency Via ASO-Targeting of PKD2
Intron 5 Increases Transcript Levels
[2025] To determine whether an increase in PKD2 expression could be
achieved by improving splicing efficiency of PKD2 intron 5 using
ASOs, the method described herein can be used. Cell lines of
interest (e.g., ARPE-19 cells, a human retinal epithelium cell line
(American Type Culture Collection (ATCC), USA), or Huh-7, a human
hepatoma cell line (NIBIOHN, Japan), or SK-N-AS, a human
neuroblastoma cell line (ATCC)) are mock-transfected, or
transfected with the targeting ASOs described in Table 5. Cells are
transfected using Lipofectamine RNAiMax transfection reagent
(Thermo Fisher) according to manufacturer's specifications.
Briefly, ASOs are plated in 96-well tissue culture plates and
combined with RNAiMax diluted in Opti-MEM. Cells are detached using
trypsin, resuspended in full medium, and approximately 25,000 cells
are added to the ASO-transfection mixture. Transfection experiments
are carried out in triplicate plate replicates. Final ASO
concentration is 80 nM. Media is changed 6h post-transfection, and
cells are harvested at 24h, using the Cells-to-Ct lysis reagent,
supplemented with DNAse (Thermo Fisher), according to
manufacturer's specifications. cDNA is generated with Cells-to-Ct
RT reagents (Thermo Fisher) according to manufacturer's
specifications. To quantify the amount of splicing at the intron of
interest, quantitative PCR is carried out using Taqman assays with
probes spanning the corresponding exon-exon junction (Thermo
Fisher), listed in Table 5. Taqman assays are carried out according
to manufacturer's specifications, on a QuantStudio 7 Flex Real-Time
PCR system (Thermo Fisher). Target gene assay values are normalized
to RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted. ASOs identified as
increasing the target gene expression by a threshold amount imply
an increase in splicing at that target intron. Together with whole
transcriptome data confirming retention of the target intron (FIG.
27), these results confirm that ASOs can improve the splicing
efficiency of a rate limiting intron.
Example 15: Identification of Intron Retention Events in PRPF3
Transcripts by RNAseq Using Next Generation Sequencing
[2026] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the PRPF3 gene to identify intron-retention events. For
this purpose, polyA+ RNA from nuclear and cytoplasmic fractions of
ARPE-19 (retinal epithelial) cells was isolated and cDNA libraries
constructed using Illumina's TruSeq Stranded mRNA library Prep Kit.
The libraries were pair-end sequenced resulting in 100-nucleotide
reads that were mapped to the human genome (February 2009,
GRCh37/hg19 assembly). The sequencing results for PRPF3 are shown
in FIG. 3. Briefly, FIG. 3 shows the mapped reads visualized using
the UCSC genome browser (operated by the UCSC Genome Informatics
Group (Center for Biomolecular Science & Engineering,
University of California, Santa Cruz, 1156 High Street, Santa Cruz,
Calif. 95064) and described by, e.g., Rosenbloom, et al., 2015,
"The UCSC Genome Browser database: 2015 update," Nucleic Acids
Research 43, Database Issue, doi: 10.1093/nar/gku1177) and the
coverage and number of reads can be inferred by the peak signals.
The height of the peaks indicates the level of expression given by
the density of the reads in a particular region. A schematic
representation of PRPF3 (drawn to scale) is provided by the UCSC
genome browser (below the read signals) so that peaks can be
matched to PRPF3 exonic and intronic regions. Based on this
display, we identified 2 introns in PRPF3 (12 and 13, indicated by
arrows, corresponding to NM_004698: intron 12, and NM_004698;
intron 13, respectively) and 1 intron in PRPF8 (31, indicated by
the arrow, corresponding to NM_006445, intron 31) (FIG. 7) that
have high read density in the nuclear fraction of ARPE-19 cells,
but have very low to no reads in the cytoplasmic fraction of these
cells (as shown for intron 12 in the bottom diagram of FIG. 3, 13
in the bottom diagram of FIG. 5, and for introns 31 in the bottom
diagram of FIG. 5). This indicates that these introns are retained
and that the intron-12 and intron-13, and, intron-31 containing
transcripts remain in the nucleus, and suggests that these retained
PRPF3 and PRPF8 RIC pre-mRNAs, respectively are non-productive, as
they are not exported out to the cytoplasm.
Example 16: Design of ASO-Walk Targeting a Retained Intron
[2027] An ASO walk was designed to target intron 12 of PRPF3 using
the method described herein (FIG. 31). A region immediately
downstream of the intron 12 5' splice site spanning nucleotides +6
to +498 and a region immediately upstream of intron 12 3' splice
site spanning nucleotides -16 to -496 of the intron were targeted
with 2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals (with the exception of ASO P3-IVS12+28).
[2028] An ASO walk was designed to target intron 13 of PRPF3 using
the method described herein (FIG. 33). A region immediately
downstream of the intron 13 5' splice site spanning nucleotides +6
to +146 and a region immediately upstream of intron 13 3' splice
site spanning nucleotides -16 to -146 of the intron were targeted
with 2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals (with the exception of ASOs P3-IVS13+15, P3-IVS13+31, and
P3-IVS13-47).
[2029] An ASO walk was designed to target intron 31 of PRPF8 using
the method described herein (FIG. 35). A region immediately
downstream of the intron 31 5' splice site spanning nucleotides +6
to +156 and a region immediately upstream of intron 31 3' splice
site spanning nucleotides -16 to -156 were targeted with 2'-O-Me
RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals.
[2030] Table 6 lists exemplary ASOs that were designed and their
target sequences.
TABLE-US-00007 TABLE 6 Target Gene Pre-mRNA Retained Sequence SEQ
ID NO. SEQ ID NO. ASOs Intron SEQ ID NO. PRPF3 PRPF3:NM_004698
5830-6064 12 6272 SEQ ID NO. SEQ ID NO. 5828 6065-6148 13 6271 5826
PRPF8 PRPF8:NM_006445 6149-6270 31 6273 SEQ ID NO. SEQ ID NO. 5829
5827
Example 17: Improved Splicing Efficiency Via ASO-Targeting of PRPF3
Intron 12 Increases Transcript Levels
[2031] To determine whether an increase in target gene intron
splicing efficiency could be achieved with ASOs, the method
described herein was used. ARPE-19 cells, a human retinal
epithelium cell line (American Type Culture Collection (ATCC), USA)
were mock-transfected, or transfected with the targeting ASOs
described in Table 6. Cells were transfected using Lipofectamine
RNAiMax transfection reagent (Thermo Fisher) according to vendor's
specifications. Briefly, ASOs were plated in 96-well tissue culture
plates and combined with RNAiMax diluted in Opti-MEM. Cells were
detached using trypsin and resuspended in full medium, and
approximately 25,000 cells were added the ASO-transfection mixture.
Transfection experiments were carried out in triplicate plate
replicates. Final ASO concentration was 80 nM. Media was changed 6h
post-transfection, and cells harvested at 24h, using the
Cells-to-Ct lysis reagent, supplemented with DNAse (Thermo Fisher),
according to vendor's specifications. cDNA was generated with
Cells-to-Ct RT reagents (Thermo Fisher) according to vendor's
specifications. To quantify the amount of splicing at the intron of
interest, quantitative PCR was carried out using Taqman assays with
probes spanning the corresponding exon-exon junction (Thermo
Fisher). Taqman assays were carried out according to vendor's
specifications, on a QuantStudio 7 Flex Real-Time PCR system
(Thermo Fisher). Target gene assay values were normalized to RPL32
(deltaCt) and plate-matched mock transfected samples (delta-delta
Ct), generating fold-change over mock quantitation (2{circumflex
over ( )}-(delta-deltaCt). Average fold-change over mock of the
three plate replicates was plotted (FIG. 37). Several ASOs were
identified that increased the target gene expression, as shown in
FIG. 37, implying an increase in splicing at that target intron.
Together with whole transcriptome data confirming retention of the
target intron (FIG. 36), these results confirm that ASOs can
improve the splicing efficiency of a rate limiting intron.
Example 18: Improved Splicing Efficiency Via ASO-Targeting of PRPF3
Intron 13 Increases Transcript Levels
[2032] To determine whether an increase in target gene intron
splicing efficiency could be achieved with ASOs, the method
described herein was used. ARPE-19 cells, a human retinal
epithelium cell line (American Type Culture Collection (ATCC), USA)
were mock-transfected, or transfected with the targeting ASOs
described in Table 6. Cells were transfected using Lipofectamine
RNAiMax transfection reagent (Thermo Fisher) according to vendor's
specifications. Briefly, ASOs were plated in 96-well tissue culture
plates and combined with RNAiMax diluted in Opti-MEM. Cells were
detached using trypsin and resuspended in full medium, and
approximately 25,000 cells were added the ASO-transfection mixture.
Transfection experiments were carried out in triplicate plate
replicates. Final ASO concentration was 80 nM. Media was changed 6h
post-transfection, and cells harvested at 24h, using the
Cells-to-Ct lysis reagent, supplemented with DNAse (Thermo Fisher),
according to vendor's specifications. cDNA was generated with
Cells-to-Ct RT reagents (Thermo Fisher) according to vendor's
specifications. To quantify the amount of splicing at the intron of
interest, quantitative PCR was carried out using Taqman assays with
probes spanning the corresponding exon-exon junction (Thermo
Fisher). Taqman assays were carried out according to vendor's
specifications, on a QuantStudio 7 Flex Real-Time PCR system
(Thermo Fisher). Target gene assay values were normalized to RPL32
(deltaCt) and plate-matched mock transfected samples (delta-delta
Ct), generating fold-change over mock quantitation (2{circumflex
over ( )}-(delta-deltaCt). Average fold-change over mock of the
three plate replicates was plotted (FIG. 37, FIG. 39). Several ASOs
were identified that increased the target gene expression, as shown
in FIG. 39, implying an increase in splicing at that target intron.
Together with whole transcriptome data confirming retention of the
target intron (FIG. 38), these results confirm that ASOs can
improve the splicing efficiency of a rate limiting intron.
Example 19: Improved Splicing Efficiency Via ASO-Targeting of PRPF8
Intron 31 Increases Transcript Levels
[2033] To determine whether an increase in target gene intron
splicing efficiency could be achieved with ASOs, the method
described herein was used. ARPE-19 cells, a human retinal
epithelium cell line (American Type Culture Collection (ATCC), USA)
were mock-transfected, or transfected with the targeting ASOs
described in Table 6. Cells were transfected using Lipofectamine
RNAiMax transfection reagent (Thermo Fisher) according to vendor's
specifications. Briefly, ASOs were plated in 96-well tissue culture
plates and combined with RNAiMax diluted in Opti-MEM. Cells were
detached using trypsin and resuspended in full medium, and
approximately 25,000 cells were added the ASO-transfection mixture.
Transfection experiments were carried out in triplicate plate
replicates. Final ASO concentration was 80 nM. Media was changed 6h
post-transfection, and cells harvested at 24h, using the
Cells-to-Ct lysis reagent, supplemented with DNAse (Thermo Fisher),
according to vendor's specifications. cDNA was generated with
Cells-to-Ct RT reagents (Thermo Fisher) according to vendor's
specifications. To quantify the amount of splicing at the intron of
interest, quantitative PCR was carried out using Taqman assays with
probes spanning the corresponding exon-exon junction (Thermo
Fisher). Taqman assays were carried out according to vendor's
specifications, on a QuantStudio 7 Flex Real-Time PCR system
(Thermo Fisher). Target gene assay values were normalized to RPL32
(deltaCt) and plate-matched mock transfected samples (delta-delta
Ct), generating fold-change over mock quantitation (2{circumflex
over ( )}-(delta-deltaCt). Average fold-change over mock of the
three plate replicates is plotted (FIG. 41). Several ASOs were
identified that increased the target gene expression, as shown in
FIG. 41, implying an increase in splicing at that target intron.
Together with whole transcriptome data confirming retention of the
target intron (FIG. 40), these results confirm that ASOs can
improve the splicing efficiency of a rate limiting intron.
Example 20: Identification of Intron Retention Events in ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA,
and STX1B Transcripts by RNAseq Using Next Generation
Sequencing
[2034] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4,
RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8,
STXBP1, PRICKLE2, PRRT2, IDUA, and STX1B gene to identify
intron-retention events. For this purpose, polyA+ RNA from nuclear
and cytoplasmic fractions of cells was isolated and cDNA libraries
constructed using Illumina's TruSeq Stranded mRNA library Prep Kit.
The libraries were pair-end sequenced resulting in 100-nucleotide
reads that were mapped to the human genome (February 2009,
GRCh37/hg19 assembly. The mapped reads were visualized using the
UCSC genome browser (operated by the UCSC Genome Informatics Group
(Center for Biomolecular Science & Engineering, University of
California, Santa Cruz, 1156 High Street, Santa Cruz, Calif. 95064)
and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome
Browser database: 2015 update," Nucleic Acids Research 43, Database
Issue, doi: 10.1093/nar/gku1177) and the coverage and number of
reads were inferred by the peak signals. The height of the peaks
indicates the level of expression given by the density of the reads
in a particular region. A schematic representation of the gene was
provided by the UCSC genome browser so that peaks could be matched
to the exonic and intronic regions. Based on this display, we
identified introns that have high read density in the nuclear
fraction of the cells, but have very low to no reads in the
cytoplasmic fraction. This indicated that these introns were
retained and that the intron-containing transcripts remain in the
nucleus, and suggested that these retained RIC pre-mRNAs are
non-productive, as they were not exported out to the cytoplasm.
Example 21: Design of ASO-Walk Targeting
[2035] An ASO walk was designed to target introns using the method
described herein. A region immediately downstream of an intron 5'
splice site spanning nucleotides, e.g., +6 to +69 and a region
immediately upstream of the intron's 3' splice site spanning
nucleotides, e.g., -16 to -79 of the intron were targeted with
2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals.
Example 22: Improved Splicing Efficiency Via ASO-Targeting
[2036] To determine whether an increase in expression of ATP1A2,
CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5,
EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B could be
achieved by improving splicing efficiency of a retained intron in
ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA,
EIF2B5-EIF2B2, NPC1, ADAR, STXBP1, PRICKLE2, PRRT2, or STX1B using
ASOs, ARPE-19 or SK-N-AS cells were mock-transfected, or
transfected with the targeting ASOs described in FIG. 46, FIG. 48,
FIG. 50, FIG. 52, FIG. 54, FIG. 56, FIG. 58, FIG. 61, FIG. 63, FIG.
65, FIG. 76, FIG. 78, FIG. 80, and Table 7. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1,
TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B2, NPC1, ADAR, STXBP1,
PRICKLE2, PRRT2, or STX1B assay values were normalized to RPL32
(deltaCt) and plate-matched mock transfected samples (delta-delta
Ct), generating fold-change over mock quantitation (2{circumflex
over ( )}-(delta-deltaCt). Average fold-change over mock of the
three plate replicates were plotted These results were used to
confirm that inducing splicing of a retained intron in the gene
using ASOs leads to an increase in gene expression.
[2037] FIG. 42 depicts a schematic of the RefSeq Genes for ADAR
intron 2 corresponding to NM_001111. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2038] FIG. 43 depicts a schematic of the RefSeq Genes for ARSA
intron 3 corresponding to NM_001085425. The Percent Intron
Retention (PIR) of the circled intron is shown.
[2039] FIG. 44 depicts a schematic of the RefSeq Genes for ARSA
intron 4 corresponding to NM_001085425. The Percent Intron
Retention (PIR) of the circled intron is shown.
[2040] FIG. 45 depicts a schematic of the RefSeq Genes for DNMT1
intron 30 corresponding to NM_001130823. The Percent Intron
Retention (PIR) of the circled intron is shown.
[2041] FIG. 46 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of DNMT1 mRNA without intron 30 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[2042] FIG. 47 depicts a schematic of the RefSeq Genes for EIF2B2
intron 1 corresponding to NM_014239. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2043] FIG. 48 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of EIF2B2 mRNA without intron 1 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[2044] FIG. 49 depicts a schematic of the RefSeq Genes for EIF2B5
intron 12 corresponding to NM_003907. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2045] FIG. 50 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of EIF2B5 mRNA without intron 12
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[2046] FIG. 51 depicts a schematic of the RefSeq Genes for EIF2B5
intron 13 corresponding to NM_003907. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2047] FIG. 52 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of EIF2B5 mRNA without intron 13
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[2048] FIG. 53 depicts a schematic of the RefSeq Genes for PEX1
intron 10 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2049] FIG. 54 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of PEX1 mRNA without intron 10 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[2050] FIG. 55 depicts a schematic of the RefSeq Genes for PEX1
intron 14 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2051] FIG. 56 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of PEX1 mRNA without intron 14 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[2052] FIG. 57 depicts a schematic of the RefSeq Genes for PRICKLE2
intron 4 corresponding to NM_198859. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2053] FIG. 58 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of PRICKLE2 mRNA without intron 4
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[2054] FIG. 59 depicts a schematic of the RefSeq Genes for PRRT2
intron 1 corresponding to NM_145239. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2055] FIG. 60 depicts a schematic of the RefSeq Genes for RAI1
intron 4 corresponding to NM_030665. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2056] FIG. 61 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of RAI1 mRNA without intron 4 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[2057] FIG. 62 depicts a schematic of the RefSeq Genes for SHANK3
intron 16 corresponding to NM_033517. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2058] FIG. 63 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of SHANK3 mRNA without intron 16
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[2059] FIG. 64 depicts a schematic of the RefSeq Genes for STXBP1
intron 18 corresponding to NM_001032221. The Percent Intron
Retention (PIR) of the circled intron is shown.
[2060] FIG. 65 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of STXBP1 mRNA without intron 18
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[2061] FIG. 66 depicts a schematic of the RefSeq Genes for SETD5
intron 4 corresponding to NM_001080517. The Percent Intron
Retention (PIR) of the circled intron is shown.
[2062] FIG. 67 depicts a schematic of the RefSeq Genes for ATP1A2
intron 22 corresponding to NM_000702. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2063] FIG. 68 depicts a schematic of the RefSeq Genes for CACNA1A
intron 36 corresponding to NM_023035. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2064] FIG. 69 depicts a schematic of the RefSeq Genes for CACNA1A
intron 37 corresponding to NM_023035. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2065] FIG. 70 depicts a schematic of the RefSeq Genes for NF2
intron 16 corresponding to NM_181832. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2066] FIG. 71 depicts a schematic of the RefSeq Genes for PEX1
intron 19 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2067] FIG. 72 depicts a schematic of the RefSeq Genes for PEX1
intron 21 corresponding to NM_000466. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2068] FIG. 73 depicts a schematic of the RefSeq Genes for STX1B
intron 6 corresponding to NM_052874. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2069] FIG. 74 depicts a schematic of the RefSeq Genes for STX1B
intron 7 corresponding to NM_052874. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2070] FIG. 75 depicts a schematic of the RefSeq Genes for NF2
intron 16 corresponding to NM_181832. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2071] FIG. 76 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of NF2 mRNA without intron 16 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[2072] FIG. 77 depicts a schematic of the RefSeq Genes for NPC1
intron 15 corresponding to NM_000271. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2073] FIG. 78 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of NPC1 mRNA without intron 15 in
ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs
over mock treated cells. Data is normalized to RPL32
expression.
[2074] FIG. 79 depicts a schematic of the RefSeq Genes for ATP1A2
intron 22 corresponding to NM_000702. The Percent Intron Retention
(PIR) of the circled intron is shown.
[2075] FIG. 80 depicts an exemplary graph showing the average (n=3)
fold change in expression levels of ATP1A2 mRNA without intron 22
in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated
ASOs over mock treated cells. Data is normalized to RPL32
expression.
[2076] FIG. 81 depicts a schematic of the RefSeq Genes for TCF4
intron 18 corresponding to NM_001243226. The Percent Intron
Retention (PIR) of the circled intron is shown.
[2077] Table 8 illustrates percentage of retained intron with
respect to the genes of interest.
TABLE-US-00008 TABLE 8 Retained Introns in Genes of Interest
Retained Intron Gene RNA Accession (Percent Intron Symbol Gene ID
Number Retention) Cells ATP1A2 477 NM_000702 Intron 22 (14%) HCN
CACNA1A 773 NM_023035 Intron 37 (26%) AST CACNA1A 773 NM_001127221
Intron 36 (56%) AST Intron 36 (55%) SETD5 55209 NM_001080517 Intron
4 (42%) HCN SHANK3 85358 NM_033517 Intron 16 (7%) AST NF2 4771
NM_181832 Intron 16 (N/A) HCN DNMT1 1786 NM_001379 Intron 29 (13%)
HCN TCF4 6925 NM_003199 Intron 17 (15%) AST TCF4 6925 NM_001243228
Intron 17 (29%) AST RAI1 10743 NM_030665 Intron 4 (13%) AST PEX1
5189 NM_000466 Intron 10 (25%) HCN Intron 14 (19%) Intron 19 (18%)
Intron 21 (14%) ARSA 410 NM_001085425 Intron 3 (N/A) HCN Intron 4
(N/A) EIF2B5 8893 NM_003907 Intron 12 (42%) HCN Intron 13 (29%)
EIF2B1 1967 NM_001414 Intron 6 (27%) HCN EIF2B2 8892 NM_014239
Intron 1 (9%) HCN NPC1 4864 NM_000271 Intron 15 (11%) HCN ADAR 103
NM_001111 Intron 2 (12%) AST MFSD8 256471 NM_152778 Intron 12 (42%)
HCN STXBP1 6812 NM_001032221 Intron 18 (N/A) AST PRICKLE2 166336
NM_198859 Intron 4 (19%) AST PRRT2 112476 NM_145239 Intron 1 (77%)
HCN STX1B 112755 NM_052874 Intron 6 (11%) HCN, AST Intron 7 (12%)
IDUA 607015 NM_000203 Intron 3 (25%) HCN Intron 4 (63%) Intron 5
(16%) Intron 6 (N/A) Intron 7 (N/A)
Example 23: Identification of Intron Retention Events in ROM1,
TEAD1, RDH5, PAX6, FSCN2, MYOC, TCF4, MFSD8, CTNS, OPTN, RLBP1,
RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 and IDUA
Transcripts by RNAseq Using Next Generation Sequencing
[2078] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the ROM1, TEAD1, RDH5, PAX6, FSCN2, TCF4, MFSD8, CTNS,
OPTN, RLBP1, RPE65, LRAT, RDH8, RDH12, RGR, CNGA3, ALMS1, PER1 and
IDUA gene to identify intron-retention events. For this purpose,
polyA+ RNA from nuclear and cytoplasmic fractions of cells was
isolated from cDNA libraries constructed using Illumina's TruSeq
Stranded mRNA library Prep Kit. ARPE-19 cells or astrocytes were
used for most analyses, and in certain cases (for RDH8 and RDH12)
human retinal transcriptome data was used (Farkas, et al., 2013,
"Transcriptome analyses of the human retina identify unprecedented
transcript diversity and 3.5 Mb of novel transcribed sequence via
significant alternative splicing and novel genes," BMC Genomics
14:486, incorporated herein by reference). The libraries were
pair-end sequenced resulting in 100-nucleotide reads that can be
mapped to the human genome. The mapped reads were visualized using
the UCSC genome browser (operated by the UCSC Genome Informatics
Group (Center for Biomolecular Science & Engineering,
University of California, Santa Cruz, 1156 High Street, Santa Cruz,
Calif. 95064) and described by, e.g., Rosenbloom, et al., 2015,
"The UCSC Genome Browser database: 2015 update," Nucleic Acids
Research 43, Database Issue, doi: 10.1093/nar/gku1177) and the
coverage and number of reads were inferred by the peak signals. The
height of the peaks indicates the level of expression given by the
density of the reads in a particular region. A schematic
representation of the gene (drawn to scale) was provided by the
UCSC genome browser (below the read signals) so that peaks can be
matched to the exonic and intronic regions. Based on this display,
we identified introns that have high read density in the nuclear
fraction of the cells, but have very low to no reads in the
cytoplasmic fraction. This indicated that these introns were
retained and that the intron-containing transcripts remain in the
nucleus, and suggested that these retained RIC pre-mRNAs are
non-productive, as they were not exported out to the cytoplasm.
Example 24: Identification of Intron Retention Events in CRX,
ABCA4, MYOC, and NXNL1 Transcript by RNAseq Using Next Generation
Sequencing
[2079] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the CRX, ABCA4, MYOC, and NXNL1 genes described herein
to identify intron-retention events. For this purpose, polyA+ RNA
from nuclear and cytoplasmic fractions of THLE-3 (human liver
epithelial) cells is isolated and cDNA libraries constructed using
Illumina's TruSeq Stranded mRNA library Prep Kit. The libraries are
pair-end sequenced resulting in 100-nucleotide reads that were
mapped to the human genome. The mapped reads are visualized using
the UCSC genome browser (operated by the UCSC Genome Informatics
Group (Center for Biomolecular Science & Engineering,
University of California, Santa Cruz, 1156 High Street, Santa Cruz,
Calif. 95064) and described by, e.g., Rosenbloom, et al., 2015,
"The UCSC Genome Browser database: 2015 update," Nucleic Acids
Research 43, Database Issue, doi: 10.1093/nar/gku1177) and the
coverage and number of reads were inferred by the peak signals. The
height of the peaks indicates the level of expression given by the
density of the reads in a particular region. A schematic
representation of the gene is provided by the UCSC genome browser
so that peaks can be matched to the exonic and intronic regions.
Based on this display, we identify introns that have high read
density in the nuclear fraction of THLE-3 cells, but have very low
to no reads in the cytoplasmic fraction of these cells. This
indicates that these introns are retained and that the
intron-containing transcripts remain in the nucleus, and suggests
that these retained RIC pre-mRNAs are non-productive, as they are
not exported out to the cytoplasm.
Example 25: Design of ASO-Walk Targeting
[2080] An ASO walk was designed to target introns using the method
described herein. A region immediately downstream of an intron 5'
splice site, e.g., spanning nucleotides +6 to +69 and a region
immediately upstream of the intron's 3' splice site, e.g., spanning
nucleotides -16 to -68 of the intron was targeted with 2'-O-Me RNA,
PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals. Table 9
lists exemplary ASOs that were designed and their target
sequences.
TABLE-US-00009 TABLE 9 Target Re- Sequence Gene Pre-mRNA ASOs
tained SEQ ID SEQ ID NO. SEQ ID NO. SEQ ID NO. Intron NO. ABCA4
ABCA4:NM_000350 34873-35104 40 61463 SEQ ID NO. SEQ ID NO. 34813
35105-35332 38 61495 34790 35333-35563 36 61445 35564-35805 44
61470 35806-35915 39 61453 RPE65 RPE65:NM_000329 35916-36082 9
61480 SEQ ID NO. SEQ ID NO. 34814 36083-36317 10 61460 34791 MYOC
MYOC:NM_000261 36318-36644 1 61458 SEQ ID NO. SEQ ID NO. 34815
36645-37107 2 61485 34792 CNGA3 CNGA3:NM_001298 37108-37333 6 61500
SEQ ID NO. SEQ ID NO. 34816 34793 CNGA3:NM_001079878 37334-37559 5
61500 SEQ ID NO. 34817 MFSD8 MFSD8:NM_152778 37560-37641 11 61492
SEQ ID NO. SEQ ID NO. 34818 37642-38420 12 61497 34794 IDUA
IDUA:NM_000203 38421-38486 3 61457 SEQ ID NO. SEQ ID NO. 34819
38487-38602 4 61468 34795 38603-38668 5 61489 38669-38741 6 61444
38742-38826 7 61452 IDUA:NR_110313 38827-38892 3 61457 SEQ ID NO.
34820 38893-39008 4 61468 39009-39074 5 61489 39075-39147 6 61444
39148-39232 7 61452 LRAT LRAT:NM_001301645 39233-40334 2 61474 SEQ
ID NO. SEQ ID NO. 34821 34796 LRAT:NM_004744 40335-41436 2 61474
SEQ ID NO. 34822 OPTN OPTN:NM_001008211 41437-41669 9 61503 SEQ ID
NO SEQ ID NO. 34823 34797 OPTN:NM_001008212 41670-41902 8 61503 SEQ
ID NO. 34824 OPTN:NM_001008213 41903-42135 9 61503 SEQ ID NO. 34825
OPTN:NM_021980 42136-42368 7 61503 SEQ ID NO. 34826 RGR
RGR:NM_002921 42369-42595 1 61446 SEQ ID NO. SEQ ID No. 34827
42596-42829 2 61476 34798 RGR:NM_001012722 42830-43056 1 61446 SEQ
ID NO. 34828 43057-43288 2 61472 RGR:NM_001012720 43289-43515 1
61446 SEQ ID NO. 34829 43516-43747 2 61472 TEAD1 TEAD1 :NM_021961
43748-43952 4 61461 SEQ ID NO. SEQ ID NO. 34830 34799 PAX6
PAX6:NM_001310160 43953-44085 2 61486 SEQ ID NO. SEQ ID NO. 34831
44086-44296 3 61466 34800 PAX6:NM_001310161 44297-44563 1 61496 SEQ
ID NO. 34832 44564-44630 3 61467 44631-44841 4 61502
PAX6:NM_001258465 44842-45041 3 61483 SEQ ID NO. 34833 45042-45273
4 61448 45274-45484 5 61502 PAX6:NM_000280 45485-45684 4 61483 SEQ
ID NO. 34834 45685-45916 5 61448 45917-46127 6 61502
PAX6:NM_001258464 46128-46327 4 61483 SEQ ID NO. 34835 46328-46559
5 61448 46560-46770 6 61502 PAX6:NM_001604 46771-46970 4 61483 SEQ
ID NO. 34836 46971-47037 6 61467 47038-47248 7 61502
PAX6:NM_001127612 47249-47448 4 61483 SEQ ID NO. 34837 47449-47680
5 61448 47681-47891 6 61502 PAX6:NM_001258462 47892-48091 4 61483
SEQ ID NO. 34838 48092-48158 6 61467 48159-48369 7 61502
PAX6:NM_001310159 48370-48569 2 61483 SEQ ID NO. 34839 48570-48801
3 61448 48802-49012 4 61502 PAX6:NM_001310158 49013-49212 4 61483
SEQ ID NO. 34840 49213-49279 6 61467 49280-49490 7 61502
PAX6:NM_001258463 49491-49690 4 61483 SEQ ID NO. 34841 49691-49757
6 61467 49758-49968 7 61502 ROM1 ROM1:NM_000327 49969-50275 1 26665
SEQ ID NO. SEQ ID NO. 34842 34801 RDH5 RDH5:NM_002905 50276-50489 1
26704 SEQ ID NO SEQ ID NO. 34843 50490-50633 2 26666 34802
RDH5:NM_001199771 50634-50846 1 26709 SEQ ID NO. 34844 50847-50991
2 26684 RDH12 RDH12:NM_152443 50992-51247 7 26693 SEQ ID NO. SEQ ID
NO. 34845 34803 NR2E3 NR2E3:NM_014249 51248-51450 1 26702 SEQ ID
NO. SEQ ID NO. 34846 51451-51061 2 26660 34804 51517-51587 3 26705
51588-51679 4 26698 51680-51916 5 26658 51917-51958 6 26676
51959-52043 7 26712 NR2E3:NM_016346 52044-52246 1 26702 SEQ ID NO.
34847 52247-52312 2 26660 52313-52383 3 26705 52384-52475 4 26698
52476-52712 5 26658 52713-52754 6 26676 52755-52998 7 26701 RLBP1
RLBP1:NM_000326 52999-53172 2 26673 SEQ ID NO. SEQ ID NO. 34848
53173-53427 5 26667 34805 CTNS CTNS:NM_004937 53428-53650 9 26690
SEQ ID NO SEQ ID NO. 34849 53651-53875 10 26692 34806
CTNS:NM_001031681 53876-54098 9 26690 SEQ ID NO. 34850 54099-54323
10 26692 PER1 PER1:NM_002616 54324-54571 1 26682 SEQ ID NO. SEQ ID
NO. 34851 54572-54634 14 26710 34807 FSCN2 FSCN2:NM_012418
54635-55021 1 26670 SEQ ID NO. SEQ ID NO. 34852 55022-55136 3 26662
34808 FSCN2:NM_001077182 55137-55523 1 26670 SEQ ID NO. 34853
55524-55638 3 26661 TCF4 TCF4:NM_001243236 55639-55880 9 26688 SEQ
ID NO SEQ ID NO. 34854 34809 TCF4:NM_001243235 55881-56122 9 26688
SEQ ID NO. 34855 TCF4:NM_001243234 56123-56366 9 26689 SEQ ID NO.
34856 TCF4:NM_001243233 56367-56608 12 26688 SEQ ID NO. 34857
TCF4:NM_001243232 56609-56852 12 26689 SEQ ID NO. 34858
TCF4:NM_001243231 56853-57094 14 26688 SEQ ID NO. 34859
TCF4:NM_003199 57095-57336 16 26688 SEQ ID NO. 34860
TCF4:NM_001306207 57337-57578 15 26688 SEQ ID NO. 34861
TCF4:NM_001306208 57579-57820 12 26688 SEQ ID NO. 34862
TCF4:NM_001243227 57821-58064 15 26689 SEQ ID NO. 34863
TCF4:NM_001243228 58065-58308 16 26689 SEQ ID NO. 34864
TCF4:NM_001243230 58309-58550 15 26688 SEQ ID NO. 34865
TCF4:NM_001243226 58551-58794 17 26689 SEQ ID NO. 34866
TCF4:NM_001083962 58795-59038 16 26689 SEQ ID NO. 34867
TCF4:NM_001330605 59039-59282 11 26689 SEQ ID NO. 34868
TCF4:NM_001330604 59283-59526 16 26689 SEQ ID NO. 34869 RDH8
RDH8:NM_015725 59527-59662 4 26680 SEQ ID NO. SEQ ID NO. 34870
34810 NXNL1 NXNL1:NM_138454 59663-60020 1 26699 SEQ ID NO. SEQ ID
NO. 34871 34811 CRX CRX:NM_000554 60021-60254 1 26675 SEQ ID NO.
SEQ ID NO. 34872 60255-60484 2 26695 34812 60485-61443 3 26686
Example 26: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases FSCN2 Transcript Levels
[2081] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 83, FIG. 85,
and FIG. 86). Several ASOs were identified that increased the
target gene expression, as shown in FIG. 83, FIG. 85, and FIG. 86,
implying an increase in splicing at that target intron. Together
with whole transcriptome data confirming retention of the target
intron (FIG. 82 and FIG. 84), these results confirm that ASOs can
improve the splicing efficiency of a rate limiting intron.
Example 27: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases MFSD8 Transcript Levels
[2082] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 89). Several
ASOs were identified that increased the target gene expression, as
shown in FIG. 89, implying an increase in splicing at that target
intron. Together with whole transcriptome data confirming retention
of the target intron (FIG. 86 and FIG. 88), these results confirm
that ASOs can improve the splicing efficiency of a rate limiting
intron.
Example 28: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases OPTN Transcript Levels
[2083] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 91). Several
ASOs were identified that increased the target gene expression, as
shown in FIG. 91, implying an increase in splicing at that target
intron. Together with whole transcriptome data confirming retention
of the target intron (FIG. 90), these results confirm that ASOs can
improve the splicing efficiency of a rate limiting intron.
Example 29: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases RDH5 Transcript Levels
[2084] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 93). Several
ASOs were identified that increased the target gene expression, as
shown in FIG. 93, implying an increase in splicing at that target
intron. Together with whole transcriptome data confirming retention
of the target intron (FIG. 92 and FIG. 94), these results confirm
that ASOs can improve the splicing efficiency of a rate limiting
intron.
Example 30: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases RLBP1 Transcript Levels
[2085] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 96). Several
ASOs were identified that increased the target gene expression, as
shown in FIG. 96, implying an increase in splicing at that target
intron. Together with whole transcriptome data confirming retention
of the target intron (FIG. 95), these results confirm that ASOs can
improve the splicing efficiency of a rate limiting intron.
Example 31: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases ABCA4 Transcript Levels
[2086] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 108 and FIG.
111). Several ASOs were identified that increased the target gene
expression, as shown in FIG. 108 and FIG. 111, implying an increase
in splicing at that target intron. These results confirm that ASOs
can improve the splicing efficiency of a rate limiting intron.
Example 32: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases IDUA Transcript Levels
[2087] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 115). Several
ASOs were identified that increased the target gene expression, as
shown in FIG. 115, implying an increase in splicing at that target
intron. Together with whole transcriptome data confirming retention
of the target intron (FIG. 114, FIG. 116, FIG. 117, FIG. 118, and
FIG. 119), these results confirm that ASOs can improve the splicing
efficiency of a rate limiting intron.
Example 33: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases CTNS Transcript Levels
[2088] To determine an increase in target gene intron splicing
efficiency with ASOs we used the method described herein. ARPE-19
cells, a human retinal epithelium cell line (American Type Culture
Collection (ATCC), USA) were mock-transfected, or transfected with
the targeting ASOs described in Table 9. Cells were transfected
using Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher). Taqman assays were carried out according to
vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR
system (Thermo Fisher). Target gene assay values were normalized to
RPL32 (deltaCt) and plate-matched mock transfected samples
(delta-delta Ct), generating fold-change over mock quantitation
(2{circumflex over ( )}-(delta-deltaCt). Average fold-change over
mock of the three plate replicates is plotted (FIG. 122). Several
ASOs were identified that increased the target gene expression, as
shown in FIG. 122, implying an increase in splicing at that target
intron. Together with whole transcriptome data confirming retention
of the target intron (FIG. 120 and FIG. 121), these results confirm
that ASOs can improve the splicing efficiency of a rate limiting
intron.
Example 34: Identification of Intron Retention Events in AMT, ADA,
PPOX, UROD, HMBS, ACADVL, PC, IVD, GALT, LDLRAP1, POGLUT1, PIK3R1,
TRIB1, TGFB1, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6,
HSD3B7, CERS2 and NCOA5 Transcripts by RNAseq Using Next Generation
Sequencing
[2089] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, GALT,
LDLRAP1, POGLUT1, PIK3R1, TRIB1, TGFB1, PNPLA3, ATP7B, FAH, ASL,
HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 genes described
herein to identify intron-retention events. For this purpose,
polyA+ RNA from nuclear and cytoplasmic fractions of THLE-3 (human
liver epithelial) cells was isolated and cDNA libraries constructed
using Illumina's TruSeq Stranded mRNA library Prep Kit. The
libraries were pair-end sequenced resulting in 100-nucleotide reads
that were mapped to the human genome (February 2009, GRCh37/hg19
assembly). The mapped reads were visualized using the UCSC genome
browser (operated by the UCSC Genome Informatics Group (Center for
Biomolecular Science & Engineering, University of California,
Santa Cruz, 1156 High Street, Santa Cruz, Calif. 95064) and
described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome
Browser database: 2015 update," Nucleic Acids Research 43, Database
Issue, doi: 10.1093/nar/gku1177) and the coverage and number of
reads were inferred by the peak signals. The height of the peaks
indicates the level of expression given by the density of the reads
in a particular region. A schematic representation of the gene was
provided by the UCSC genome browser so that peaks could be matched
to the exonic and intronic regions. Based on this display, introns
were identified that have high read density in the nuclear fraction
of THLE-3 cells, but have very low to no reads in the cytoplasmic
fraction of these cells (see Table 10 and 11 and FIGs. for percent
intron retention (PIR) data obtained). This indicated that these
introns were retained and that the intron-containing transcripts
remain in the nucleus, and suggested that these retained RIC
pre-mRNAs are non-productive, as they were not exported out to the
cytoplasm.
Example 35: Identification of Intron Retention Events in APOA5,
HNF4A, GCK, HNF1A, HAMP, and THPO Transcripts by RNAseq Using Next
Generation Sequencing
[2090] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts,
e.g., those produced by the APOA5, HNF4A, GCK, HNF1A, HAMP, and
THPO genes described herein, to identify intron-retention events.
For this purpose, polyA+ RNA from nuclear and cytoplasmic fractions
of THLE-3 (human liver epithelial) cells was isolated and cDNA
libraries constructed using Illumina's TruSeq Stranded mRNA library
Prep Kit. The libraries were pair-end sequenced resulting in
100-nucleotide reads that were mapped to the human genome (February
2009, GRCh37/hg19 assembly). The mapped reads were visualized using
the UCSC genome browser (operated by the UCSC Genome Informatics
Group (Center for Biomolecular Science & Engineering,
University of California, Santa Cruz, 1156 High Street, Santa Cruz,
Calif. 95064) and described by, e.g., Rosenbloom, et al., 2015,
"The UCSC Genome Browser database: 2015 update," Nucleic Acids
Research 43, Database Issue, doi: 10.1093/nar/gku1177) and the
coverage and number of reads were inferred by the peak signals. The
height of the peaks indicated the level of expression given by the
density of the reads in a particular region. A schematic
representation of the gene was provided by the UCSC genome browser
so that peaks could be matched to the exonic and intronic regions.
Based on this display, introns were identified that have high read
density in the nuclear fraction of THLE-3 cells, but have very low
to no reads in the cytoplasmic fraction of these cells (see Table
10 and 11 and FIGs. for percent intron retention (PIR) data
obtained). This indicated that these introns were retained and that
the intron-containing transcripts remained in the nucleus, and
suggested that these retained RIC pre-mRNAs were non-productive, as
they were not exported out to the cytoplasm.
Example 36: Design of ASO-Walk Targeting a Retained Intron
[2091] An ASO walk was designed to target a retained intron using
the method described herein. A region immediately downstream of the
intron 5' splice site, e.g., spanning nucleotides +6 to +69 and a
region immediately upstream of intron 3' splice site, e.g.,
spanning nucleotides -16 to -79 of the intron was targeted with
2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals. Table 10 lists retained introns in genes of interest in
THLE-3 cells. Table 11 lists exemplary ASOs that were designed and
their target sequences.
TABLE-US-00010 TABLE 10 Gene Gene RNA Accession Retained Intron
(Percent Symbol ID Number Intron Retention) AMT 275 NM_000481
Intron 4(39%) Intron 6(15%) ADA 100 NM_000022 Intron 4(11%) Intron
11(12%) Intron 7(N/A) PPOX 5498 NM_000309 Intron 5(59%) Intron
4(22%) Intron 8(18%) Intron 12(18%) UROD 7389 NM_000374 Intron
3(20%) Intron 4(17%) Intron 5(14%) Intron 6(19%) Intron 7(18%) HMBS
3145 NM_000190 Intron 8(15%) Intron 9(12%) ACADVL 37 NM_000018
Intron 3(N/A) Intron 5(N/A) Intron 8(N/A) Intron 9(N/A) Intron
10(15%) Intron 13(15%) PC 5091 NM_022172 Intron 16(25%) IVD 3712
NM_002225 Intron 7(22%) Intron 9(13%) Intron 11(23%) APOA5 116519
No reads No reads GALT 2592 NM_000155 Intron 5(13%) Intron 7(21%)
Intron 10(15%) Intron 2(N/A) Intron 3(N/A) Intron 4(N/A) Intron
8(N/A) Intron 9(N/A) LDLRAP1 26119 NM_015627 Intron 1(12%) Intron
8(12%) GCK 2645 No reads No reads POTGLUT1 56983 NM_152305 Intron
1(18%) POTGLUT1 56983 NR 024265 Intron 4(47%) Intron 5(44%) PIK3R1
5295 NM_181523 Intron 3(7%) Intron 9(11%) HNF1A 6927 No reads No
reads TRIB1 10221 NM_025195 Intron 2(48%) Intron 1(N/A) TGFB1 7040
NM_000660 Intron 4(N/A) HAMP 57817 No reads No reads THPO 7066 No
reads No reads PNPLA3 80339 NM_025225 Intron 1(8%) Intron 4(34%)
Intron 5(16%) Intron 7(41%) ATP7B 540 NM_000053 Intron 7(N/A)
Intron 13(8%) FAH 2184 NM_000137 Intron 1(N/A) ASL 435 NM_000048
Intron 7(24%) Intron 8(N/A) Intron 9(23%) Intron 16(16%) HFE 3077
NM_000410 Intron 2(N/A) ALMS1 7840 NM_015120 Intron 21(60%) PPARD
5467 NM_006238 Intron 3(20%) Intron 4(18%) Intron 5(12%) Intron
6(17%) Intron 7(N/A) IL6 3569 NM_000600 Intron 1(N/A) Intron 2(10%)
Intron 3(10%) Intron 4(N/A) HSD3B7 80270 NM_001142777 Intron 1(74%)
Intron 2(25%) Intron 3(14%) Intron 4(22%) HSD3B7 80270 NM_025193
Intron 5(12%) Intron 6(24%) CERS2 29956 NM_022075 Intron 6(13%)
NCOA5 57727 NM_020967 Intron 2(22%)
TABLE-US-00011 TABLE 11 Gene Pre-mRNA ASOs Retained Target Sequence
SEQ ID NO. SEQ ID NO. SEQ ID NOs. Intron SEQ ID NO. LDLRAP1
LDLRAP1: 61634-62185 8 139857 SEQ ID NM_015627 62186-62428 1 139962
NO. 61504 SEQ ID NO. 61535 UROD UROD: NM_000374 62429-62467 3
139913 SEQ ID SEQ ID NO. 61536 62468-62562 4 139889 NO. 61505
62563-62653 5 139914 62654-62782 6 139963 62783-62855 7 139966
UROD: NR_036510 62856-62894 3 139913 SEQ ID NO. 61537 62895-62989 4
139889 62990-63080 5 139914 63081-63209 6 139963 63210-63282 7
139966 CERS2 CERS2: NM_022075 63283-63342 6 139950 SEQ ID SEQ ID
NO. 61538 NO. 61506 CERS2: NM_181746 63343-63402 6 139950 SEQ ID
NO. 61539 PPOX PPOX: NM_000309 63403-63462 8 139964 SEQ ID SEQ ID
NO. 61540 63463-63563 5 139976 NO. 61507 63664-63613 12 139983
63614-63752 4 139924 PPOX: 63753-63812 8 139964 NM_001122764
63813-63913 5 139976 SEQ ID NO. 61541 63914-63963 12 139983
63964-64102 4 139924 ALMS1 ALMS1: 64103-64315 21 139902 SEQ ID
NM_015120 NO. 61508 SEQ ID NO. 61542 AMT AMT: 64316-64530 3 139986
SEQ ID NM_001164710 64531-64613 5 139968 NO. 61509 SEQ ID NO. 61543
AMT: 64614-64730 3 139937 NM_001164711 64731-64813 5 139968 SEQ ID
NO. 61544 AMT: 64814-64930 4 139937 NM_001164712 64931-65013 6
139968 SEQ ID NO. 61545 AMT: NM_000481 65014-65130 4 139937 SEQ ID
NO. 61546 65131-65213 6 139968 AMT: NR_028435 65214-65330 4 139937
SEQ ID NO. 61547 65331-65413 6 139968 POGLUT1 POGLUT1: 65414-65595
1 61503 SEQ ID NM_152305 65596-65836 4 139853 NO. 61510 SEQ ID NO.
61548 65837-66062 5 139935 POGLUT1: 66063-66234 1 61503 NR_024265
66235-66513 4 139942 SEQ ID NO. 61549 66514-66767 5 139892 THPO
THPO: 66768-67028 2 139928 SEQ ID NM_001289998 67029-67119 3 139903
NO. 61511 SEQ ID NO. 61550 67120-67207 4 139896 67208-67446 5
139854 67447-67747 6 139884 THPO: 67748-67976 1 139869 NM_001177598
67977-68067 2 139903 SEQ ID NO. 61551 68068-68155 3 139896
68156-68394 4 139854 68395-68695 5 139960 THPO: 68696-68956 2
139928 NM_001290022 68957-69047 3 139903 SEQ ID NO. 61552
69048-69135 4 139896 69136-69373 5 139854 69374-69675 6 139946
THPO: 69676-69936 2 139928 NM_001290003 69937-70027 3 139903 SEQ ID
NO. 61553 70028-70115 4 139896 70116-70354 5 139854 70355-70655 6
139884 THPO: 70656-70884 1 139869 NM_001289997 70885-70975 2 139903
SEQ ID NO. 61554 70976-71063 3 139896 71064-71302 4 139854
71303-71391 5 139865 THPO: NM_000460 71392-71620 1 139869 SEQ ID
NO. 61555 71621-71711 2 139903 71711-71799 3 139896 71800-72038 4
139854 72039-72339 5 139884 THPO: 72340-72568 1 139869 NM_001177597
72569-72659 2 139903 SEQ ID NO. 61556 72660-72747 3 139896
72748-72986 4 139854 72987-73287 5 139946 THPO: 73288-73508 1
139949 NM_001290028 73509-73599 2 139903 SEQ ID NO. 61557
73600-73687 3 139896 73688-73926 4 139854 73927-74227 5 139884
THPO: 74228-74488 2 139928 NM_001290027 74489-74579 3 139903 SEQ ID
NO. 61558 74580-74667 4 139896 74668-74906 5 139854 74907-74995 6
139865 THPO: 74996-75256 2 139928 NM_001290026 75257-75347 3 139903
SEQ ID NO. 61559 75348-75435 4 139896 75436-75674 5 139854
75675-75975 6 139960 PIK3R1 PIK3R1: 75976-76047 9 139893 SEQ ID
NM_181523 76048-76163 3 139921 NO. 61512 SEQ ID NO. 61560 PIK3R1:
76164-76235 3 139893 NM_181524 SEQ ID NO. 61561 PIK3R1: 76236-76307
3 139893 NM_181504 SEQ ID NO. 61562 PIK3R1: 76308-76379 2 139893
NM_001242466 SEQ ID NO. 61563 PPARD PPARD: 76380-76636 3 139917 SEQ
ID NM_006238 76637-76879 4 139981 NO. 61513 SEQ ID NO. 61564
76880-77131 5 139975 77132-77291 6 139860 77292-77982 7 139898
PPARD: 77983-78239 3 139917 NM_177435 78240-78482 4 139981 SEQ ID
NO. 61565 78483-78734 5 139975 78735-79015 6 139862 PPARD:
79016-79706 8 139898 NM_001171818 79707-79963 4 139917 SEQ ID NO.
61566 79964-80206 5 139981 80207-80458 6 139975 80459-80618 7
139860 PPARD: 80619-80848 2 139877 NM_001171819 80849-81091 3
139981 SEQ ID NO. 61567 81092-81343 4 139975 81344-81503 5 139860
81504-82194 6 139898 PPARD: 82195-82461 3 139993 NM_001171820
82462-82621 4 139860 SEQ ID NO. 61568 82622-83312 5 139898 HFE HFE:
NM_139007 83313-83586 1 139922 SEQ ID SEQ ID NO. 61569 NO. 61514
HFE: NM_139006 83587-83730 2 139887 SEQ ID NO. 61570 HFE: NM_139004
83731-84016 2 140003 SEQ ID NO. 61571 HFE: NM_139003 84017-84296 2
139927 SEQ ID NO. 61572 HFE: 84297-84440 2 139887 NM_001300749 SEQ
ID NO. 61573 HFE: NM_139009 84441-84570 2 139969 SEQ ID NO. 61574
HFE: NM_139008 84571-84844 1 139922 SEQ ID NO. 61575 HFE: NM_000410
84845-84988 2 139887 SEQ ID NO. 61576 IL6 IL6: NM_001318095
84989-85209 1 139939 SEQ ID SEQ ID NO. 61577 85210-85394 2 139916
NO. 61515 85395-85725 3 139918 IL6: NM_000600 85726-85808 1 139901
SEQ ID NO. 61578 85809-86044 2 139906 86045-86229 3 139916
86230-86560 4 139918 GCK GCK: NM_033508 86561-86788 1 139948 SEQ ID
SEQ ID NO. 61579 86789-86998 2 139984 NO. 61516 86999-87216 3
139882 87217-87444 4 139934 87445-87647 5 139972 87648-87700 6
139911 87701-87934 7 139880 87935-88177 8 139920 88178-88385 9
139890 88386-88646 10 139958 GCK: NM_000162 88647-88937 1 139987
SEQ ID NO. 61580 88938-89155 2 139882 89156-89383 3 139934
89384-89586 4 139972 89587-89639 5 139911 89640-89873 6 139880
89874-90116 7 139920 90117-90324 8 139890 90325-90585 9 139958 GCK:
NM_033507 90586-90831 1 139873 SEQ ID NO. 61581 90832-91049 2
139882 91050-91277 3 139934 91278-91480 4 139972 91481-91533 5
139911 91534-91767 6 139880 91768-92010 7 139920 92011-92218 8
139890 92219-92479 9 139958 ASL ASL: NM_000048 92480-92597 8 139997
SEQ ID SEQ ID NO. 61582 92598-92672 9 139907 NO. 61517 92673-92815
16 139874 92816-92855 7 139868 ASL: 92856-92930 8 139907
NM_001024943 92931-93073 15 139874 SEQ ID NO. 61583 93074-93113 6
139868 93114-93231 7 139997 ASL: 93232-93306 8 139907 NM_001024944
93307-93346 6 139868 SEQ ID NO. 61584 93347-93489 14 139874
93490-93607 7 139997 ASL: 93608-93750 14 139874 NM_001024946
93751-93896 6 139856 SEQ ID NO. 61585 93897-386184 7 139907 TRIB1
TRIM: NM_025195 93972-94373 1 139990 SEQ ID SEQ ID NO. 61586
94374-95069 2 139989 NO. 61518 TRIB1: 95070-95327 1 139989
NM_001282985 95328-96023 2 139989 SEQ ID NO. 61587 GALT GALT:
NM_000155 96024-96104 2 139992 SEQ ID SEQ ID NO. 61588 96105-96137
3 139919 NO. 61519 96138-96188 4 139919 96189-96243 5 139855
96244-96338 7 139938 96339-96387 8 139996 96388-96491 9 139926
96492-96719 10 139940 GALT: 96720-96871 2 139952 NM_001258332
96872-96926 3 139855 SEQ ID NO. 61589 96927-97021 5 139938
97022-97070 6 139996 97071-97174 7 139926 97175-97402 8 139940 PC
PC: NM_000920 97403-97533 17 139998 SEQ ID SEQ ID NO. 61590 NO.
61520 PC: NM_001040716 97534-97664 18 139998 SEQ ID NO. 61591 PC:
NM_022172 97665-97795 16 139998 SEQ ID NO. 61592 APOA5 APOA5:
97796-97900 1 139910 SEQ ID NM_052968 97901-97958 2 140002 NO.
61521 SEQ ID NO. 61593 97959-98393 3 139923 APOA5: 98394-98497 1
139875 NM_001166598 98498-98550 2 139900 SEQ ID NO. 61594
98551-98985 3 139923 HMBS HMBS: NM_000190 98986-99057 10 139858 SEQ
ID SEQ ID NO. 61595 99058-99135 11 139970 NO. 61522 HMBS:
99136-99266 10 139957 NM_001258208 SEQ ID NO. 61596 HMBS:
99267-99338 10 139858 NM_001024382 99339-99416 11 139970 SEQ ID NO.
61597 HMBS: 99417-99547 10 139957 NM_001258209 SEQ ID NO. 61598
HNF1A HNF1A: 99548-99864 1 139956 SEQ ID NM_000545 99865-100124 2
139864 NO. 61523 SEQ ID NO. 61599 100125-100281 3 139866
100282-100533 4 139936
100534-100610 5 139941 100611-100812 6 139933 100813-101046 7
139991 101047-101101 8 139965 101102-101576 9 139971 HNF1A:
101577-101893 1 139956 NM_001306179 101894-102153 2 139864 SEQ ID
NO. 61600 102154-102310 3 139866 102311-103012 4 139936
103013-102639 5 139941 102640-102841 6 139933 102842-103075 7
139991 103076-103131 8 139908 103132-103608 9 139995 ATP7B ATP7B:
103609-103870 8 139930 SEQ ID NM_001243182 103871-104118 14 139886
NO. 61524 SEQ ID NO. 61601 ATP7B: NM_000053 104119-104366 13 139886
SEQ ID NO. 61602 104367-104628 7 139930 ATP7B: 104629-104876 9
139886 NM_001005918 SEQ ID NO. 61603 ATP7B: 104877-105124 11 139886
NM_001330579 105125-105389 5 139947 SEQ ID NO. 61604 ATP7B:
105390-105637 12 139886 NM_001330578 105638-105873 7 139897 SEQ ID
NO. 61605 FAH FAH: NM_000137 105874-106128 1 139885 SEQ ID SEQ ID
NO. 61606 NO. 61525 IVD IVD: 106129-106278 8 139967 SEQ ID
NM_001159508 106279-107101 10 139878 NO. 61526 SEQ ID NO. 61607
107102-107322 6 139883 IVD: NM_002225 107323-107472 9 139967 SEQ ID
NO. 61608 107473-108295 11 139878 108296-108516 7 139883 HSD3B7
HSD3B7: 108517-108618 1 139852 SEQ ID NM_001142777 108619-108747 2
139973 NO. 61527 SEQ ID NO. 61609 108748-108828 3 140001
108829-108873 4 139909 108874-109328 5 139945 HSD3B7: 109329-109427
1 139954 NM_001142778 109428-109524 2 139899 SEQ ID NO. 61610
109525-109605 3 140001 109606-109650 4 139909 109651-110105 5
139945 HSD3B7: 110106-110204 1 139954 NM_025193 110205-110301 2
139899 SEQ ID NO. 61611 110302-110382 3 140001 110383-110427 4
139909 110428-110492 5 139912 110493-110925 6 139881 ACADVL ACADVL:
110926-110961 2 139870 SEQ ID NM_001270448 110962-111015 4 139879
NO. 61528 SEQ ID NO. 61612 111016-111068 7 139943 111069-111200 8
139951 111201-111303 9 139980 111304-111349 12 139988 111350-111393
17 139999 111394-111472 18 139925 ACADVL: 111473-111508 4 139870
NM_001270447 111509-111562 6 139879 SEQ ID NO. 61613 111563-111615
9 139943 111616-111747 10 139951 111748-111850 11 139980
111851-111896 14 139988 111897-111940 19 139999 111941-112019 20
139925 ACADVL: 112020-112055 3 139870 NM_000018 112056-112109 5
139879 SEQ ID NO. 61614 112110-112162 8 139943 112163-112294 9
139951 112295-112397 10 139980 112398-112443 13 139988
112444-112487 18 139999 112488-112566 19 139925 ACADVL:
112567-112661 2 139915 NM 001033859 112662-112715 4 139879 SEQ ID
NO. 61615 112716-112768 7 139943 112769-112900 8 139951
112901-113003 9 139980 113004-113049 12 139988 113050-113093 17
139999 113094-113172 18 139925 HAMP HAMP: 113173-113424 1 140000
SEQ ID NM_021175 113425-113474 2 139929 NO. 61529 SEQ ID NO. 61616
TGFB1 TGFB1: 113475-113528 4 139961 SEQ ID NM_000660 NO. 61530 SEQ
ID NO. 61617 HNF4A HNF4A: 113529-113770 2 139895 SEQ ID
NM_001287184 113771-114002 3 139959 NO. 61531 SEQ ID NO. 61618
114003-114202 4 139931 114203-114380 5 139994 114381-114614 6
140004 114615-114851 7 139863 114852-115179 8 139944 HNF4A:
115180-115421 2 139895 NM_001287183 115422-115653 3 139959 SEQ ID
NO. 61619 115654-115853 4 139931 115854-116031 5 139994
116032-116265 6 140004 116266-116502 7 139863 116503-116758 8
139932 116759-117011 9 139861 117012-117834 10 139867 HNF4A:
117835-118047 1 139978 NM 175914 118048-118279 2 139959 SEQ ID NO.
61620 118280-118479 3 139931 118480-118657 4 139994 118658-118891 5
140004 118892-119128 6 139863 119129-119384 7 139932 119385-119637
8 139861 119638-120460 9 139867 HNF4A: 120461-120673 1 139978
NM_001030004 120674-120905 2 139959 SEQ ID NO. 61621 120906-121105
3 139931 121106-121283 4 139994 121284-121517 5 140004
121518-121754 6 139863 121755-122082 7 139944 HNF4A: 122083-122324
2 139895 NM_001287182 122325-122556 3 139959 SEQ ID NO. 61622
122557-122756 4 139931 122757-122934 5 139994 122935-123168 6
140004 123169-123405 7 139863 123406-123661 8 139932 123662-123911
9 139894 123912-124730 10 139982 HNF4A: 124731-124943 1 139978
NM_001030003 124944-125175 2 139959 SEQ ID NO. 61623 125176-125375
3 139931 125376-125553 4 139994 125554-125787 5 140004
125788-126024 6 139863 126025-126280 7 139932 126281-126530 8
139894 126531-127349 9 139982 HNF4A: 129035-129283 1 139904
NM_000457 129284-129515 2 139959 SEQ ID NO. 61624 129516-129715 3
139931 129716-129893 4 139994 129894-130127 5 140004 130128-130364
6 139863 130365-130620 7 139932 130621-130873 8 139861
130874-131696 9 139867 HNF4A: 131697-131939 1 139876 NM_001258355
131940-132151 2 139953 SEQ ID NO. 61625 132152-132383 3 139959
132384-132583 4 139931 132584-132761 5 139994 132762-132995 6
140004 132996-133232 7 139863 133233-133488 8 139932 133489-133741
9 139861 133742-134564 10 139867 HNF4A: 134565-134813 1 139904
NM_178850 134814-135045 2 139959 SEQ ID NO. 61626 135046-135245 3
139931 135246-135423 4 139994 135424-135657 5 140004 135658-135894
6 139863 135895-136222 7 139944 HNF4A: 136223-136471 1 139904
NM_178849 136472-136703 2 139959 SEQ ID NO. 61627 136704-136903 3
139931 136904-137081 4 139994 137082-137315 5 140004 137316-137552
6 139863 137553-137808 7 139932 137809-138058 8 139894
138059-138877 9 139982 ADA ADA: 127350-127517 10 139985 SEQ ID
NM_001322051 127518-127695 4 139888 NO. 61532 SEQ ID NO. 61628 ADA:
NM_000022 127696-127863 11 139985 SEQ ID NO. 61629 127864-128041 4
139888 ADA: 128042-128209 10 139985 NM_001322050 128210-128438 4
139872 SEQ ID NO. 61630 ADA: NR_136160 128439-128606 10 139985 SEQ
ID NO. 61631 128607-128784 4 139888 NCOA5 NCOA5: 128785-129034 2
139955 SEQ ID NM_020967 NO. 61533 SEQ ID NO. 61632 PNPLA3 PNPLA3:
138878-139162 1 139891 SEQ ID NM_025225 139173-139394 4 139905 NO.
61534 SEQ ID NO. 61633 139395-139621 5 139974 139622-139851 7
139859
Example 37: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases AMT Transcript Levels
[2092] To determine whether an increase in expression of AMT could
be achieved by improving splicing efficiency of a retained intron
using ASOs, the methods described herein were used. ARPE-19 cells
were mock-transfected, or transfected with AMT targeting ASOs, or a
non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 123A and B). Taqman qPCR results showed
that several targeting ASOs increase AMT gene transcript level
compared to the mock-transfected. Ct values from AMT
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several AMT targeting ASOs
increase gene transcript levels. These results show that inducing
splicing of a retained intron in the gene using ASOs leads to an
increase in gene expression. Altogether, these results show that
improving the splicing efficiency of a rate limiting intron in the
AMT gene using ASOs led to an increase in AMT gene expression.
Example 38: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases GALT Transcript Levels
[2093] To determine whether an increase in expression of GALT could
be achieved by improving splicing efficiency of a retained intron
using ASOs, the methods described herein were used. ARPE-19 cells
were mock-transfected, or transfected with GALT targeting ASOs, or
a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 124C and D; and FIGS. 124G and 4H).
Taqman qPCR results showed that several targeting ASOs increase
GALT gene transcript level compared to the mock-transfected. Ct
values from GALT targeting-ASO-transfected cells are normalized to
RPL32 and plotted relative to the normalized qPCR product from
mock-treated cells. Results of this analysis indicated that several
GALT targeting ASOs increase gene transcript levels. These results
show that inducing splicing of a retained intron in the gene using
ASOs leads to an increase in gene expression. Altogether, these
results show that improving the splicing efficiency of a rate
limiting intron in the GALT gene using ASOs led to an increase in
GALT gene expression.
Example 39: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases PC Transcript Levels
[2094] To determine whether an increase in expression of PC could
be achieved by improving splicing efficiency of a retained intron
using ASOs, the methods described herein were used. ARPE-19 cells
were mock-transfected, or transfected with PC targeting ASOs, or a
non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 125A and B). Taqman qPCR results showed
that several targeting ASOs increase PC gene transcript level
compared to the mock-transfected. Ct values from PC
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several PC targeting ASOs
increase gene transcript levels. These results show that inducing
splicing of a retained intron in the gene using ASOs leads to an
increase in gene expression. Altogether, these results show that
improving the splicing efficiency of a rate limiting intron in the
PC gene using ASOs led to an increase in PC gene expression.
Example 40: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases FAH Transcript Levels
[2095] To determine whether an increase in expression of FAH could
be achieved by improving splicing efficiency of a retained intron
using ASOs, the methods described herein were used. ARPE-19 cells
were mock-transfected, or transfected with FAH targeting ASOs, or a
non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 126A and B). Taqman qPCR results showed
that several targeting ASOs increase FAH gene transcript level
compared to the mock-transfected. Ct values from FAH
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several FAH targeting ASOs
increase gene transcript levels. These results show that inducing
splicing of a retained intron in the gene using ASOs leads to an
increase in gene expression. Altogether, these results show that
improving the splicing efficiency of a rate limiting intron in the
FAH gene using ASOs led to an increase in FAH gene expression.
Example 41: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases PPARD Transcript Levels
[2096] To determine whether an increase in expression of PPARD
could be achieved by improving splicing efficiency of a retained
intron using ASOs, the methods described herein were used. ARPE-19
cells were mock-transfected, or transfected with PPARD targeting
ASOs, or a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 127A and B, FIGS. 127E and F). Taqman
qPCR results showed that several targeting ASOs increase PPARD gene
transcript level compared to the mock-transfected. Ct values from
PPARD targeting-ASO-transfected cells are normalized to RPL32 and
plotted relative to the normalized qPCR product from mock-treated
cells. Results of this analysis indicated that several PPARD
targeting ASOs increase gene transcript levels. These results show
that inducing splicing of a retained intron in the gene using ASOs
leads to an increase in gene expression. Altogether, these results
show that improving the splicing efficiency of a rate limiting
intron in the PPARD gene using ASOs led to an increase in PPARD
gene expression.
Example 42: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases HMBS Transcript Levels
[2097] To determine whether an increase in expression of HMBS could
be achieved by improving splicing efficiency of a retained intron
using ASOs, the methods described herein were used. ARPE-19 cells
were mock-transfected, or transfected with HMBS targeting ASOs, or
a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 129A and B). Taqman qPCR results showed
that several targeting ASOs increase HMBS gene transcript level
compared to the mock-transfected. Ct values from HMBS
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several HMBS targeting ASOs
increase gene transcript levels. These results show that inducing
splicing of a retained intron in the gene using ASOs leads to an
increase in gene expression. Altogether, these results show that
improving the splicing efficiency of a rate limiting intron in the
HMBS gene using ASOs led to an increase in HMBS gene
expression.
Example 43: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases ALMS1 Transcript Levels
[2098] To determine whether an increase in expression of ALMS1
could be achieved by improving splicing efficiency of a retained
intron using ASOs, the methods described herein were used. ARPE-19
cells were mock-transfected, or transfected with ALMS1 targeting
ASOs, or a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 131A and B). Taqman qPCR results showed
that several targeting ASOs increase ALMS1 gene transcript level
compared to the mock-transfected. Ct values from ALMS1
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several ALMS1 targeting
ASOs increase gene transcript levels. These results show that
inducing splicing of a retained intron in the gene using ASOs leads
to an increase in gene expression. Altogether, these results show
that improving the splicing efficiency of a rate limiting intron in
the ALMS1 gene using ASOs led to an increase in ALMS1 gene
expression.
Example 44: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases ASL Transcript Levels
[2099] To determine whether an increase in expression of ASL could
be achieved by improving splicing efficiency of a retained intron
using ASOs, the methods described herein were used. ARPE-19 cells
were mock-transfected, or transfected with ASL targeting ASOs, or a
non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 132A and B, FIGS. 132C and D, FIGS. 132E
and F). Taqman qPCR results showed that several targeting ASOs
increase ASL gene transcript level compared to the
mock-transfected. Ct values from ASL targeting-ASO-transfected
cells are normalized to RPL32 and plotted relative to the
normalized qPCR product from mock-treated cells. Results of this
analysis indicated that several ASL targeting ASOs increase gene
transcript levels. These results show that inducing splicing of a
retained intron in the gene using ASOs leads to an increase in gene
expression. Altogether, these results show that improving the
splicing efficiency of a rate limiting intron in the ASL gene using
ASOs led to an increase in ASL gene expression.
Example 45: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases ATP7B Transcript Levels
[2100] To determine whether an increase in expression of ATP7B
could be achieved by improving splicing efficiency of a retained
intron using ASOs, the methods described herein were used. ARPE-19
cells were mock-transfected, or transfected with ATP7B targeting
ASOs, or a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 133B and C). Taqman qPCR results showed
that several targeting ASOs increase ATP7B gene transcript level
compared to the mock-transfected. Ct values from ATP7B
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several ATP7B targeting
ASOs increase gene transcript levels. These results show that
inducing splicing of a retained intron in the gene using ASOs leads
to an increase in gene expression. Altogether, these results show
that improving the splicing efficiency of a rate limiting intron in
the ATP7B gene using ASOs led to an increase in ATP7B gene
expression.
Example 46: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases HSD3B7 Transcript Levels
[2101] To determine whether an increase in expression of HSD3B7
could be achieved by improving splicing efficiency of a retained
intron using ASOs, the methods described herein were used. ARPE-19
cells were mock-transfected, or transfected with HSD3B7 targeting
ASOs, or a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 135B and C, FIGS. 135E and F). Taqman
qPCR results showed that several targeting ASOs increase HSD3B7
gene transcript level compared to the mock-transfected. Ct values
from HSD3B7 targeting-ASO-transfected cells are normalized to RPL32
and plotted relative to the normalized qPCR product from
mock-treated cells. Results of this analysis indicated that several
HSD3B7 targeting ASOs increase gene transcript levels. These
results show that inducing splicing of a retained intron in the
gene using ASOs leads to an increase in gene expression.
Altogether, these results show that improving the splicing
efficiency of a rate limiting intron in the HSD3B7 gene using ASOs
led to an increase in HSD3B7 gene expression.
Example 47: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases Transcript Levels
[2102] To determine whether an increase in expression of a target
gene could be achieved by improving intron splicing efficiency with
ASOs we used the method described herein. ARPE-19 cells, a human
retinal epithelium cell line (American Type Culture Collection
(ATCC), USA), or Huh-7, a human hepatoma cell line (NIBIOHN,
Japan), or SK-N-AS, a human neuroblastoma cell line (ATCC) were
mock-transfected, or transfected with the targeting ASOs described
in FIGS. 123-137 and Tables 10 and 11. Cells were transfected using
Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher) of the retained intron listed in Tables 10 and 11.
Taqman assays were carried out according to vendor's
specifications, on a QuantStudio 7 Flex Real-Time PCR system
(Thermo Fisher). Target gene assay values were normalized to RPL32
(deltaCt) and plate-matched mock transfected samples (delta-delta
Ct), generating fold-change over mock quantitation (2{circumflex
over ( )}-(delta-deltaCt). Average fold-change over mock of the
three plate replicates was plotted (FIG. 123B, FIG. 124D, FIG.
124H, FIG. 125B, FIG. 126B, FIG. 127F, FIG. 129B, FIG. 131B, FIG.
132B, FIG. 132D, FIG. 132F, FIG. 133C, FIC. 135C and FIG. 135F).
Several ASOs were identified that increase the target gene
expression, implying an increase in splicing at that target intron.
Together with whole transcriptome data showing retention of the
target intron (FIG. 123-137), these results confirm that ASOs can
improve the splicing efficiency of a rate limiting intron.
Example 48: Identification of Intron Retention Events in
Transcripts by RNAseq Using Next Generation Sequencing
[2103] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by a gene described herein to identify intron-retention
events. For this purpose, polyA+ RNA from nuclear and cytoplasmic
fractions of renal epithelial cells was isolated and cDNA libraries
constructed using Illumina's TruSeq Stranded mRNA library Prep Kit.
The libraries were pair-end sequenced resulting in 100-nucleotide
reads that were mapped to the human genome (February 2009,
GRCh37/hg19 assembly) The mapped reads were visualized using the
UCSC genome browser (operated by the UCSC Genome Informatics Group
(Center for Biomolecular Science & Engineering, University of
California, Santa Cruz, 1156 High Street, Santa Cruz, Calif. 95064)
and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome
Browser database: 2015 update," Nucleic Acids Research 43, Database
Issue, doi: 10.1093/nar/gku1177) and the coverage and number of
reads were inferred by the peak signals. The height of the peaks
indicates the level of expression given by the density of the reads
in a particular region. A schematic representation of the gene
(drawn to scale) was provided by the UCSC genome browser (below the
read signals) so that peaks can be matched to the exonic and
intronic regions. Based on this display, we identified introns that
have high read density in the nuclear fraction of renal epithelial
cells, but have very low to no reads in the cytoplasmic fraction of
these cells (see Table 12 for percent intron retention (PIR) data
for introns identified in genes described herein). This indicated
that these introns were retained and that the intron-containing
transcripts remain in the nucleus, and suggested that these
retained RIC pre-mRNAs are non-productive, as they were not
exported out to the cytoplasm.
Example 49: Identification of Intron Retention Events in Gene
Transcripts by RNAseq Using Next Generation Sequencing for Retained
Introns not Yet Identified
[2104] Whole transcriptome shotgun sequencing was carried out using
next generation sequencing to reveal a snapshot of transcripts
produced by a gene described herein to identify unknown
intron-retention events. For this purpose, polyA+ RNA from nuclear
and cytoplasmic fractions of renal epithelial cells were isolated
and cDNA libraries were constructed using Illumina's TruSeq
Stranded mRNA library Prep Kit. The libraries were pair-end
sequenced resulting in 100-nucleotide reads that were mapped to the
human genome (February 2009, GRCh37/hg19 assembly) The mapped reads
were visualized using the UCSC genome browser (operated by the UCSC
Genome Informatics Group (Center for Biomolecular Science &
Engineering, University of California, Santa Cruz, 1156 High
Street, Santa Cruz, Calif. 95064) and described by, e.g.,
Rosenbloom, et al., 2015, "The UCSC Genome Browser database: 2015
update," Nucleic Acids Research 43, Database Issue, doi:
10.1093/nar/gku1177) and the coverage and number of reads can be
inferred by the peak signals. The height of the peaks indicated the
level of expression given by the density of the reads in a
particular region. A schematic representation of the gene (drawn to
scale) was provided by the UCSC genome browser (below the read
signals) so that peaks could be matched to the gene exonic and
intronic regions. Based on this display, retained introns were
inferred as those that have high read density in the nuclear
fraction of renal epithelial cells, but have very low to no reads
in the cytoplasmic fraction of these cells. This indicated that the
introns are retained and that the retained intron-containing
transcripts remained in the nucleus, and suggests that these
retained RIC pre-mRNAs are non-productive, as they are not exported
out to the cytoplasm.
Example 50: Design of ASO-Walk Targeting a Retained Intron
[2105] An ASO walk was designed to target a retained intron using
the method described herein. A region immediately downstream of the
intron 5' splice site, e.g., spanning nucleotides +6 to +69 and a
region immediately upstream of intron 3' splice site, e.g.,
spanning nucleotides -16 to -79 of the intron was targeted with
2'-O-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide
intervals. Table 12 lists exemplary ASOs that were designed and
their target sequences.
TABLE-US-00012 TABLE 12 Pre-mRNA (RNA Accession Retained Target
Gene Gene Number) ASOs Intron (% Sequence SEQ ID NO. Symbol SEQ ID
NO. SEQ ID NOs. Retention) SEQ ID NO. PPARD 5467 PPARD: NM_006238
140024-140280 Intron 3 150740 SEQ ID NO. SEQ ID NO. 140009
140281-140523 Intron 4 150751 140005 140524-140776 Intron 5 150743
140777-140935 Intron 6 150747 140936-141626 Intron 7 150746 PPARD:
NM_177435 141627-141883 Intron 3 150740 SEQ ID NO. 1400010
141884-142126 Intron 4 150751 142127-142378 Intron 5 150743
142379-142659 Intron 6 150741 PPARD: 142660-143350 Intron 8 150746
NM_001171818 143351-143607 Intron 4 150740 SEQ ID NO. 1400011
143608-143850 Intron 5 150751 143851-144102 Intron 6 150743
144103-144262 Intron 7 150747 NM_001171819 145263-144492 Intron 2
150739 SEQ ID NO. 1400012 144493-144735 Intron 3 150751
144736-144987 Intron 4 150743 144988-145147 Intron 5 150747
145148-145838 Intron 6 150746 PPARD: 145839-146105 Intron 3 150748
NM_001171820 146106-146265 Intron 4 150747 SEQ ID NO. 1400013
146266-146956 Intron 5 150746 PAX2 5076 PAX2: 146957-147177 Intron
2 150742 SEQ ID NO. NM_001304569 140006 SEQ ID NO. 1400014 PAX2:
147178-147467 Intron 1 150744 NM_000278 SEQ ID NO. 1400015 PAX2:
147468-147757 Intron 1 (N/A) 150744 NM_003990 SEQ ID NO. 1400016
PAX2: 147758-148047 Intron 1 150744 NM_003988 SEQ ID NO. 1400017
PAX2: 148048-148337 Intron 1 150744 NM_003987 SEQ ID NO. 1400018
PAX2: 148338-148627 Intron 1 150744 NM_003989 SEQ ID NO. 1400019
CTNS 1497 CTNS: 148628-148850 Intron 9 150752 SEQ ID NO. NM_004937
(10%) 140007 SEQ ID NO. 140020 148851-149075 Intron 10 150738 CTNS:
149076-149298 Intron 9 150752 NM_001031681 (10%) SEQ ID NO. 140021
149299-149523 Intron 10 150738 CYP24A1 1591 CYP24A1: 149524-149599
Intron 10 150750 SEQ ID NO. NM_000782 (23%) 140008 SEQ ID NO.
140022 149600-150072 Intron 11 150749 (50%) CYP24A1: 150073-150264
Intron 9 150745 NM_001128915 150265-150737 Intron 10 150749 SEQ ID
NO. 140023
Example 51: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases CTNS Transcript Levels
[2106] To determine whether an increase in expression of CTNS could
be achieved by improving splicing efficiency of a retained intron
using ASOs, the methods described herein were used. ARPE-19 cells
were mock-transfected, or transfected with CTNS targeting ASOs, or
a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 138B and C). Taqman qPCR results showed
that several targeting ASOs increase CTNS gene transcript level
compared to the mock-transfected. Ct values from CTNS
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several CTNS targeting ASOs
increase gene transcript levels. These results show that inducing
splicing of a retained intron in the gene using ASOs leads to an
increase in gene expression. Altogether, these results show that
improving the splicing efficiency of a rate limiting intron in the
CTNS gene using ASOs led to an increase in CTNS gene
expression.
Example 52: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases CYP24A1 Transcript Levels
[2107] To determine whether an increase in expression of CYP24A1
could be achieved by improving splicing efficiency of a retained
intron using ASOs, the methods described herein were used. ARPE-19
cells were mock-transfected, or transfected with CYP24A1 targeting
ASOs, or a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 139B and C). Taqman qPCR results showed
that several targeting ASOs increase CYP24A1 gene transcript level
compared to the mock-transfected. Ct values from CYP24A1
targeting-ASO-transfected cells are normalized to RPL32 and plotted
relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several CYP24A1 targeting
ASOs increase gene transcript levels. These results show that
inducing splicing of a retained intron in the gene using ASOs leads
to an increase in gene expression. Altogether, these results show
that improving the splicing efficiency of a rate limiting intron in
the CYP24A1 gene using ASOs led to an increase in CYP24A1 gene
expression.
Example 53: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases PPARD Transcript Levels
[2108] To determine whether an increase in expression of PPARD
could be achieved by improving splicing efficiency of a retained
intron using ASOs, the methods described herein were used. ARPE-19
cells were mock-transfected, or transfected with PPARD targeting
ASOs, or a non-targeting ASO control, independently, using RNAiMAX
(Invitrogen) delivery reagents. Experiments were performed using 80
nM ASOs for 24 hrs (FIGS. 141A and B, FIGS. 141E and F). Taqman
qPCR results showed that several targeting ASOs increase PPARD gene
transcript level compared to the mock-transfected. Ct values from
PPARD targeting-ASO-transfected cells are normalized to RPL32 and
plotted relative to the normalized qPCR product from mock-treated
cells. Results of this analysis indicated that several PPARD
targeting ASOs increase gene transcript levels. These results show
that inducing splicing of a retained intron in the gene using ASOs
leads to an increase in gene expression. Altogether, these results
show that improving the splicing efficiency of a rate limiting
intron in the PPARD gene using ASOs led to an increase in PPARD
gene expression.
Example 54: Improved Splicing Efficiency Via ASO-Targeting of a
Retained Intron Increases Transcript Levels
[2109] To determine whether an increase in expression of a target
gene could be achieved by improving intron splicing efficiency with
ASOs we used the method described herein. ARPE-19 cells, a human
retinal epithelium cell line (American Type Culture Collection
(ATCC), USA), or Huh-7, a human hepatoma cell line (NIBIOHN,
Japan), or SK-N-AS, a human neuroblastoma cell line (ATCC) were
mock-transfected, or transfected with the targeting ASOs described
in FIGS. 138-141 and Table 12. Cells were transfected using
Lipofectamine RNAiMax transfection reagent (Thermo Fisher)
according to vendor's specifications. Briefly, ASOs were plated in
96-well tissue culture plates and combined with RNAiMax diluted in
Opti-MEM. Cells were detached using trypsin and resuspended in full
medium, and approximately 25,000 cells were added the
ASO-transfection mixture. Transfection experiments were carried out
in triplicate plate replicates. Final ASO concentration was 80 nM.
Media was changed 6h post-transfection, and cells harvested at 24h,
using the Cells-to-Ct lysis reagent, supplemented with DNAse
(Thermo Fisher), according to vendor's specifications. cDNA was
generated with Cells-to-Ct RT reagents (Thermo Fisher) according to
vendor's specifications. To quantify the amount of splicing at the
intron of interest, quantitative PCR was carried out using Taqman
assays with probes spanning the corresponding exon-exon junction
(Thermo Fisher) of the retained intron listed in Table 12. Taqman
assays were carried out according to vendor's specifications, on a
QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher). Target
gene assay values were normalized to RPL32 (deltaCt) and
plate-matched mock transfected samples (delta-delta Ct), generating
fold-change over mock quantitation (2{circumflex over (
)}-(delta-deltaCt). Average fold-change over mock of the three
plate replicates was plotted (FIG. 138C, FIG. 139C, FIG. 141B and
FIG. 141F). Several ASOs were identified that increase the target
gene expression, implying an increase in splicing at that target
intron. Together with whole transcriptome data showing retention of
the target intron (FIGS. 138-141), these results confirm that ASOs
can improve the splicing efficiency of a rate limiting intron.
[2110] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220118000A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220118000A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References