U.S. patent application number 17/607908 was filed with the patent office on 2022-07-07 for restoration of the cftr function by splicing modulation.
This patent application is currently assigned to YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM. The applicant listed for this patent is SpliSense Ltd., YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM LTD.. Invention is credited to Ofra BARCHAD-AVITZUR, Bat Sheva KEREM, Yifat OREN, Efrat OZERI-GALAI.
Application Number | 20220213479 17/607908 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220213479 |
Kind Code |
A1 |
KEREM; Bat Sheva ; et
al. |
July 7, 2022 |
RESTORATION OF THE CFTR FUNCTION BY SPLICING MODULATION
Abstract
The present invention provides oligonucleotides capable of
binding to and modulating the splicing of the pre-mRNA of the CFTR
gene, including compositions comprising the oligonucleotides, and
uses thereof, such as for suppressing the inclusion of a cryptic
exon between exon 22 and 23 as a result of the mutation 3849 +10 Kb
C-to-T, optionally in combination with additional CF
therapeutics.
Inventors: |
KEREM; Bat Sheva; (Mevaseret
Zion, IL) ; OZERI-GALAI; Efrat; (Jerusalem, IL)
; OREN; Yifat; (Jerusalem, IL) ; BARCHAD-AVITZUR;
Ofra; (Jerusalem, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF
JERUSALEM LTD.
SpliSense Ltd. |
Jerusalem
Jerusalem |
|
IL
IL |
|
|
Assignee: |
YISSUM RESEARCH DEVELOPMENT COMPANY
OF THE HEBREW UNIVERSITY OF JERUSALEM
Jerusalem
IL
SpliSense Ltd.
Jerusalem
IL
|
Appl. No.: |
17/607908 |
Filed: |
May 5, 2020 |
PCT Filed: |
May 5, 2020 |
PCT NO: |
PCT/IL2020/050495 |
371 Date: |
November 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62843469 |
May 5, 2019 |
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International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/7088 20060101 A61K031/7088; A61K 45/06
20060101 A61K045/06; A61P 11/00 20060101 A61P011/00 |
Claims
1-31. (canceled)
32. A synthetic oligonucleotide molecule characterized by at least
partly suppressing the inclusion of intron 22 cryptic exon in the
mature mRNA of a CFTR gene having a 3849 +10 Kb C-to-T mutation, a)
wherein the sequence of said synthetic oligonucleotide molecule
comprises the sequence as set forth in any one of SEQ ID NOs: 4, 6,
10, or 17 or an active fragment thereof; or b) wherein the
nucleotide sequence of said synthetic oligonucleotide molecule
comprises the sequence as set forth in any one of SEQ ID NOs: 1-3,
5, 8, 40-48, or an active fragment thereof and is characterized by
increasing the percentage of correctly spliced mature CFTR mRNA by
at least about 10%; and decreasing the level of aberrantly spliced
mature CFTR mRNA by at least about 20%, and wherein said synthetic
oligonucleotide molecule or active fragment thereof consists of
17-21 consecutive bases.
33. The synthetic oligonucleotide molecule of claim 32, wherein
said active fragment comprises a nucleotide sequence as set forth
in SEQ ID NO: 40.
34. The synthetic oligonucleotide molecule of claim 32, comprising
the sequence GAUGGAAGA (SEQ ID NO: 38).
35. The synthetic oligonucleotide molecule of claim 32, consisting
of 18 or 19 consecutive nucleotide bases.
36. The synthetic oligonucleotide molecule of claim 32, comprising
a chemically modified backbone comprising: a phosphate-ribose
backbone, a phosphate-deoxyribose backbone, a
2'-O-methyl-phosphorothioate (2'OMP) backbone, a phosphorodiamidate
morpholino backbone, a peptide nucleic acid backbone, a
2-methoxyethyl phosphorothioate backbone, a 2'-Methoxy Ethyl
(2'MOE) backbone, an alternating locked nucleic acid backbone,
constrained ethyl backbone, a phosphorothioate backbone, N3'-P5'
phosphoroamidates, 2'-deoxy-2'-fluoro-.beta.-d-arabino nucleic
acid, cyclohexene nucleic acid backbone nucleic acid, tricyclo-DNA
(tcDNA) nucleic acid backbone, or a combination thereof.
37. The synthetic oligonucleotide molecule of claim 32, having at
least 80% complementarity to a nucleotide sequence within SEQ ID
NO: 37.
38. The synthetic oligonucleotide molecule of claim 32, having 100%
complementarity to a nucleotide sequence within SEQ ID NO: 37.
39. A pharmaceutical composition comprising the synthetic
oligonucleotide molecule of claim 32 and a pharmaceutically
acceptable carrier.
40. The pharmaceutical composition of claim 39, further comprising
a CFTR modifier; a CF drug, or a combination thereof, wherein said
CFTR modifier is selected from the group consisting of: CFTR
potentiator, CFTR corrector, Translational Read-Through agent, and
CFTR amplifier.
41. The pharmaceutical composition of claim 40, wherein said CFTR
modifier is ivacaftor, lumacaftor, tezacaftor, elexacaftor, VX-659,
VX-152, or VX-440, or any combination thereof.
42. The pharmaceutical composition of claim 40, wherein said CF
drug is an antibiotic drug, a bronchodilator, a corticosteroid, or
any combination thereof.
43. The pharmaceutical composition of claim 39, formulated for
oral, nasal, inhalation, abdominal, subcutaneous, intra-peritoneal
or intravenous administration.
44. A method for treating Cystic Fibrosis (CF) in a subject in need
thereof, comprising administering to said subject a therapeutically
effective amount of a synthetic oligonucleotide that at least
partly suppresses the inclusion of intron 22 cryptic exon in the
mature CFTR mRNA of a CFTR gene having a 3849 +10 Kb C-to-T
mutation, a) wherein the sequence of said synthetic oligonucleotide
molecule comprises the sequence as set forth in any one of SEQ ID
NOs: 4, 6, 10, or 17 or an active fragment thereof; or b) wherein
the nucleotide sequence of said synthetic oligonucleotide molecule
comprises the sequence as set forth in any one of SEQ ID NOs: 1-3,
5, 8, 40-48, or an active fragment thereof and increases the
percentage of correctly spliced mature CFTR mRNA by at least about
10%; and decreases the level of aberrantly spliced mature CFTR mRNA
by at least about 20%, and wherein said synthetic oligonucleotide
molecule or active fragment thereof consists of 17-21 consecutive
bases, thereby treating CF in the subject.
45. The method of claim 44, wherein said active fragment comprises
a nucleotide sequence as set forth in SEQ ID NO: 40.
46. The method of claim 44, wherein said synthetic oligonucleotide
molecule comprises the sequence GAUGGAAGA (SEQ ID NO: 38).
47. The method of claim 44, wherein said synthetic oligonucleotide
comprises a backbone selected from the group consisting of: a
phosphate-ribose backbone, a phosphate-deoxyribose backbone, a
phosphorothioate-deoxyribose backbone, a
2'-O-methyl-phosphorothioate backbone (2'OMP), a phosphorodiamidate
morpholino backbone, a peptide nucleic acid backbone, a
2-methoxyethyl phosphorothioate backbone, a 2'-Methoxy Ethyl
(2'MOE) backbone, an alternating locked nucleic acid backbone, a
phosphorothioate backbone, N3'-P5' phosphoroamidates,
2'-deoxy-2'-fluoro-.beta.-d-arabino nucleic acid, cyclohexene
nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic
acid backbone, and a combination thereof.
48. The method of claim 44, wherein said synthetic oligonucleotide
molecule has at least 80% complementarity to a sequence within SEQ
ID NO: 36 or to a sequence within SEQ ID NO: 37.
49. The method of claim 44, further comprising administering to
said subject a therapeutically effective amount of one or more CFTR
modifiers comprising a CFTR-splicing-modulating agent, a
Translational Read-Through agent, a CFTR amplifier, a CFTR
potentiator, or a CFTR corrector.
50. The method of claim 44, wherein said CFTR modifier is selected
from the group consisting of: a different synthetic oligonucleotide
molecule capable of suppressing intron 22 cryptic exon inclusion in
the mature CFTR mRNA, Ataluren, ELX-02, ivacaftor, QBW251, PTI-808,
VX-561, lumacaftor, tezacaftor, elexacaftor, VX-659, VX-445, VX-152
and VX-440, GLPG2222, FDL169, PTI-801, and any combination
thereof.
51. The method of claim 44, wherein said subject is heterozygous
for said 3849 +10 Kb C-to-T mutation.
52. The method of claim 44, wherein said treating comprises
improving at least one clinical parameter of CF selected from the
group consisting of: lung function, time to the first pulmonary
exacerbation, change in weight, change in height, a change in Body
Mass Index (BMI), change in the concentration of sweat chloride,
number and/or duration of pulmonary exacerbations, total number of
days of hospitalization for pulmonary exacerbations, and the need
for antibiotic therapy for sinopulmonary signs or symptoms.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Patent Application No. 62/843,469 titled "RESTORATION
OF THE CFTR FUNCTION BY SPLICING MODULATION", filed May 5, 2019,
the contents of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to oligonucleotides capable of
binding to a Cystic Fibrosis Trans-membrane conductance Regulator
(CFTR) pre-mRNA, and methods of using same, such as for suppressing
the inclusion of a cryptic exon between exon 22 and 23 as a result
of the mutation 3849 +10 Kb C-to-T, and for treating Cystic
Fibrosis (CF).
BACKGROUND
[0003] Cystic fibrosis (CF) is a common, severe autosomal recessive
disease caused by mutations in the CFTR gene. The CFTR gene encodes
for a chloride channel responsible for chloride transport in
epithelial cells. The major manifestations of CF are in the lungs,
with more than 90% mortality related to the respiratory disease.
The disease in the respiratory tract is linked to the insufficient
CFTR function in the airway epithelium.
[0004] As of today, approximately 2000 different mutations
disrupting the CFTR functions have been identified worldwide,
grouped into five distinct classes based on their effect on the
CFTR function. Class I includes mutations that lead to
non-functional CFTR (large deletions and stop codon mutations).
Class II mutations (including the common .DELTA.F508) lead to
aberrantly folded CFTR protein that is recognized by the cell
quality control mechanism and subsequently degraded, resulting in
the absence of mature CFTR protein at the apical cell membrane.
Class III mutations lead to full-length CFTR protein being
incorporated into the cell membrane, but with defective regulation
so that no CFTR function is present. These three classes usually
lead to a classic CF phenotype with pancreatic insufficiency,
although the severity of lung disease is highly variable. CFTR
mutations leading to defective chloride conductance are grouped
into Class IV. Class V mutations involve transcription
dysregulation, resulting in a decreased amount of otherwise normal
CFTR. The latter two classes are often associated with a milder
phenotype and pancreatic sufficiency. Specifically, CFTR that
results from a class IV mutation inserts into the plasma membrane
but exhibits reduced single-channel chloride ion conductance
because of reduced chloride permeation and open channel
probability. Found in <1% of patients with CF, class V mutations
produce normal plasma membrane CFTR. The quantity, however, is
generally reduced as a result of transcriptional dysregulation.
Class V mutations are frequently influence by the splicing
machinery and generate both aberrantly and correctly spliced mRNA,
the levels of which vary among different patients and even among
different organs of the same patients. Ultimately, the splice
variants result in a reduced number of functioning CFTR in the
plasma membrane.
[0005] About 10-15% of CFTR mutations affect the correct splicing
of the gene transcripts. Among these is the splicing mutation 3849
+10 kb C-to-T which leads to inclusion of an 84 base pair cryptic
exon in the mature messenger RNA (mRNA) (denoted "intron 22 cryptic
exon inclusion" mutation). This mutation is the 12th most common
CFTR mutation in the world, which occurs in hundreds of CF patients
worldwide (Kerem et al., 1997). Correction of the aberrant splicing
of the CFTR gene by "anti-sense" oligonucleotides (AOs) was
attempted by Friedman et al, 1999 and recently described in
international PCT Application Publication WO 2014/045283 to the
present inventors.
[0006] Anti-sense oligonucleotides (AOs or ASOs) administration is
one of the most promising therapeutic approaches for the treatment
of genetic disorders caused by splicing mutations. AOs are short
synthetic molecules which can anneal to motifs predicted to be
involved in the pre-mRNA splicing. The method is based on
splice-switching. The AOs binding to selected sites is expected to
mask the targeted region and promote normal splicing. AOs are
highly specific for their targets and do not affect any other
sequences in the cells. Several types of chemically modified AO
molecules are commonly used including: 2'-O-methyl-phosphorothioate
(2'OMP), phosphorodiamidate morpholino oligomer (PMO), peptide
nucleic acids (PNAs), 2-methoxyethyl phosphorothioate (MOE),
constrained ethyl (cET), LIgand-Conjugated Antisense (LICA) and
alternating locked nucleic acids (LNAs).
[0007] The AOs modifications maintain their stabilization, improve
their target affinity, and provide favorable pharmacokinetic
properties and biological stability. It has been conclusively shown
that splice-switching AOs can redirect dystrophin pre-mRNA
processing in murine models for Duchenne Muscular Dystrophy (DMD)
so that an exon carrying a premature protein termination signal
(nonsense mutation) can be excluded from the mature gene transcript
resulting in a shorter but still functional dystrophin isoform.
Progress in dystrophin exon skipping has been rapid, with
proof-of-concept studies being reported, and with the publication
of results from systemic administration to patients in both 2OMP
and PMO chemistries. In 2016, the FDA granted accelerated approval
to first drug for Duchenne muscular dystrophy (eteplirsen), which
is a PMO AO for exon 51 skipping.
[0008] In addition to induced exon skipping, AOs can be designed to
mask splice-silencing elements that reduce exon recognition and
subsequent inclusion in the mature mRNA. Spinal Muscular Atrophy
(SMA) is a common autosomal recessive condition caused by the loss
of the SMN1 gene together with a C>T variation in SMN2 exon 7,
leading to abnormal splicing in which SMN2 exon 7 is skipped,
resulting in a non-functional gene product. AOs have been designed
to mask nearby flanking SMN2 splice silencer elements to promote
synthesis of full-length transcripts. An intrathecally
administration of morpholino oligomer to neonatal mouse pups with
severe SMA was highly successful, significantly extending their
survival.
[0009] This led to the development of an AO-based drug, Spinraza
(Nusinersen), that was recently approved by the US Food and Drug
Administration (FDA) and the European Medicines Agency (EMA). The
approval followed a very successful completion of a phase-III
clinical trial in patients with infantile-onset SMA. The treated
infants experienced a statistically significant improvement in the
achievement of motor milestones. The results of this study
demonstrate the great potential of AO-based splicing modulation for
the treatment of genetic diseases and emphasize the importance of
local delivery for efficient treatment with minimal toxicity.
[0010] There remains a constant need in the field of Cystic
Fibrosis management for novel, potent therapeutics, designed to
overcome the numerous mutations in the CFTR gene identified thus
far, and restore CFTR function.
SUMMARY
[0011] The present invention provides compositions comprising
oligonucleotides capable of binding to a CFTR pre-mRNA, thereby
modulating splicing and restoring or enhancing the function of the
CFTR gene product. The present invention thus identifies sequences
within the CFTR pre-mRNA which are targeted in order to modulate
the splicing cascade of the CFTR pre-mRNA. Modulating CFTR pre-mRNA
splicing, as demonstrated in the present invention, can avoid
improper recognition of intron sequences as exons. As a result of
the modulation of splicing, a functional CFTR protein is produced
in sufficient levels by an otherwise aberrant CFTR allele. The
present invention, in some embodiments thereof, further provides
modified oligonucleotides for modulating splicing and restoring or
enhancing the function of the CFTR gene product.
[0012] The present invention is based, in part, on the finding that
artificial "anti-sense" oligonucleotide molecules are able to
target and bind predetermined sequences at the pre-mRNA molecule of
the CFTR gene, and that the binding can modulate the splicing of
the pre-mRNA molecule into a mature mRNA which is subsequently
translated into a functional CFTR protein in sufficient levels. The
targets within a CFTR pre-mRNA molecule are those discovered to be
involved in splicing, either indirectly, by affecting the splicing
of adjacent as well as more remote sequences, or directly, by
affecting their own splicing.
[0013] Advantageously, the present invention provides anti-sense
oligonucleotide molecules with improved efficacy. The
oligonucleotides are highly effective in raising the percentage of
correctly spliced CFTR mRNA. The oligonucleotides are short, having
no more than 21 nucleotides, having advantages such as improved
cell/tissue penetrating capacity.
[0014] According to a first aspect, there is provided a synthetic
oligonucleotide molecule consisting of 17-21 consecutive bases
having at least 80% complementarity to a pre-mRNA transcript of a
CFTR gene having a 3849 +10 Kb C-to-T mutation, and characterized
by at least partly suppressing the inclusion of intron 22 cryptic
exon in the mature CFTR mRNA, increasing the percentage of
correctly spliced mature CFTR mRNA by at least about 10%; and
decreasing the level of aberrantly spliced mature CFTR mRNA by at
least about 20%.
[0015] According to another aspect, there is provided a
pharmaceutical composition comprising the synthetic oligonucleotide
molecule of the invention, and a pharmaceutically acceptable
carrier.
[0016] According to another aspect, there is provided a method for
treating CF in a subject in need thereof, comprising administering
to the subject a therapeutically effective amount of a synthetic
oligonucleotide, wherein the synthetic oligonucleotide suppresses
the inclusion of intron 22 cryptic exon in the mature CFTR mRNA,
increases the percentage of correctly spliced mature CFTR mRNA by
at least about 10%; and decreases the level of aberrantly spliced
mature CFTR mRNA by at least about 20%, thereby treating CF in the
subject.
[0017] According to another aspect, there is provided a kit
comprising: (a) at least one synthetic oligonucleotide molecule;
and at least one of: (b) at least one CFTR modifier; or (c) at
least one CF drug, wherein the synthetic oligonucleotide is
selected from the group consisting of SEQ ID Nos. 1-25, and 41-44,
and wherein the CFTR modifier is selected from the group consisting
of: CFTR potentiator, CFTR corrector, Translational Read-Through
agent, and CFTR amplifier.
[0018] In some embodiments, the synthetic oligonucleotide molecule
has at least 80% complementarity to a nucleotide sequence within
SEQ ID NO: 37.
[0019] In some embodiments, the synthetic oligonucleotide molecule
has 100% complementarity to a nucleotide sequence within SEQ ID NO:
37.
[0020] The synthetic oligonucleotide molecule of any one of claims
1 to 3, consisting of 19 consecutive nucleotide bases.
[0021] In some embodiments, the synthetic oligonucleotide molecule
consists of 18 consecutive nucleotide bases.
[0022] In some embodiments, the synthetic oligonucleotide molecule
comprises a chemically modified comprising: a phosphate-ribose
backbone, a phosphate-deoxyribose backbone, a
2'-O-methyl-phosphorothioate backbone, a phosphorodiamidate
morpholino backbone, a peptide nucleic acid backbone, a
2-methoxyethyl phosphorothioate backbone, an alternating locked
nucleic acid backbone, constrained ethyl backbone, a
phosphorothioate backbone, N3'-P5' phosphoroamidates,
2'-deoxy-2'-fluoro-.beta.-d-arabino nucleic acid, cyclohexene
nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic
acid backbone, and a combination thereof.
[0023] In some embodiments, the synthetic oligonucleotide molecule
comprises a backbone comprising a 2'-O-Methyl phosphorothioate
(2'OMP) modification or a 2'-Methoxy Ethyl (2'MOE)
modification.
[0024] In some embodiments, the synthetic oligonucleotide molecule
comprises a nucleotide sequence set forth in one of SEQ ID Nos.:
1-25, and 41-44.
[0025] In some embodiments, the synthetic oligonucleotide molecule
comprises a nucleotide sequence set forth in one of SEQ ID Nos.:
1-5, and 41.
[0026] In some embodiments, the synthetic oligonucleotide molecule
comprises the sequence GAUGGAAGA (SEQ ID NO: 38).
[0027] In some embodiments, the pharmaceutical composition is
formulated for oral, nasal, inhalation, abdominal, subcutaneous,
intra-peritoneal or intravenous administration.
[0028] In some embodiments, the pharmaceutical composition is for
use in the treatment of cystic fibrosis (CF), in a subject in need
thereof.
[0029] In some embodiments, the method further comprising
administering to the subject a therapeutically effective amount of
one or more CFTR modifiers.
[0030] In some embodiments, the CFTR modifier is selected from the
group consisting of: a CFTR-splicing-modulating agent,
Translational Read-Through agent, a CFTR amplifier, a CFTR
potentiator, and a CFTR corrector.
[0031] In some embodiments, the CFTR modifier is selected from the
group consisting of: a different synthetic oligonucleotide molecule
capable of suppressing intron 22 cryptic exon inclusion in the
mature CFTR mRNA, Ataluren, ELX-02, ivacaftor, QBW251, PTI-808,
VX-561, lumacaftor, tezacaftor, elexacaftor, VX-659, VX-445, VX-152
and VX-440, GLPG2222, FDL169, PTI-801, and any combination
thereof.
[0032] In some embodiments, the CFTR modifier is ivacaftor,
lumacaftor, tezacaftor, elexacaftor, VX-659, VX-152, or VX-440, or
any combination thereof.
[0033] In some embodiments, the subject comprises a 3849 +10 Kb
C-to-T mutation in the CFTR gene.
[0034] In some embodiments, the subject is heterozygous to the 3849
+10 Kb C-to-T mutation.
[0035] In some embodiments, treating comprises improving at least
one clinical parameter of CF selected from the group consisting of:
lung function, time to the first pulmonary exacerbation, change in
weight, change in height, a change in Body Mass Index (BMI), change
in the concentration of sweat chloride, number and/or duration of
pulmonary exacerbations, total number of days of hospitalization
for pulmonary exacerbations, and the need for antibiotic therapy
for sinopulmonary signs or symptoms.
[0036] In some embodiments, the CF drug is an antibiotic drug, a
bronchodilator, a corticosteroid, or any combination thereof.
[0037] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
[0038] Further embodiments and the full scope of applicability of
the present invention will become apparent from the detailed
description given hereinafter. However, it should be understood
that the detailed description and specific examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 is an illustration showing the effect of the 3849 +10
kb C to T mutation in intron 22 of the CFTR gene. Top--upon normal
splicing, exon 22 and exon 23 become adjacent. Bottom--a splicing
mutation in intron 22 (denoted "3849 +10 kb C-to-T" mutation) leads
to inclusion of an excess of 84 bases in the mature CFTR mRNA
(denoted "intron 22 cryptic exon"). The mutation creates a
premature in-frame stop codon, leading to mRNA degradation by the
nonsense mediated mRNA decay (NMD) mechanism.
[0040] FIG. 2 is an illustration showing the binding sites
(underlined) of oligonucleotides SPL84-1--SPL84-26 within the CFTR
allele carrying the 3849 +10 kb C to T mutation.
[0041] FIG. 3A is a vertical bar graph showing the change in levels
of aberrantly spliced CFTR transcripts in FRT 3849 mut cells
following antisense oligonucleotide (ASO) transfection (10 nM).
Following a 24-hour treatment with the indicated ASO, RNA was
extracted and the levels of aberrantly spliced CFTR transcripts
were measured by qRT-PCR. The values shown are the average fold
change (mean.+-.s.e.m.) relative to cells treated with control ASO
from 2-7 independent experiments. Values are normalized against
transcripts of HPRT gene.
[0042] FIG. 3B is a vertical bar graph showing the percentage of
correctly spliced CFTR transcripts in FRT 3849 mut cells following
ASO transfection (10 nM). Following a 24-hour treatment with the
indicated ASO, RNA was extracted and the ratio between the
correctly and aberrantly spliced CFTR transcripts were measured by
RT-PCR. The values shown are the average percentage of correctly
spliced transcripts (mean.+-.s.e.m.) from 3-8 independent
experiments.
[0043] FIG. 4A is a vertical bar graph showing the change in levels
of aberrantly spliced CFTR transcripts in FRT 3849 mut cells
following ASO transfection (2.5 nM), compared to the ASOs described
in Friedman et al., 1999. Following a 24-hour treatment with the
indicated ASO, RNA was extracted and the levels of aberrantly
spliced CFTR transcripts were measured by qRT-PCR. The values shown
are the average fold change relative to cells treated with control
ASO (mean.+-.s.e.m.) from 3 independent experiments. Values were
normalized against transcripts of HPRT gene.
[0044] FIG. 4B is a vertical bar graph showing the percentage of
correctly spliced CFTR transcripts in FRT 3849 mut cells following
ASO transfection (2.5 nM), compared to the ASOs described in
Friedman et al., 1999. Following a 24-hour treatment with the
indicated ASO, RNA was extracted and the levels of correctly and
aberrantly spliced CFTR transcripts were measured by RT-PCR. The
values shown are the percentage of correctly spliced transcripts
(mean.+-.s.e.m.) from 3 independent experiments.
[0045] FIG. 5A is a vertical bar graph showing the change in levels
of aberrantly spliced CFTR transcripts in FRT 3849 mut cells
following ASO transfection (10 nM), compared to the ASOs described
in WO 2014/045283. Following a 24-hour treatment with the indicated
ASO, RNA was extracted and the levels of aberrantly spliced CFTR
transcripts were measured by qRT-PCR. The values shown are the
average fold change relative to cells treated with control ASO
(mean.+-.s.e.m.) from 3 independent experiments. Values were
normalized against transcripts of HPRT gene.
[0046] FIG. 5B is a vertical bar showing the percentage of
correctly spliced CFTR transcripts in FRT 3849 mut cells following
ASO transfection (10 nM), compared to the ASOs described in WO
2014/045283. Following a 24-hour treatment with the indicated ASO,
RNA was extracted and the levels of correctly and aberrantly
spliced CFTR transcripts were measured by RT-PCR. The values shown
are the percentage of correctly spliced transcripts
(mean.+-.s.e.m.) from 3 independent experiments.
[0047] FIG. 6 is a graph showing the effect of different ASOs in
different concentrations on the aberrantly spliced CFTR transcripts
in FRT 3849 mut cells following a 24-hour treatment with the
indicated ASO at the indicated concentration, RNA was extracted and
the levels of aberrantly spliced CFTR transcripts were measured by
qRT-PCR. The values shown are the average fold change relative to
cells treated with control ASO (mean.+-.s.e.m.). Values were
normalized against transcripts of HPRT gene.
[0048] FIGS. 7A-7B are micrographs of western blot analyses showing
the effect of different ASOs on the formation of full length mature
CFTR protein. (7A) FRT 3849 mut cells were transfected with the
indicated ASO. (7B) HEK 293T cells transiently expressing the CFTR
cDNA mutated with the 3849 +10 Kb C-to-T mutation and containing
the flanking intronic regions were transfected with the indicated
ASO. The M3A7 antibody recognizes the C terminal of the protein
allowing identification of only full length CFTR proteins.
[0049] FIG. 8A is a graph showing a representative Fluorescent
Imaging Plate Reader (FLIPR) trace demonstrating CFTR activation
induced by the effect of ASO SPL84-17 in FRT 3849 mut cells.
[0050] FIG. 8B is a vertical bar graph showing the average effect
of certain ASOs on CFTR function. The activity of the CFTR channel
was analyzed using the FLIPR assay in FRT 3849 mut cells following
transfection with the indicated ASO (10 nM). mean.+-.s.e.m.
[0051] FIG. 9A includes curves showing the effect of ASOs on CFTR
activation in primary Human Nasal Epithelial (HNE) cells from the
heterozygous CF patient with the genotype 3849 +10 Kb
C-to-T/F508del. Representative traces of electrophysiological
responses in Ussing chamber following free uptake of the indicated
ASO.
[0052] FIG. 9B is a scatter dot plot of absolute values of
.DELTA.I.sub.SCCFTRinh172(.mu.A/cm.sup.2) from Ussing chamber
following free uptake of the indicated ASO in the same HNE cells as
in FIG. 9A. The grey thick horizontal line is marking the median.
.DELTA.I.sub.SCCFTRinh172 was calculated for: 4 filters treated
with SPL84-22, SPL84-23 and SPL84-17 derived from 2 samples
obtained from the patient, 2 filters treated with SPL84-2 and one
filter treated with SPL84-25 (marked with *). The horizontal dashed
line indicates 50% of the level of mean .DELTA.I.sub.SCCFTRinh172
in HNE cultures from healthy WT/WT individuals.
[0053] FIG. 10A includes curves showing the effect of ASO on CFTR
activation in primary HNE cells from a patient homozygote for the
3849 +10 Kb C-to-T mutation. Representative traces of
electrophysiological responses in Ussing chamber following
treatment with the indicated ASOs.
[0054] FIG. 10B is a scatter dot plot of absolute values of
.DELTA.I.sub.SCCFTRinh172(.mu.A/cm.sup.2) from Ussing chamber
following free uptake of the indicated ASO in the same HNE cells as
in FIG. 10A. The grey thick horizontal line is marking the median.
.DELTA.I.sub.SCCFTRinh172 was calculated for: 2 filters, except for
one filter treated with SPL84-2 (marked with *). The horizontal
dashed line indicates the level of mean .DELTA.I.sub.SCCFTRinh172
in HNE cultures from healthy WT/WT individuals.
[0055] FIG. 11 is a vertical bar graph showing the summary of
activation of CFTR in heterozygote patients. Mean(.+-.SEM) values
of % of WT
(.DELTA.I.sub.SCCFTRinh172-Patient*100/Average.DELTA..sub.SCCFTRinh172-WT-
) were calculated from median values of .DELTA.I.sub.SCCFTRinh172
per patient. The level of WT was set according to the mean
.DELTA.I.sub.SCFSK/I in HNE cultures from healthy WT/WT
individuals. The values are calculated from 9 filters treated with
SPL84-17 and SPL84-22 derived from 3 patients, 24 filters treated
with SPL84-23 derived from 5 patients, 5 filters treated with
SPL84-2 derived from 2 patients and 1 filter treated with SPL84-25
derived from 1 patient.
[0056] FIGS. 12A-12B include a vertical bar graph and a micrograph
of gel electrophoresis showing the effect of ASO SPL84-23 on the
splicing pattern of 3849 +10 kb C>T mutation. HNE cells from
patients homozygous (N=1) or heterozygous (N=4) for the 3849 +10 kb
C-to-T mutation were treated with 200 nM SPL84-23. RNA was
extracted and the levels of aberrantly and correctly spliced CFTR
transcripts were measured. FIG. 12A shows qRT-PCR analysis for the
effect of SPL84-23 on aberrantly spliced CFTR transcripts. The
values shown are the average fold change (mean.+-.SEM) relative to
cells treated with control ASO. Values were normalized against
transcripts of GUSb gene. Statistical analysis was performed using
student's t-test (1 tail, paired). ***p<0.001. FIG. 12B shows
RT-PCR on the homozygous samples in agarose gels.
[0057] FIG. 13 is a graph showing the effect of chemical
modifications on ASO SPL84-23 on the splicing pattern of 3849 +10
kb C>T mutation. Scatter dot plot of absolute values of
.DELTA.I.sub.SCCFTRinh172(.mu.A/cm.sup.2). The horizontal lines are
marking the medians. .DELTA.I.sub.SCCFTRinh172 was calculated for
cells treated with the indicated concentrations of ASO modified
with the 2'OMP or 2'MOE. The horizontal dashed line indicates the
level of mean .DELTA.I.sub.SCCFTRinh172 in HNE cultures sampled
from healthy WT/WT individuals.
[0058] FIG. 14 is a vertical bar graph showing optimization of the
ASO length on aberrantly spliced variants of CFTR transcripts.
Short versions of AS084-23 were screened for their effect on
splicing by free uptake on primary patient HNE cells
(SPL84-23-1-SPL84-23-9). On average in the three 3849 heterozygote
patients analyzed, the effect of SPL84-23-1 (19 mer) was comparable
to SPL84-23.
[0059] FIGS. 15A-15B are graphs showing optimization of the ASO
length. Primary HNE cells were obtained from an heterozygous
patient (3849/F508del). SPL84-23-1 (19 mer) has a similar effect on
the CFTR function as the longer SPL84-23 ASO in primary HNE cells.
(15A) represents absolute values of
.DELTA.I.sub.SCCFTRinh172(.mu.A/cm.sup.2), and (15B) represents the
ratio compared to the WT (% of WT (I.sub.SCCFTRinh172)).
[0060] FIGS. 16A-16B are graphs showing optimization of the ASO
length. Primary HBE cells were obtained from an heterozygous
patient (3849/F508del). SPL84-23-1 (19 mer) has a similar effect on
the CFTR function as the longer SPL84-23 ASO in primary HNE cells.
(16A) represents absolute values of
.DELTA.I.sub.SCCFTRinh172(.mu.A/cm.sup.2), and (16B) represents the
ratio compared to the WT (% of WT (I.sub.SCCFTRinh172)).
DETAILED DESCRIPTION
[0061] In some embodiments, there is provided a synthetic
oligonucleotide molecule consisting of 18-21 consecutive bases
having at least 80% complementarity to a pre-mRNA transcript of a
CFTR gene having a 3849 +10 Kb C-to-T mutation, and characterized
by at least partly suppressing the inclusion of intron 22 cryptic
exon in the mature CFTR mRNA, increasing the percentage of
correctly spliced mature CFTR mRNA by at least about 10%; and
decreasing the level of aberrantly spliced mature CFTR mRNA by at
least about 20%.
[0062] In some embodiments, there is provided a method for treating
CF in a subject in need thereof, comprising administering to the
subject a therapeutically effective amount of the synthetic
oligonucleotide of the invention, thereby treating CF in the
subject.
[0063] In some embodiments, the treating comprises: suppressing the
inclusion of intron 22 cryptic exon in the mature CFTR mRNA,
increasing the percentage of correctly spliced mature CFTR mRNA by
at least about 10%, decreasing the level of aberrantly spliced
mature CFTR mRNA by at least about 20%, or any combination
thereof.
[0064] The present invention provides oligonucleotides and
compositions comprising same, capable of binding to a CFTR
pre-mRNA, thereby modulating splicing and restoring the function of
the CFTR gene product. The present invention thus identifies
sequences within the CFTR pre-mRNA which are targeted in order to
modulate the splicing cascade of the CFTR pre-mRNA. Modulating CFTR
pre-mRNA splicing, as demonstrated in the present invention, can
avoid improper recognition of intron sequences as exons. As a
result of the modulation of splicing, a functional CFTR protein is
produced by an otherwise aberrant CFTR allele.
[0065] In some embodiments, the herein disclosed oligonucleotide is
a synthetic oligonucleotide.
[0066] The present invention stems in part from the finding that
artificial "anti-sense" polynucleotide molecules are able to target
and bind predetermined sequences at the pre-mRNA molecule of the
CFTR gene, and that the binding modulates the splicing of the
pre-mRNA molecule into mature mRNA, which subsequently translates
into a functional CFTR protein. The targets within a CFTR pre-mRNA
molecule are those discovered to be involved in splicing, either
indirectly, by affecting the splicing of adjacent as well as remote
sequences, or directly, by affecting their own splicing.
[0067] The present invention provides, in one aspect, a synthetic
oligonucleotide molecule, consisting of 17-21 consecutive bases
that are complementary to a pre-mRNA transcript of a CFTR gene,
wherein the synthetic oligonucleotide molecule at least partly
suppresses the inclusion of intron 22 cryptic exon in the mature
CFTR mRNA, increases the percentage of correctly spliced mature
CFTR mRNA by at least about 10%; and decreases the level of
aberrantly spliced mature CFTR mRNA by at least about 20%.
[0068] In another aspect, the present invention provides a
synthetic oligonucleotide molecule consisting of 17-21 consecutive
bases that are complementary to a pre-mRNA transcript of a CFTR
gene having a 3849 +10 Kb C-to-T mutation, wherein the synthetic
oligonucleotide molecule at least partly suppresses the inclusion
of intron 22 cryptic exon in the mature CFTR mRNA, decreases the
level of aberrantly spliced mature CFTR mRNA by at least about
20%.
[0069] In some embodiments, the oligonucleotide increases the
percentage of correctly spliced mature CFTR mRNA by at least about
12%. In some embodiments, the oligonucleotide increases the
percentage of correctly spliced mature CFTR mRNA by at least about
10%.
[0070] The present invention provides, in additional aspect, a
synthetic oligonucleotide molecule, consisting of 17-21 consecutive
bases that are complementary to a pre-mRNA transcript of a CFTR
gene having a 3849 +10 Kb C-to-T mutation, wherein the synthetic
oligonucleotide molecule at least partly suppresses the inclusion
of intron 22 cryptic exon in the mature CFTR mRNA, increases the
percentage of correctly spliced mature CFTR mRNA by at least about
10%; and decreases the level of aberrantly spliced mature CFTR mRNA
by at least about 20%, wherein the oligonucleotide comprises a
2'-O-methyl-phosphorothioate backbone and/or 2'-Methoxy Ethyl
(2'MOE) backbone. The phrase "suppress intron 22 cryptic exon
inclusion" as used herein refers to lowering the occurrence of the
addition of 84 nucleotides (SEQ ID NO: 35) found within intron 22
of the CFTR gene to the mature CFTR mRNA.
[0071] The phrase "increases the percentage of correctly spliced
mature CFTR mRNA" as used herein refers to the percentage of
correctly spliced mature CFTR mRNA compared to the 100% of
correctly spliced mature CFTR mRNA found in a healthy cell or
subject not having a mutation in either alleles of the CFTR gene.
For example, an increase from 1% before treatment by the ASOs
provided by the present invention to 11% after the treatment by the
ASOs provided by the present invention is considered an increase of
10%. An increase from 10% before treatment by the ASOs provided by
the present invention to 11% after the treatment by the ASOs
provided by the present invention is considered an increase of
1%.
[0072] In certain embodiments, the CFTR transcript comprises a
mutation that increases inclusion of an intron 22 cryptic exon. In
certain embodiments, the mutation is a 3849 +10 Kb C to T mutation.
In some embodiments, the intron 22 cryptic exon comprises the
sequence of SEQ ID NO: 35 or a fragment thereof.
[0073] In certain embodiments, the oligonucleotide molecule is
complementary to a nucleotide sequence within SEQ ID NO: 37. In
some embodiments, the oligonucleotide molecule is complementary to
a nucleotide sequence not more than 1000, 900, 800, 700, 600, 500,
400, 300, 200 or 100 bases upstream of a mutation that increase
inclusion of an intron 22 cryptic exon. Each possibility represents
a separate embodiment of the invention. In some embodiments, the
oligonucleotide molecule is complementary to a nucleotide sequence
not more than 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100
bases downstream of a mutation that increase inclusion of an intron
22 cryptic exon. Each possibility represents a separate embodiment
of the invention.
[0074] In some embodiments, the oligonucleotide has at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
99%, or 100% complementarity to a nucleotide sequence within SEQ ID
NO: 37.
[0075] In certain embodiments, the oligonucleotide molecule is
complementary to a nucleotide sequence corresponding to pre-mRNA
molecule comprising a sequence about 100 base pairs before the
intron 22 cryptic exon to about 100 base pairs after the intron 22
cryptic exon transcribed from a CFTR gene having a 3849 +10 Kb
C-to-T mutation. In certain embodiments, the oligonucleotide
molecule is complementary to a nucleotide sequence within SEQ ID
NO: 37.
[0076] The terms "complementary" or "complementarity" refer to the
ability of nucleic acids, e.g., oligonucleotide, polynucleotide,
etc., to form base pairs with one another. Base pairs are typically
formed by hydrogen bonds between nucleotide units in antiparallel
polynucleotide strands. Complementary polynucleotide strands can
base pair in the Watson-Crick manner (e.g., A to T, A to U, C to
G), or in any other manner that allows for the formation of
duplexes. As persons skilled in the art are aware, when using RNA
as opposed to DNA, uracil rather than thymine is the base that is
considered to be complementary to adenosine. However, when a U is
denoted in the context of the present invention, the ability to
substitute a T is implied, unless otherwise stated.
[0077] In certain embodiments, the oligonucleotide molecule
increases the percentage of correctly spliced mature CFTR mRNA by
about 20%. In certain embodiments, the oligonucleotide molecule
increases the percentage of correctly spliced mature CFTR mRNA by
about 30%. In certain embodiments, the oligonucleotide molecule
increases the percentage of correctly spliced mature CFTR mRNA by
about 40%. In certain embodiments, the oligonucleotide molecule
increases the percentage of correctly spliced mature CFTR mRNA by
about 50%. In certain embodiments, the oligonucleotide molecule
increases the percentage of correctly spliced mature CFTR mRNA by
about 60%.
[0078] The phrase "increases the level of correctly spliced mature
CFTR mRNA" as used herein refers to the increase in the level of
correctly spliced mature CFTR mRNA after treatment by the ASOs
provided by the present invention compared to the level before
treatment or after mock-treatment. In certain embodiments, the
increase in the level of correctly spliced mature CFTR mRNA is
compared to mock-treatment by a control ASO. In certain
embodiments, the control ASO consists of the nucleotide sequence in
SEQ ID NO: 33.
[0079] In certain embodiments, the oligonucleotide molecule
decreases the level of aberrantly spliced mature CFTR mRNA by about
30%. In certain embodiments, the oligonucleotide molecule decreases
the level of aberrantly spliced mature CFTR mRNA by about 40%. In
certain embodiments, the oligonucleotide molecule decreases the
level of aberrantly spliced mature CFTR mRNA by about 50%. In
certain embodiments, the oligonucleotide molecule decreases the
level of aberrantly spliced mature CFTR mRNA by about 60%. In
certain embodiments, the oligonucleotide molecule decreases the
level of aberrantly spliced mature CFTR mRNA by about 70%. In
certain embodiments, the oligonucleotide molecule decreases the
level of aberrantly spliced mature CFTR mRNA by about 80%.
[0080] The phrase "decreases the level of aberrantly spliced mature
CFTR mRNA" as used herein refers to the percentage decrease in the
level of aberrantly spliced mature CFTR mRNA after treatment by the
ASOs provided by the present invention compared to the level before
treatment or after mock-treatment. In certain embodiments, the
decrease in the level of aberrantly spliced mature CFTR mRNA is
compared to mock-treatment by a control ASO. In certain
embodiments, the control ASO consists of the nucleotide sequence in
SEQ ID NO: 33. The phrase "increases the percentage of correctly
spliced mature CFTR mRNA" as used herein refers to the percent
increase in the ratio of correctly spliced to aberrantly spliced
CFTR mRNA compared to untreated or mock-treatment.
[0081] In certain embodiments, the oligonucleotide molecule
consists of 18 or 19 consecutive nucleotide bases. In certain
embodiments, the oligonucleotide molecule consists of 18
consecutive nucleotide bases. In certain embodiments, the
oligonucleotide molecule consists of 19 consecutive nucleotide
bases. In certain embodiments, the oligonucleotide molecule
consists of 20 consecutive nucleotide bases. In certain
embodiments, the oligonucleotide molecule consists of 21
consecutive nucleotide bases.
[0082] In certain embodiments, the base is selected from the group
consisting of adenine, guanine, cytosine, uracil and optionally
thymine. In other certain embodiments, the base is selected from
the group consisting of adenine, guanine, cytosine, and uracil.
Each possibility represents a separate embodiment of the present
invention.
[0083] In some embodiments, the oligonucleotide is chemically
modified. In some embodiments, the chemical modification is a
modification of a backbone of the oligonucleotide. In some
embodiments, the chemical modification is a modification of a sugar
of the oligonucleotide. In some embodiments, the chemical
modification is a modification of a nucleobase of the
oligonucleotide. In some embodiments, the chemical modification
increases stability of the oligonucleotide in a cell. In some
embodiments, the chemical modification increases stability of the
oligonucleotide in vivo. In some embodiments, the chemical
modification increases the oligonucleotide's ability to modulate
splicing. In some embodiments, the chemical modification increases
the oligonucleotide's ability to induce suppress the inclusion of
intron 22 cryptic exon. In some embodiments, the chemical
modification increases the half-life of the oligonucleotide. In
some embodiments, the chemical modification inhibits polymerase
extension from the 3' end of the oligonucleotide. In some
embodiments, the chemical modification inhibits recognition of the
oligonucleotide by a polymerase. In some embodiments, the chemical
modification inhibits double-strand trigged degradation. In some
embodiments, the chemically modified oligonucleotide does not
trigger nucleic acid double-stranded degradation upon binding a
CFTR pre-mRNA. In some embodiments, the chemical modification
inhibits RISC-mediated degradation. In some embodiments, the
chemical modification inhibits RISC-mediated degradation or any
parallel nucleic acid degradation pathway.
[0084] In some embodiments, the oligonucleotide is devoid of a
labeling moiety. In some embodiments, the oligonucleotide is not
labeled. In some embodiments, the oligonucleotide does not emit a
detectable signal or does not comprise moieties capable of being
recognized so as to enable nucleic acid detection (e.g.,
digoxigenin and fluorescently labeled anti-DIG antibody). In some
embodiments, a detectable signal comprises a dye or an emitting
energy which provides detection of a compound, e.g., a
polynucleotide, in vivo, ex vivo, or in vitro. In some embodiments,
a detectable signal comprises: a fluorescent signal, a chromatic
signal, or a radioactive signal.
[0085] In some embodiments, the oligonucleotide is devoid of
radioactive nucleobase(s); digoxigenin, streptavidin, biotin, a
fluorophore, hapten label, CLICK label, amine label, or thiol
label.
[0086] In certain embodiments, the consecutive nucleotide bases are
linked by a backbone selected from the group consisting of a
phosphate-ribose backbone, a phosphate-deoxyribose backbone, a
2'-O-methyl-phosphorothioate backbone, a phosphorodiamidate
morpholino backbone, a peptide nucleic acid backbone, a
2-methoxyethyl phosphorothioate backbone, an alternating locked
nucleic acid backbone, constrained ethyl backbone, and a
phosphorothioate backbone. Each possibility represents a separate
embodiment of the invention.
[0087] According to some embodiments, the oligonucleotide comprises
a 2'-O-methyl-phosphorothioate backbone. According to other
embodiments, the oligonucleotide comprises a 2'-Methoxy Ethyl
(2'MOE) modification.
[0088] In some embodiments, the oligonucleotide comprises a
2'-O-methyl-phosphorothioate modification. In some embodiments, the
oligonucleotide comprises a 2'MOE modification. In some
embodiments, the modification is throughout the molecule. In some
embodiments, the modification is at the 3' end of the molecule. In
some embodiments, the modification is at the 5' end of the
molecule. In some embodiments, the molecule comprises at least 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 modifications. Each possibility represents a
separate embodiment of the invention.
[0089] According to some embodiments, the oligonucleotide
modification is capable of completely restoring CFTR function
compared to non-mutated CFTR. According to certain embodiments, the
oligonucleotide modification is capable of restoring at least 70%,
65%, 80%, 85%, 90%, or 95% of CFTR function compared to non-mutated
CFTR. Each possibility represents a separate embodiment of the
invention.
[0090] In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 1. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 2. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 3. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 4. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 5. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 6. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 7. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 8. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 9. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 10. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 11. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 12. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 13. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 14. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 15. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 16. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 17. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 18. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 19. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 20. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 21. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 22. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 23. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 24. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 25. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 40. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 41. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 42. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 43. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 44. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 45. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 46. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 47. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 48. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 49. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 50. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 51. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 52. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 53. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 54. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 55. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 56. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 57. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 58. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 59. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 60. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 61. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 62. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 63. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 64. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 65. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 66. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 67. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 68. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 69. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 70. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 71.
[0091] In certain embodiments, the oligonucleotide molecule
comprises a nucleotide sequence set forth in one of SEQ ID NO: 1 to
SEQ ID NO: 5, and SEQ ID NO: 41. In certain embodiments, the
oligonucleotide molecule consists of a nucleotide sequence set
forth in SEQ ID NO: 1, in SEQ ID NO: 2, SEQ ID NO: 3, in SEQ ID NO:
4, in SEQ ID NO: 5, or in SEQ ID NO: 41. Each possibility
represents a separate embodiment of the invention.
[0092] In some embodiments, the oligonucleotide is specific to a
CFTR pre-mRNA. As used herein, the term "specific" refers to both
base pair specificity and also gene specificity. In some
embodiments, the oligonucleotide is specific to the CFTR gene. In
some embodiments, the oligonucleotide is specific to an intronic
sequence of CFTR. In some embodiments, the oligonucleotide is
specific to a cryptic exon in CFTR. In some embodiments, the
oligonucleotide is specific to a nucleic acid sequence of intron 22
of CFTR. In some embodiments, the nucleic acid sequence of intron
22 is intron 22 cryptic exon of CFTR.
[0093] In some embodiments, the oligonucleotide binds the CFTR
pre-mRNA with perfect complementarity. In some embodiments, the
oligonucleotide does not bind any gene other than CFTR with perfect
complementarity. In some embodiments, the oligonucleotide does not
bind any gene other than CFTR with a complementarity of greater
than 70, 75, 80, 85, 90, 95, 97, 99 or 100%. Each possibility
represents a separate embodiment of the invention. In some
embodiments, the oligonucleotide does not bind any gene other than
CFTR with a complementarity of greater than 90%. In some
embodiments, the oligonucleotide binds SEQ ID NO: 37 with perfect
complementarity. In some embodiments, the oligonucleotide does not
bind any sequence other than SEQ ID NO: 37 with perfect
complementarity. In some embodiments, the oligonucleotide does not
bind any sequence other than SEQ ID NO: 37 with complementarity of
greater than 70, 75, 80, 85, 90, 95, 97, 99 or 100%. Each
possibility represents a separate embodiment of the invention. In
some embodiments, the oligonucleotide does not bind any sequence
other than SEQ ID NO: 37 with a complementarity of greater than
90%. In some embodiments, the oligonucleotide does not bind with
perfect complementarity to anywhere in the genome of a cell other
than within CFTR. In some embodiments, the oligonucleotide does not
bind with complementarity of greater than 70, 75, 80, 85, 90, 95,
97, 99 or 100% to anywhere in the genome of a cell other than
within CFTR. Each possibility represents a separate embodiment of
the invention. In some embodiments, the cell is a mammalian cell.
In some embodiments, the mammal is a human.
[0094] In some embodiments, the oligonucleotide modulates
expression of CFTR. In some embodiments, the oligonucleotide
modulates splicing of CFTR. In some embodiments, the
oligonucleotide modulates splicing, e.g., suppresses inclusion, of
intron 22 cryptic exon of CFTR. In some embodiments, the
oligonucleotide does not cause an off-target effect. In some
embodiments, off-target is a target other than CFTR. In some
embodiments, off-target is a target other than splicing, e.g.,
suppressing the inclusion, of intron 22 cryptic exon of CFTR. In
some embodiments, the oligonucleotide does not substantially or
significantly modulate expression of a gene other than CFTR. In
some embodiments, the oligonucleotide does not substantially or
significantly modulate splicing of a gene other than CFTR. In some
embodiments, the oligonucleotide does not substantially or
significantly modulate splicing of an exon other than intron 22
cryptic exon of CFTR. In some embodiments, substantial modulation
of expression is a change in expression of at least 5, 10, 15, 20,
25, 30, 35, 40, 45 or 50%. Each possibility represents a separate
embodiment of the invention. In some embodiments, substantial
modulation of expression is a change in expression of at least
20%.
[0095] In some embodiments, an oligonucleotide as disclosed herein
targets, complements, suppresses, or any combination thereof, the
inclusion of intron 22 cryptic exon to the mature CFTR mRNA
transcribed from a mutated allele of the CFTR gene. In some
embodiments, an oligonucleotide as disclosed herein does not
target, complement, suppresses, or any combination thereof,
splicing modulation of CFTR pre-mRNA transcribed from a wild type
allele of the CFTR gene. In some an oligonucleotide as disclosed
herein targets, complements, suppresses, or any combination thereof
at least 2 fold more efficiently, at least 3 fold more efficiently,
at least 5 fold more efficiently, at least 7 fold more efficiently,
at least 10 fold more efficiently, at least 20 fold more
efficiently, at least 50 fold more efficiently, or at least 100
fold more efficiently, the inclusion of intron 22 cryptic exon into
the mature CFTR mRNA transcribed from a mutated allele of the CFTR
gene compared to the wild type allele of the CFTR gene, or any
value and range therebetween. Each possibility represents a
separate embodiment of the invention. In some an oligonucleotide as
disclosed herein targets, complements, suppresses, or any
combination thereof 2-10 fold more efficiently, 3-50 fold more
efficiently, 5-100 fold more efficiently, 7-20 fold more
efficiently, 2-40 fold more efficiently, 2-25 fold more
efficiently, 50-150 fold more efficiently, or 2-100 fold more
efficiently, the inclusion of intron 22 cryptic exon into the
mature CFTR mRNA transcribed from a mutated allele of the CFTR gene
compared to the wild type allele of the CFTR gene. Each possibility
represents a separate embodiment of the invention.
[0096] In some embodiments, an oligonucleotide of the invention
fully complements with a mutated allele of the CFTR gene. As used
herein, the term "fully complements" refers to 100% hybridization,
meaning the mutated CFTR allele and the oligonucleotide represent a
reversed and complementary nucleic acid sequence versions of one
another, as would be apparent to one of ordinary skill in the art
of molecular biology. In some embodiments, an oligonucleotide of
the invention partially complements with the wild type allele of
the CFTR gene. As used herein, the term "partially" refers to any
value or range lower than 100%. In some embodiments, the
oligonucleotide of the invention and the wild type CFTR allele
represent a reversed and complementary nucleic acid sequence
version of one another which differ by at least one nucleotide,
e.g., comprising at least one mismatched nucleotide.
[0097] In some embodiments, the oligonucleotide of the invention,
and method of using same, provide the exclusion of a cryptic exon
from the mature CFTR mRNA transcribed from a mutated allele of the
CFTR gene. In some embodiments, the mature mRNA transcribed from
the wild type allele is devoid of the cryptic exon.
[0098] In some embodiments, the cryptic exon is intron 22 cryptic
exon. In some embodiments, the cryptic exon is 80-90 bases
long.
[0099] In some embodiments, the oligonucleotide comprises an active
fragment of any one of SEQ ID Nos.: 1-25, and 41-44.
[0100] In some embodiments, the oligonucleotide comprises an active
fragment of any one of SEQ ID Nos.: 1-5, and 41.
[0101] As used herein, the term "active fragment" refers to a
fragment that is 100% identical to a contiguous portion of the full
nucleotide sequence of the oligonucleotide, providing that at
least: 30%, 40%, 50%, 60%, 70%, 80% or 90% of the activity of the
original oligonucleotide sequence is retained, or any value and
range therebetween. Each possibility represents a separate
embodiment of the present invention.
[0102] In some embodiments, the subject is heterozygous to the 3849
+10 Kb C-to-T mutation. In some embodiments, a subject treated
according to the method of the invention, comprises or is
characterized by having a mixture of a wild type full-length and
fully functional CFTR protein encoded from the wild type allele and
a full-length and fully functional CFTR protein encoded from the
pre-mRNA from which the inclusion of intron 22 cryptic exon
inclusion was suppressed using the oligonucleotide of the
invention. In some embodiments, the oligonucleotide of the
invention does not reduce the level of the wild type full-length
and fully functional CFTR protein in a subject, e.g., heterozygous
to the mutation disclosed hereinabove.
[0103] The present invention further provides, in another aspect, a
pharmaceutical composition comprising a synthetic oligonucleotide
molecule as described above, and a pharmaceutically acceptable
carrier.
[0104] The term "pharmaceutically acceptable carrier" as used
herein refers to any of the standard pharmaceutical carriers known
in the field such as sterile solutions, tablets, coated tablets,
and capsules. Typically, such carriers contain excipients such as
starch, milk, sugar, certain types of clay, gelatin, stearic acids,
or salts thereof, magnesium or calcium stearate, talc, vegetable
fats or oils, gums, glycols, or other known excipients. Such
carriers may also include flavor and color additives or other
ingredients. Examples of pharmaceutically acceptable carriers
include, but are not limited to, the following: water, saline,
buffers, inert, nontoxic solids (e.g., mannitol, talc).
Compositions comprising such carriers are formulated by well-known
conventional methods. Depending on the intended mode of
administration and the intended use, the compositions may be in the
form of solid, semi-solid, or liquid dosage forms, such, for
example, as powders, granules, crystals, liquids, suspensions,
liposomes, nano-particles, nano-emulsions, pastes, creams, salves,
etc., and may be in unit-dosage forms suitable for administration
of relatively precise dosages.
[0105] In certain embodiments, the pharmaceutical composition is
formulated for oral, administration. In certain embodiments, the
pharmaceutical composition is formulated for nasal administration.
In certain embodiments, the pharmaceutical composition is
formulated for administration by inhalation. In certain
embodiments, the pharmaceutical composition is formulated for
abdominal administration. In certain embodiments, the
pharmaceutical composition is formulated subcutaneous
administration. In certain embodiments, the pharmaceutical
composition is formulated for intra-peritoneal administration. In
certain embodiments, the pharmaceutical composition is formulated
for intravenous administration.
[0106] In some embodiments, the pharmaceutical composition is
formulated for systemic administration. In some embodiments, the
pharmaceutical composition is formulated for administration to a
subject. In some embodiments, the subject is a human subject. It
will be understood by a skilled artisan that a pharmaceutical
composition intended to administration to a subject should not have
off-target effects, i.e. effects other than the intended
therapeutic ones. In some embodiments, the pharmaceutical
composition is devoid of a substantial effect on a gene other than
CFTR. In some embodiments, the pharmaceutical composition is devoid
of any substantial effect other than suppressing the inclusion of
intron 22 cryptic exon to the mature CFTR. In some embodiments, a
substantial effect is one with a phenotypic result. In some
embodiments, a substantial effect is a deleterious effect. In some
embodiments, deleterious is with respect to the health and/or
wellbeing of the subject.
[0107] In some embodiments, the composition administered by
inhalation. In some embodiments, the composition is an inhalation
composition. in some embodiments, the composition is a
pharmaceutical composition.
[0108] Being a long-known and well-studied disease, certain drugs
and agents are known in the art for the treatment of Cystic
Fibrosis patients. Administrating a synthetic polynucleotide
molecule according to the present invention with one or more of
these drugs may be beneficial in achieving significant therapeutic
results.
[0109] In certain embodiments, the pharmaceutical composition
further comprises one or more CFTR modifiers.
[0110] In some embodiments, the method further comprises
administering to the subject a therapeutically effective amount of
one or more CFTR modifiers.
[0111] In some embodiments, the CFTR modifier increases the
duration of the CFTR gate being open, chloride flow through the
CFTR gate, CFTR protein proper folding, the number of CFTR anchored
to the cell membrane, or any combination thereof. Each possibility
represents a separate embodiment of the invention.
[0112] In some embodiments, the modifier is selected from:
potentiator, corrector, and amplifier.
[0113] As used herein, the term "potentiator" refers to any agent
that increases the probability that a defective CFTR will be open
and therefore allows chloride ions to pass through the channel
pore.
[0114] As used herein, the term "corrector" refers to any agent
that assists in proper CFTR channel folding so as to enable its
trafficking to the cell membrane.
[0115] As used herein, the term "amplifier" refers to any agent
that induces a cell to increase its CFTR protein production rates
or yields, therefore resulting in an increased amount of the CFTR
protein.
[0116] In certain embodiments, the CFTR modifier is selected from:
a CFTR-splicing-modulating agent, Translational Read-Through agent,
a CFTR amplifier, a CFTR potentiator and a CFTR corrector. In
certain embodiments, the CFTR-splicing-modulating agent is a
different synthetic oligonucleotide molecule capable of suppressing
intron 22 cryptic exon inclusion in the mature CFTR mRNA; the
Translational Read-Through agent is selected from the group
consisting of 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic
acid (Ataluren) and ELX-02; the CFTR amplifier is PTI-428; the CFTR
potentiator is selected from the group consisting of
N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
amide (Ivacaftor), QBW251, PTI-808 and VX-561 (deuterated
ivacaftor); the CFTR potentiator is
N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
amide (Ivacaftor); or the CFTR corrector is selected from the group
consisting of
3-{6-{[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl]amino}-3-
-methylpyridin-2-yl}benzoic acid (Lumacaftor),
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-.about.{N}-[1-[(2-{R})-2,3-dihydrox-
ypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-
-1-carboxamide (Tezacaftor), VX-659, VX-445, VX-152 and VX-440,
GLPG2222, FDL169 and PTI-801.
[0117] In certain embodiments, the pharmaceutical composition
comprises at least about 1 nM of the synthetic oligonucleotide
molecule. In certain embodiments, the pharmaceutical composition
comprises at least about 2.5 nM of the synthetic oligonucleotide
molecule. In certain embodiments, the pharmaceutical composition
comprises at least about 10 nM of the synthetic oligonucleotide
molecule. In certain embodiments, the pharmaceutical composition
comprises 2.5 nM to 10 nM of the synthetic oligonucleotide
molecule.
[0118] In certain embodiments, the pharmaceutical composition
comprises 1 nM to 1 .mu.M of the synthetic oligonucleotide
molecule. In certain embodiments, the pharmaceutical composition
comprises 1 nM to 0.5 .mu.M of the synthetic oligonucleotide
molecule. In certain embodiments, the pharmaceutical composition
comprises 1 nM to 100 nM of the synthetic oligonucleotide
molecule.
[0119] The present invention further provides, in another aspect, a
synthetic oligonucleotide molecule as described above, or a
pharmaceutical composition as described above, for use in the
modulation of splicing of a CFTR pre-mRNA transcribed from a CFTR
gene having a 3849 +10 Kb C-to-T mutation.
[0120] The phrase "modulation of splicing" as used herein refers to
affecting a change in the level of any RNA or mRNA variant produced
by the CFTR native pre-mRNA. For example, modulation may mean e.g.
causing an increase or decrease in the level of abnormal CFTR mRNA,
causing an increase or decrease in the level of normal, full-length
CFTR mRNA, and/or causing an increase or decrease in the level of
abnormal CFTR RNA or mRNA comprising a premature termination codon
(non-sense codon). It is therefore evident that any change in ratio
between certain CFTR splicing variants is also considered to be the
result of splicing modulation. Each possibility represents a
separate embodiment of the present invention. In certain
embodiments, modulation means increasing the level of normal,
full-length CFTR mRNA and/or decreasing the level of abnormal CFTR
mRNA.
[0121] In certain embodiments, the use is for reducing the level of
an mRNA molecule comprising the intron 22 cryptic exon. In certain
embodiments, the use is for reducing the level of an mRNA molecule
comprising the nucleotide sequence set forth in SEQ ID NO: 36. In
certain embodiments, the use is for increasing the level of normal,
full-length CFTR mRNA. In certain embodiments, the use is for
increasing the level of an mRNA molecule comprising the nucleotide
sequence set forth in SEQ ID NO: 34. In certain embodiments, the
use is for correcting or improving chloride transport through the
CFTR channel. In certain embodiments, the use is for increasing the
production of functional CFTR protein. Each possibility represents
a separate embodiment of the present invention.
[0122] The present invention further provides, in another aspect, a
synthetic oligonucleotide molecule as described above, or a
pharmaceutical composition as described above, for use in a method
for improving at least one clinical parameter of Cystic
Fibrosis.
[0123] The invention further provides, in another aspect, a method
for improving at least one clinical parameter of Cystic Fibrosis in
a patient in need thereof, comprising the step of administering a
therapeutically effective amount of a synthetic polynucleotide
molecule as described above to the patient.
[0124] The term "a therapeutically effective amount" as used herein
refers to an amount necessary for improving at least one clinical
parameter of Cystic Fibrosis or reducing the severity of at least
one clinical parameter of Cystic Fibrosis in a patient. The
therapeutically effective amounts may differ according to the
patient's status, the synthetic polynucleotide molecule's
administration route, excipient usage and co-usage of other active
agents.
[0125] In certain embodiments, the clinical parameter is selected
from the group consisting of lung function, time to the first
pulmonary exacerbation, change in weight, change in height, a
change in Body Mass Index (BMI), change in the concentration of
sweat chloride, number and/or duration of pulmonary exacerbations,
total number of days of hospitalization for pulmonary
exacerbations, and the need for antibiotic therapy for
sinopulmonary signs or symptoms. Each possibility represents a
separate embodiment of the invention.
[0126] As used herein, the terms "treatment" or "treating" of a
disease, disorder, or condition encompasses alleviation of at least
one symptom thereof, a reduction in the severity thereof, or
inhibition of the progression thereof. Treatment need not mean that
the disease, disorder, or condition is totally cured. To be an
effective treatment, a useful composition herein needs only to
reduce the severity of a disease, disorder, or condition, reduce
the severity of symptoms associated therewith, or provide
improvement to a patient or subject's quality of life.
[0127] As used herein, the term "condition" includes anatomic and
physiological deviations from the normal that constitute an
impairment of the normal state of the living animal or one of its
parts, that interrupts or modifies the performance of the bodily
functions.
[0128] As used herein, the terms "subject" or "individual" or
"animal" or "patient" or "mammal," refers to any subject,
particularly a mammalian subject, for whom therapy is desired, for
example, a human.
[0129] Being a genetic disease, Cystic Fibrosis currently cannot
yet be cured, but its clinical manifestations and/or symptoms can
be treated by the oligonucleotides of the present invention, for a
marked increase and/or improvement in a patient's clinical status
and quality of life.
[0130] The term "improving" as used herein refers to a favorable
change, i.e. an increase or a decrease of at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, or at least 50% in a
clinical parameter of Cystic Fibrosis.
[0131] Different routes of AOs delivery have been examined in
animal models and applied in clinical trials, chosen primarily
according to the target tissue. For example, 2OMP was administrated
to DMD patients (PRO-051) by local intramuscular injection (van
Deutekom et al., 2007), and by abdominal subcutaneous injections
(Goemans et al., 2011). 2OMP was also administrated to a SMA mouse
model by intracerebroventricular injection (Williams et al., 2009;
Hua et al., 2010). PMO was administrated to a DMD mouse model by
intramuscular injection (Gebski, Mann, Fletcher, & Wilton,
2003), and repeated weakly intraperitoneal injections (Goyenvalle
et al., 2010). PMO was also administrated to a SMA mouse model by
intracerebroventricular injection (Porensky et al., 2012), and to
DMD patients (AVI-4658) by local intramuscular injection (Kinali et
al., 2009), or intravenously administration (Cirak et al., 2011;
Mendell et al., 2013).
[0132] In certain embodiments, the method further comprises
administering at least one additional anti-Cystic-Fibrosis agent to
the patient. In certain such embodiments, the additional
anti-Cystic-Fibrosis agent is selected from the group consisting of
a CFTR-splicing-modulating agent, a CFTR potentiator and a CFTR
corrector. Each possibility represents a separate embodiment of the
present invention. In certain embodiments, the administration of
the therapeutically effective amount of a synthetic polynucleotide
molecule of the present invention and the administration of the at
least one additional anti-Cystic-Fibrosis agent are independently
oral, nasal, aerosol, inhalational, abdominal, subcutaneous,
intra-peritoneal or intravenous administration. Each possibility
represents a separate embodiment of the present invention. It
should be understood that the selection of an administration route
depends on the nature of the therapeutic agent and the site of its
intended effect, and thus certain agents may be administrated via
the same or different administration routes.
[0133] In certain embodiments, the administration of the synthetic
oligonucleotide molecule or of the pharmaceutical composition is
oral, nasal, inhalational, abdominal, subcutaneous,
intra-peritoneal or intravenous administration.
[0134] In certain embodiments, the synthetic oligonucleotide
molecule is administered in a concentration of at least about 1 nM.
In certain embodiments, the synthetic oligonucleotide molecule is
administered in a concentration of at least about 2.5 nM. In
certain embodiments, the synthetic oligonucleotide molecule is
administered in a concentration of at least about 10 nM. In certain
embodiments, the synthetic oligonucleotide molecule is administered
in a concentration of 2.5 nM to 10 nM.
[0135] The present invention further provides, in another aspect,
the use of a synthetic oligonucleotide molecule as described above,
or of a pharmaceutical composition as described above, in preparing
a medicament.
[0136] In certain embodiments, the medicament is for treating or
ameliorating a symptom of Cystic Fibrosis. In some embodiments, the
medicament improves at least one clinical parameter of Cystic
Fibrosis. According to some embodiments, the clinical parameter is
selected from the group consisting of lung function, time to the
first pulmonary exacerbation, change in weight, change in height, a
change in Body Mass Index (BMI), change in the concentration of
sweat chloride, number and/or duration of pulmonary exacerbations,
total number of days of hospitalization for pulmonary
exacerbations, and the need for antibiotic therapy for
sinopulmonary signs or symptoms. Each possibility represents a
separate embodiment of the invention.
[0137] The present invention further provides, in another aspect, a
kit comprising a synthetic oligonucleotide molecule as described
above.
[0138] In certain embodiments, the kit further comprises an
additional anti-Cystic-Fibrosis agent.
[0139] In certain embodiments, the synthetic oligonucleotide and
the additional anti-Cystic-Fibrosis agent are comprised in one
pharmaceutical composition. In certain embodiments, the synthetic
oligonucleotide and the additional anti-Cystic-Fibrosis agent are
comprised in different pharmaceutical compositions. In certain
embodiments, the synthetic oligonucleotide and the additional
anti-Cystic-Fibrosis agent are independently formulated for oral,
nasal, inhalation, abdominal, subcutaneous, or intra-peritoneal
administration. Each possibility represents a different embodiment
of the invention.
[0140] In certain embodiments, the synthetic oligonucleotide is in
a concentration of at least about 1 nM. In certain embodiments, the
synthetic oligonucleotide is in a concentration of at least about
2.5 nM. In certain embodiments, the synthetic oligonucleotide is in
a concentration of at least about 10 nM. In certain embodiments,
the synthetic oligonucleotide molecule is in a concentration of 2.5
nM to 10 nM.
[0141] The present invention further provides, in another aspect, a
synthetic oligonucleotide molecule, consisting of 18-50 consecutive
bases that are complementary to a pre-mRNA transcript of a CFTR
gene having a 3849 +10 Kb C-to-T mutation and at least partly
suppresses the inclusion of intron 22 cryptic exon in the mature
CFTR mRNA, comprising a nucleotide sequence set forth in one of SEQ
ID NO: 1 to SEQ ID NO: 25, and SEQ ID NO: 41 to SEQ ID NO: 44.
[0142] The present invention further provides, in another aspect, a
synthetic oligonucleotide molecule, consisting of 17-50 consecutive
bases that are complementary to a pre-mRNA transcript of a CFTR
gene having a 3849 +10 Kb C-to-T mutation and at least partly
suppresses the inclusion of intron 22 cryptic exon in the mature
CFTR mRNA, comprising a nucleotide sequence set forth in one of SEQ
ID NO: 1 to SEQ ID NO: 25 and SEQ ID NO: 40 to SEQ ID NO: 71.
[0143] In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in one of
SEQ ID NO: 1 to SEQ ID NO: 10. In certain embodiments, the
oligonucleotide molecule comprises or consists of a nucleotide
sequence set forth in one of SEQ ID NO: 1 to SEQ ID NO: 5, and SEQ
ID NO: 41. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 1, in SEQ ID NO: 3, in SEQ ID NO: 4, in SEQ ID NO: 5, or SEQ ID
NO: 41. In certain embodiments, the oligonucleotide molecule
comprises or consists of a nucleotide sequence set forth in SEQ ID
NO: 7.
[0144] In certain embodiments, the oligonucleotide molecule
comprises a nucleotide sequence set forth in one of SEQ ID NO: 40
to SEQ ID NO: 71. In certain embodiments, the oligonucleotide
molecule comprises a nucleotide sequence set forth in one of SEQ ID
NO: 40 to SEQ ID NO: 48. In certain embodiments, the
oligonucleotide molecule comprises a nucleotide sequence set forth
in one of SEQ ID NO: 49 to SEQ ID NO: 53. In certain embodiments,
the oligonucleotide molecule comprises a nucleotide sequence set
forth in one of SEQ ID NO: 54 to SEQ ID NO: 58. In certain
embodiments, the oligonucleotide molecule comprises a nucleotide
sequence set forth in one of SEQ ID NO: 59 to SEQ ID NO: 63. In
certain embodiments, the oligonucleotide molecule comprises a
nucleotide sequence set forth in one of SEQ ID NO: 54 to SEQ ID NO:
71.
[0145] The present invention further provides, in another aspect, a
synthetic oligonucleotide sequence consisting of 18-20 consecutive
bases comprising the sequence GAUGGAAGA (SEQ ID NO: 38), wherein
the synthetic oligonucleotide sequence is complementary to a
pre-mRNA transcript of a CFTR gene having a 3849 +10 Kb C-to-T
mutation.
[0146] The present invention further provides, in another aspect, a
synthetic oligonucleotide sequence consisting of 17-20 consecutive
bases comprising the sequence GAUGGAAGA (SEQ ID NO: 38), wherein
the synthetic oligonucleotide sequence is complementary to a
pre-mRNA transcript of a CFTR gene having a 3849 +10 Kb C-to-T
mutation.
[0147] In certain embodiments, the oligonucleotide sequence is
complementary to a nucleotide sequence within SEQ ID NO: 37. In
certain embodiments, the oligonucleotide sequence is selected from
the group consisting of: SEQ ID Nos: 1, 2, 4 and 7. In certain
embodiments, the synthetic oligonucleotide sequence comprises the
sequence CAACAGAUGGAAGA (SEQ ID NO: 39). In certain embodiments,
the sequence is selected from the group consisting of: SEQ ID Nos:
1, 2 and 4.
[0148] In one embodiment, the present invention provides combined
preparations. In one embodiment, "a combined preparation" defines
especially a "kit of parts" in the sense that the combination
partners as defined above can be dosed independently or by use of
different fixed combinations with distinguished amounts of the
combination partners i.e., simultaneously, concurrently, separately
or sequentially. In some embodiments, the parts of the kit of parts
can then, e.g., be administered simultaneously or chronologically
staggered, that is at different time points and with equal or
different time intervals for any part of the kit of parts. The
ratio of the total amounts of the combination partners, in some
embodiments, can be administered in the combined preparation.
[0149] In some embodiments, the kit of the invention comprises: at
least one oligonucleotide; and at least one of: at least one CFTR
modifier; or at least one CF drug, wherein the oligonucleotide is
selected from SEQ ID Nos.: 1-25, and 41-44, and wherein the CFTR
modifier is selected from: CFTR potentiator, CFTR corrector, and
CFTR amplifier.
[0150] In some embodiments, the CF drug is an antibiotic drug, a
bronchodilator, a corticosteroid, or any combination thereof.
[0151] Types and doses of CF drugs, such as an antibiotic, a
bronchodilator, and a corticosteroid, would be apparent to one of
ordinary skill in the art. Non-limiting examples of CF drugs, such
as antibiotics include, but are not limited to, cloxacillin,
dicloxacillin, cephalosporin, trimethoprim, sulfamethoxazole,
erythromycin, amoxicillin, clavulanate, ampicillin, tetracycline,
linezolid, tobramycin or aztreonam lysine, fluoroquinolone,
gentamicin, and monobactam with antipseudomonal activity.
[0152] In some embodiments, the components of the kit disclosed
above are sterile. As used herein, the term "sterile" refers to a
state of being free from biological contaminants. Any method of
sterilization is applicable and would be apparent to one of
ordinary skill in the art.
[0153] In some embodiments, the components of the kit are packaged
within a container.
[0154] In some embodiments, the container is made of a material
selected from the group consisting of thin-walled film or plastic
(transparent or opaque), paperboard-based, foil, rigid plastic,
metal (e.g., aluminum), glass, etc.
[0155] In some embodiments, the content of the kit is packaged, as
described below, to allow for storage of the components until they
are needed.
[0156] In some embodiments, some or all components of the kit may
be packaged in suitable packaging to maintain sterility.
[0157] In some embodiments, the components of the kit are stored in
separate containers within the main kit containment element e.g.,
box or analogous structure, may or may not be an airtight
container, e.g., to further preserve the sterility of some or all
of the components of the kit.
[0158] In some embodiments, the instructions may be recorded on a
suitable recording medium or substrate. For example, the
instructions may be printed on a substrate, such as paper or
plastic, etc.
[0159] In some embodiments, the instructions may be present in the
kit as a package insert, in the labeling of the container of the
kit or components thereof (i.e., associated with the packaging or
sub-packaging) etc. In other embodiments, the instructions are
present as an electronic storage data file present on a suitable
computer readable storage medium, e.g. CD-ROM, diskette, etc. In
other embodiments, the actual instructions are not present in the
kit, but means for obtaining the instructions from a remote source,
e.g. via the internet, are provided. An example of this embodiment
is a kit that includes a web address where the instructions can be
viewed and/or from which the instructions can be downloaded. As
with the instructions, this means for obtaining the instructions is
recorded on a suitable substrate.
[0160] According to some embodiments, there is provided a method
for producing a compound suitable for treating CF.
[0161] In some embodiments, the method comprises obtaining a
compound that binds to intro 22 of the CFTR pre-mRNA. In some
embodiments, the method comprises obtaining a compound that binds
to SEQ ID NO: 37. In some embodiments, the method comprises
assaying the inclusion of intron 22 cryptic exon in the mature CFTR
mRNA in the presence of the obtained compound, and selecting at
least one compound that suppresses the inclusion of intron 22
cryptic exon in the mature CFTR mRNA, thereby producing a compound
suitable for treating CF.
[0162] In some embodiments, the compound is an oligonucleotide. In
some embodiments, the oligonucleotide is an oligonucleotide as
disclosed and as described herein.
[0163] Methods of assaying cryptic exon inclusion are common.
Non-limiting examples of such methods include, but are not limited
to, PCR, qPCR, gene sequencing, northern-blot, dot-blot, in situ
hybridization, or others all of which would be apparent to one of
ordinary skill in the art.
[0164] In the discussion unless otherwise stated, adjectives such
as "substantially" and "about" modifying a condition or
relationship characteristic of a feature or features of an
embodiment of the invention, are understood to mean that the
condition or characteristic is defined to within tolerances that
are acceptable for operation of the embodiment for an application
for which it is intended. Unless otherwise indicated, the word "or"
in the specification and claims is considered to be the inclusive
"or" rather than the exclusive or, and indicates at least one of,
or any combination of items it conjoins.
[0165] It should be understood that the terms "a" and "an" as used
above and elsewhere herein refer to "one or more" of the enumerated
components. It will be clear to one of ordinary skill in the art
that the use of the singular includes the plural unless
specifically stated otherwise. Therefore, the terms "a," "an" and
"at least one" are used interchangeably in this application.
[0166] For purposes of better understanding the present teachings
and in no way limiting the scope of the teachings, unless otherwise
indicated, all numbers expressing quantities, percentages or
proportions, and other numerical values used in the specification
and claims, are to be understood as being modified in all instances
by the term "about". Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the following specification
and attached claims are approximations that may vary depending upon
the desired properties sought to be obtained. At the very least,
each numerical parameter should at least be construed in light of
the number of reported significant digits and by applying ordinary
rounding techniques.
[0167] In the description and claims of the present application,
each of the verbs, "comprise", "include" and "have" and conjugates
thereof, are used to indicate that the object or objects of the
verb are not necessarily a complete listing of components, elements
or parts of the subject or subjects of the verb.
[0168] Other terms as used herein are meant to be defined by their
well-known meanings in the art.
[0169] Unless specifically stated or obvious from context, as used
herein, the term "or" is understood to be inclusive.
[0170] Throughout this specification and claims, the word
"comprise", or variations such as "comprises" or "comprising",
indicate the inclusion of any recited integer or group of integers
but not the exclusion of any other integer or group of
integers.
[0171] As used herein, the term "consists essentially of", or
variations such as "consist essentially of" or "consisting
essentially of", as used throughout the specification and claims,
indicate the inclusion of any recited integer or group of integers,
and the optional inclusion of any recited integer or group of
integers that do not materially change the basic or novel
properties of the specified method, structure or composition.
[0172] As used herein, the terms "comprises", "comprising",
"containing", "having" and the like can mean "includes",
"including", and the like; "consisting essentially of" or "consists
essentially" likewise has the meaning ascribed in U.S. patent law
and the term is open-ended, allowing for the presence of more than
that which is recited so long as basic or novel characteristics of
that which is recited is not changed by the presence of more than
that which is recited, but excludes prior art embodiments. In one
embodiment, the terms "comprises," "comprising, "having" are/is
interchangeable with "consisting".
[0173] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
[0174] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments
unless the embodiment is inoperative without those elements.
[0175] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to". This term encompasses the terms "consisting of" and
"consisting essentially of". As used herein, the singular form "a",
"an", "the" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a compound" or
"at least one compound" may include a plurality of compounds,
including mixtures thereof.
[0176] The following examples are meant to be construed as
non-limiting to the scope of the invention and are to serve merely
as illustrative embodiments.
[0177] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0178] Generally, the nomenclature used herein, and the laboratory
procedures utilized in the present invention include chemical,
molecular, biochemical, and cell biology techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); "Cell Biology: A Laboratory Handbook",
Volumes I-III Cellis, J. E., ed. (1994); The Organic Chemistry of
Biological Pathways by John McMurry and Tadhg Begley (Roberts and
Company, 2005); Organic Chemistry of Enzyme-Catalyzed Reactions by
Richard Silverman (Academic Press, 2002); Organic Chemistry (6th
Edition) by Leroy "Skip" G Wade; Organic Chemistry by T. W. Graham
Solomons and, Craig Fryhle.
TABLE-US-00001 TABLE 1 Anti-Sense Oligonucleotides (ASOs). SEQ
Length ID Nucleotide (nucle- Name NO: sequence 5'.fwdarw.3' otides)
SPL84-23 1 CUGCAACAGAUGGAAGACUC 20 SPL84-22 2 CAACAGAUGGAAGACUCUU
19 SPL84-17 3 CUCCAGAAAUCAAGAUGAC 19 SPL84-25 4 UACUGCAACAGAUGGAAGA
19 SPL84-2 5 AUCAAGAUGACAAGUCAACU 20 SPL84-16 6 GUGGUCUCCAGAAAUCAAG
19 SPL84-21 7 GAUGGAAGACUCUUGUAAU 19 SPL84-1 8
CAAGAUGACAAGUCAACUGAA 21 SPL84-7 9 GAAAUCAAGAUGACAAGUCAAC 22
SPL84-19 10 ACCUUGUGGUCUCCAGAAA 19 SPL84-18 11 CCAGAAAUCAAGAUGACAAG
20 SPL84-13 12 CACCAUUUUAAUACUGCAACA 21 SPL84-24 13
UGGAAGACUCUUGUAAUUAUU 21 SPL84-3 14 GAUGACAAGUCAACUGAAAUU 21
SPL84-15 15 CUUUCAGGGUGUCUUACUC 19 SPL84-14 16 UCAGGGUGUCUUACUCACC
19 SPL84-20 17 AUUACCUUGUGGUCUCCAGA 20 SPL84-5 18
GACAAGUCAACUGAAAUUUAG 21 SPL84-8 19 CAACUGAAAUUUAGAUCCACA 21
SPL84-6 20 AGUCAACUGAAAUUUAGAUCC 21 SPL84-9 21
GUGUCUUACUCACCAUUUUAA 21 SPL84-10 22 GUGUCUUACUCACCAUUU 18 SPL84-12
23 CUCACCAUUUUAAUACUGC 19 SPL84-4 24 GACAAGUCAACUGAAAUU 18 SPL84-11
25 CUUACUCACCAUUUUAAUAC 20 5' Kole 26 GUCUUACUCACCAUUUUA 18 3' Kole
27 CAAGUCAACUGAAAUUUAG 19 stop Kole 28 CUUGUAAUUAUUUUUACAU 19
ASO84-1 29 AAAUCAAGAUGACAAGUCAACUGAA 25 ASO84-2 30
CUUGUGGUCUCCAGAAAUCAAGAUG 25 ASO84-3 31 AACAGAUGGAAGACUCUUGUAAUUA
25 ASO84-3 32 UCAGGGUGUCUUACUCACCAUUUUA 25 Control 33
GACCACUUGCCACCCAUC 18 SPL84-23-1 40 CUGCAACAGAUGGAAGACU 19
SPL84-23-2 41 UGCAACAGAUGGAAGACUC 19 SPL84-23-3 42
CUGCAACAGAUGGAAGAC 18 SPL84-23-4 43 GCAACAGAUGGAAGACUC 18
SPL84-23-5 44 UGCAACAGAUGGAAGACU 18 SPL84-23-6 45 CUGCAACAGAUGGAAGA
17 SPL84-23-7 46 CAACAGAUGGAAGACUC 17 SPL84-23-8 47
UGCAACAGAUGGAAGAC 17 SPL84-23-9 48 GCAACAGAUGGAAGACU 17 49
CUCCAGAAAUCAAGAUGA 18 50 UCCAGAAAUCAAGAUGAC 18 51 CUCCAGAAAUCAAGAUG
17 52 CCAGAAAUCAAGAUGAC 17 53 UCCAGAAAUCAAGAUGA 17 54
CAACAGAUGGAAGACUCU 18 55 AACAGAUGGAAGACUCUU 18 56 ACAGAUGGAAGACUCUU
17 57 AACAGAUGGAAGACUCU 17 58 UACUGCAACAGAUGGAAG 18 59
ACUGCAACAGAUGGAAGA 18 60 UACUGCAACAGAUGGAA 17 61 ACUGCAACAGAUGGAAG
17 62 AUCAAGAUGACAAGUCAAC 19 63 UCAAGAUGACAAGUCAACU 19 64
AUCAAGAUGACAAGUCAA 18 65 CAAGAUGACAAGUCAACU 18 66
UCAAGAUGACAAGUCAAC 18 67 AUCAAGAUGACAAGUCA 17 68 AAGAUGACAAGUCAACU
17 69 UCAAGAUGACAAGUCAA 17 70 CAAGAUGACAAGUCAAC 17 SPL84-26 71
AAUUAUUUUUCAUUACCUUG 20
TABLE-US-00002 TABLE 2 CFTR-related sequences (all in sense
orientation). SEQ ID Name NO: Chr. 7 position Mature CFTR mRNA 34
aauuggaagc aaaugacauc acagcagguc agagaaaaag gguugagcgg
caggcacccagaguaguagg ucuuuggcau uaggagcuug agcccagacg gcccuagcag
ggaccccagc gcccgagaga ccaugcagag gucgccucug gaaaaggcca gcguugucuc
caaacuuuuu uucagcugga ccagaccaau uuugaggaaa ggauacagac agcgccugga
auugucagac auauaccaaa ucccuucugu ugauucugcu gacaaucuau cugaaaaauu
gaaagagaa ugggauagag agcuggcuuc aaagaaaaau ccuaaacuca uuaaugcccu
ucggcgaugu uuuuucugga gauuuauguu cuauggaauc uuuuuauauu uaggggaagu
caccaaagca guacagccuc ucuuacuggg aagaaucaua gcuuccuaug acccggauaa
caaggaggaa cgcucuaucg cgauuuaucu aggcauaggc uuaugccuuc ucuuuauugu
gaggacacug cuccuacacc cagccauuuu uggccuucau cacauuggaa ugcagaugag
aauagcuaug uuuaguuuga uuuauaagaa gacuuuaaag cugucaagcc guguucuaga
uaaaauaagu auuggacaac uuguuagucu ccuuuccaac aaccugaaca aauuugauga
aggacuugca uuggcacauu ucguguggau cgcuccuuug caaguggcac uccucauggg
gcuaaucugg gaguuguuac aggcgucugc cuucugugga cuugguuucc ugauaguccu
ugcccuuuuu caggcugggc uagggagaau gaugaugaag uacagagauc agagagcugg
gaagaucagu gaaagacuug ugauuaccuc agaaaugauu gaaaauaucc aaucuguuaa
ggcauacugc ugggaagaag caauggaaaa aaugauugaa aacuuaagac aaacagaacu
gaaacugacu cggaaggcag ccuaugugag auacuucaau agcucagccu ucuucuucuc
aggguucuuu gugguguuuu uaucugugcu ucccuaugca cuaaucaaag gaaucauccu
ccggaaaaua uucaccacca ucucauucug cauuguucug cgcauggcgg ucacucggca
auuucccugg gcuguacaaa caugguauga cucucuugga gcaauaaaca aaauacagga
uuucuuacaa aagcaagaau auaagacauu ggaauauaac uuaacgacua cagaaguagu
gauggagaau guaacagccu ucugggagga gggauuuggg gaauuauuug agaaagcaaa
acaaaacaau aacaauagaa aaacuucuaa uggugaugac agccucuucu ucaguaauuu
cucacuucuu gguacuccug uccugaaaga uauuaauuuc aagauagaaa gaggacaguu
guuggcgguu gcuggaucca cuggagcagg caagacuuca cuucuaaugg ugauuauggg
agaacuggag ccuucagagg guaaaauuaa gcacagugga agaauuucau ucuguucuca
guuuuccugg auuaugccug gcaccauuaa agaaaauauc aucuuuggug uuuccuauga
ugaauauaga uacagaagcg ucaucaaagc augccaacua gaagaggaca ucuccaaguu
ugcagagaaa gacaauauag uucuuggaga agguggaauc acacugagug gaggucaacg
agcaagaauu ucuuuagcaa gagcaguaua caaagaugcu gauuuguauu uauuagacuc
uccuuuugga uaccuagaug uuuuaacaga aaaagaaaua uuugaaagcu gugucuguaa
acugauggcu aacaaaacua ggauuuuggu cacuucuaaa auggaacauu uaaagaaagc
ugacaaaaua uuaauuuugc augaagguag cagcuauuuu uaugggacau uuucagaacu
ccaaaaucua cagccagacu uuagcucaaa acucauggga ugugauucuu ucgaccaauu
uagugcagaa agaagaaauu caauccuaac ugagaccuua caccguuucu cauuagaagg
agaugcuccu gucuccugga cagaaacaaa aaaacaaucu uuuaaacaga cuggagaguu
uggggaaaaa aggaagaauu cuauucucaa uccaaucaac ucuauacgaa aauuuuccau
ugugcaaaag acucccuuac aaaugaaugg caucgaagag gauucugaug agccuuuaga
gagaaggcug uccuuaguac cagauucuga gcagggagag gcgauacugc cucgcaucag
cgugaucagc acuggcccca cgcuucaggc acgaaggagg cagucugucc ugaaccugau
gacacacuca guuaaccaag gucagaacau ucaccgaaag acaacagcau ccacacgaaa
agugucacug gccccucagg caaacuugac ugaacuggau auauauucaa gaagguuauc
ucaagaaacu ggcuuggaaa uaagugaaga aauuaacgaa gaagacuuaa aggagugcuu
uuuugaugau auggagagca uaccagcagu gacuacaugg aacacauacc uucgauauau
uacuguccac aagagcuuaa uuuuugugcu aauuuggugc uuaguaauuu uucuggcaga
gguggcugcu ucuuugguug ugcuguggcu ccuuggaaac acuccucuuc aagacaaagg
gaauaguacu cauaguagaa auaacagcua ugcagugauu aucaccagca ccaguucgua
uuauguguuu uacauuuacg ugggaguagc cgacacuuug cuugcuaugg gauucuucag
aggucuacca cuggugcaua cucuaaucac agugucgaaa auuuuacacc acaaaauguu
acauucuguu cuucaagcac cuaugucaac ccucaacacg uugaaagcag gugggauucu
uaauagauuc uccaaagaua uagcaauuuu ggaugaccuu cugccucuua ccauauuuga
cuucauccag uuguuauuaa uugugauugg agcuauagca guugucgcag uuuuacaacc
cuacaucuuu guugcaacag ugccagugau aguggcuuuu auuauguuga gagcauauuu
ccuccaaacc ucacagcaac ucaaacaacu ggaaucugaa ggcaggaguc caauuuucac
ucaucuuguu acaagcuuaa aaggacuaug gacacuucgu gccuucggac ggcagccuua
cuuugaaacu cuguuccaca aagcucugaa uuuacauacu gccaacuggu ucuuguaccu
gucaacacug cgcugguucc aaaugagaau agaaaugauu uuugucaucu ucuucauugc
uguuaccuuc auuuccauuu uaacaacagg agaaggagaa ggaagaguug guauuauccu
gacuuuagcc augaauauca ugaguacauu gcagugggcu guaaacucca gcauagaugu
ggauagcuug augcgaucug ugagccgagu cuuuaaguuc auugacaugc caacagaagg
uaaaccuacc aagucaacca aaccauacaa gaauggccaa cucucgaaag uuaugauuau
ugagaauuca cacgugaaga aagaugacau cuggcccuca gggggccaaa ugacugucaa
agaucucaca gcaaaauaca cagaaggugg aaaugccaua uuagagaaca uuuccuucuc
aauaaguccu ggccagaggg ugggccucuu gggaagaacu ggaucaggga agaguacuuu
guuaucagcu uuuuugagac uacugaacac ugaaggagaa auccagaucg augguguguc
uugggauuca auaacuuugc aacaguggag gaaagccuuu ggagugauac cacagaaagu
auuuauuuuu ucuggaacau uuagaaaaaa cuuggauccc uaugaacagu ggagugauca
agaaauaugg aaaguugcag augagguugg gcucagaucu gugauagaac aguuuccugg
gaagcuugac uuuguccuug uggauggggg cuguguccua agccauggcc acaagcaguu
gaugugcuug gcuagaucug uucucaguaa ggcgaagauc uugcugcuug augaacccag
ugcucauuug gauccaguaa cauaccaaau aauuagaaga acucuaaaac aagcauuugc
ugauugcaca guaauucucu gugaacacag gauagaagca augcuggaau gccaacaauu
uuuggucaua gaagagaaca aagugcggca guacgauucc auccagaaac ugcugaacga
gaggagccuc uuccggcaag ccaucagccc cuccgacagg gugaagcucu uuccccaccg
gaacucaagc aagugcaagu cuaagcccca gauugcugcu cugaaagagg agacagaaga
agaggugcaa gauacaaggc uuuagagagc agcauaaaug uugacauggg acauuugcuc
auggaauugg agcucguggg acagucaccu cauggaauug gagcucgugg aacaguuacc
ucugccucag aaaacaagga ugaauuaagu uuuuuuuuaa aaaagaaaca uuugguaagg
gaauugagg acacugauau gggucuugau aaauggcuuc cuggcaauag ucaaauugug
ugaaagguac uucaaauccu ugaagauuua ccacuugugu uuugcaagcc agauuuuccu
gaaaacccuu gccaugugcu aguaauugga aaggcagcuc uaaaugucaa ucagccuagu
ugaucagcuu auugucuagu gaaacucguu aauuuguagu guuggagaag aacugaaauc
auacuucuua ggguuaugau uaaguaauga uaacuggaaa cuucagcggu uuauauaagc
uuguauuccu uuuucucucc ucuccccaug auguuuagaa acacaacuau auuguuugcu
aagcauucca acuaucucau uuccaagcaa guauuagaau accacaggaa ccacaagacu
gcacaucaaa auaugcccca uucaacaucu agugagcagu caggaaagag aacuuccaga
uccuggaaau caggguuagu auuguccagg ucuaccaaaa aucucaauau uucagauaau
cacaauacau cccuuaccug ggaaagggcu guuauaaucu uucacagggg acaggauggu
ucccuugaug aagaaguuga uaugccuuuu cccaacucca gaaagugaca agcucacaga
ccuuugaacu agaguuuagc uggaaaagua uguuagugca aauugucaca ggacagcccu
ucuuuccaca gaagcuccag guagagggug uguaaguaga uaggccaugg gcacuguggg
uagacacaca ugaaguccaa gcauuuagau guauagguug auggugguau guuuucaggc
uagauguaug uacuucaugc ugucuacacu aagagagaau gagagacaca cugaagaagc
accaaucaug aauuaguuuu auaugcuucu guuuuauaau uuugugaagc aaaauuuuuu
cucuaggaaa uauuuauuuu aauaauguuu caaacauaua uaacaaugcu guauuuuaaa
agaaugauua ugaauuacau uuguauaaaa uaauuuuuau auuugaaaua uugacuuuuu
auggcacuag uauuucuaug aaauauuaug uuaaaacugg gacaggggag aaccuagggu
gauauuaacc aggggccaug aaucaccuuu uggucuggag ggaagccuug gggcugaugc
aguuguugcc cacagcugua ugauucccag ccagcacagc cucuuagaug caguucugaa
gaagauggua ccaccagucu gacuguuucc aucaagggua cacugccuuc ucaacuccaa
acugacucuu aagaagacug cauuauauuu auuacuguaa gaaaauauca cuugucaaua
aaauccauac auuuguguga aa Intron 22 cryptic exon * 35
TTGACTTGTCATCTTGATTTCTGGAGACCACAAGGTAATGA
AAAATAATTACAAGAGTCTTCCATCTGTTGCAGTATTAAAA TG Exons 1-27 + cryptic
36 aauuggaagc aaaugacauc acagcagguc agagaaaaag gguugagcgg exon 22
caggcaccca gaguaguagg ucuuuggcau uaggagcuug agcccagacg gcccuagcag
ggaccccagc gcccgagaga ccaugcagag gucgccucug gaaaaggcca gcguugucuc
caaacuuuuu uucagcugga ccagaccaau uuugaggaaa ggauacagac agcgccugga
auugucagac auauaccaaa ucccuucugu ugauucugcu gacaaucuau cugaaaaauu
ggaaagagaa ugggauagag agcuggcuuc aaagaaaaau ccuaaacuca uuaaugcccu
ucggcgaugu uuuuucugga gauuuauguu cuauggaauc uuuuuauauu uaggggaagu
caccaaagca guacagccuc ucuuacuggg aagaaucaua gcuuccuaug acccggauaa
caaggaggaa cgcucuaucg cgauuuaucu aggcauaggc uuaugccuuc ucuuuauugu
gaggacacug cuccuacacc cagccauuuu uggccuucau cacauuggaa ugcagaugag
aauagcuaug uuuaguuuga uuuauaagaa gacuuuaaag cugucaagcc guguucuaga
uaaaauaagu auuggacaac uuguuagucu ccuuuccaac aaccugaaca aauuugauga
aggacuugca uuggcacauu ucguguggau cgcuccuuug caaguggcac uccucauggg
gcuaaucugg gaguuguuac aggcgucugc cuucugugga cuugguuucc ugauaguccu
ugcccuuuuu caggcugggc uagggagaau gaugaugaag uacagagauc agagagcugg
gaagaucagu gaaagacuug ugauuaccuc agaaaugauu gaaaauaucc aaucuguuaa
ggcauacugc ugggaagaag caauggaaaa aaugauugaa aacuuaagac aaacagaacu
gaaacugacu cggaaggcag ccuaugugag auacuucaau agcucagccu ucuucuucuc
aggguucuuu gugguguuuu uaucugugcu ucccuaugca cuaaucaaag gaaucauccu
ccggaaaaua uucaccacca ucucauucug cauuguucug cgcauggcgg ucacucggca
auuucccugg gcuguacaaa caugguauga cucucuugga gcaauaaaca aaauacagga
uuucuuacaa aagcaagaau auaagacauu ggaauauaac uuaacgacua cagaaguagu
gauggagaau guaacagccu ucugggagga gggauuuggg gaauuauuug agaaagcaaa
acaaaacaau aacaauagaa aaacuucuaa uggugaugac agccucuucu ucaguaauuu
cucacuucuu gguacuccug uccugaaaga uauuaauuuc aagauagaaa gaggacaguu
guuggcgguu gcuggaucca cuggagcagg caagacuuca cuucuaaugg ugauuauggg
agaacuggag ccuucagagg guaaaauuaa gcacagugga agaauuucau ucuguucuca
guuuuccugg auuaugccug gcaccauuaa agaaaauauc aucuuuggug uuuccuauga
ugaauauaga uacagaagcg ucaucaaagc augccaacua gaagaggaca ucuccaaguu
ugcagagaaa gacaauauag uucuuggaga agguggaauc acacugagug gaggucaacg
agcaagaauu ucuuuagcaa gagcaguaua caaagaugcu gauuuguauu uauuagacuc
uccuuuugga uaccuagaug uuuuaacaga aaaagaaaua uuugaaagcu gugucuguaa
acugauggcu aacaaaacua ggauuuuggu cacuucuaaa auggaacauu uaaagaaagc
ugacaaaaua uuaauuuugc augaagguag cagcuauuuu uaugggacau uuucagaacu
ccaaaaucua cagccagacu uuagcucaaa acucauggga ugugauucuu ucgaccaauu
uagugcagaa agaagaaauu caauccuaac ugagaccuua caccguuucu cauuagaagg
agaugcuccu gucuccugga cagaaacaaa aaaacaaucu uuuaaacaga cuggagaguu
uggggaaaaa aggaagaauu cuauucucaa uccaaucaac ucuauacgaa aauuuuccau
ugugcaaaag acucccuuac aaaugaaugg caucgaagag gauucugaug agccuuuaga
gagaaggcug uccuuaguac cagauucuga gcagggagag gcgauacugc cucgcaucag
cgugaucagc acuggcccca cgcuucaggc acgaaggagg cagucugucc ugaaccugau
gacacacuca guuaaccaag gucagaacau ucaccgaaag acaacagcau ccacacgaaa
agugucacug gccccucagg caaacuugac ugaacuggau auauauucaa gaagguuauc
ucaagaaacu ggcuuggaaa uaagugaaga aauuaacgaa gaagacuuaa aggagugcuu
uuuugaugau auggagagca uaccagcagu gacuacaugg aacacauacc uucgauauau
uacuguccac aagagcuuaa uuuuugugcu aauuuggugc uuaguaauuu uucuggcaga
gguggcugcu ucuuugguug ugcuguggcu ccuuggaaac acuccucuuc aagacaaagg
gaauaguacu cauaguagaa auaacagcua ugcagugauu aucaccagca ccaguucgua
uuauguguuu uacauuuacg ugggaguagc cgacacuuug cuugcuaugg gauucuucag
aggucuacca cuggugcaua cucuaaucac agugucgaaa auuuuacacc acaaaauguu
acauucuguu cuucaagcac cuaugucaac ccucaacacg uugaaagcag gugggauucu
uaauagauuc uccaaagaua uagcaauuuu ggaugaccuu cugccucuua ccauauuuga
cuucauccag uuguuauuaa uugugauugg agcuauagca guugucgcag uuuuacaacc
cuacaucuuu guugcaacag ugccagugau aguggcuuuu auuauguuga gagcauauuu
ccuccaaacc ucacagcaac ucaaacaacu ggaaucugaa ggcaggaguc caauuuucac
ucaucuuguu acaagcuuaa aaggacuaug gacacuucgu gccuucggac ggcagccuua
cuuugaaacu cuguuccaca aagcucugaa uuuacauacu gccaacuggu ucuuguaccu
gucaacacug cgcugguucc aaaugagaau agaaaugauu uuugucaucu ucuucauugc
uguuaccuuc auuuccauuu uaacaacagg agaaggagaa ggaagaguug guauuauccu
gacuuuagcc augaauauca ugaguacauu gcagugggcu guaaacucca gcauagaugu
ggauagcuug augcgaucug ugagccgagu cuuuaaguuc auugacaugc caacagaagg
uaaaccuacc aagucaacca aaccauacaa gaauggccaa cucucgaaag uuaugauuau
ugagaauuca cacgugaaga aagaugacau cuggcccuca gggggccaaa ugacugucaa
agaucucaca gcaaaauaca cagaaggugg aaaugccaua uuagagaaca uuuccuucuc
aauaaguccu ggccagaggu ugacuuguca ucuugauuuc uggagaccac aagguaauga
aaaauaauua caagagucuu ccaucuguug caguauuaaa auggugggcc ucuugggaag
aacuggauca gggaagagua cuuuguuauc agcuuuuuug agacuacuga acacugaagg
agaaauccag aucgauggug ugucuuggga uucaauaacu uugcaacagu ggaggaaagc
cuuuggagug auaccacaga aaguauuuau uuuuucugga acauuuagaa aaaacuugga
ucccuaugaa caguggagug aucaagaaau auggaaaguu gcagaugagg uugggcucag
aucugugaua gaacaguuuc cugggaagcu ugacuuuguc cuuguggaug ggggcugugu
ccuaagccau ggccacaagc aguugaugug cuuggcuaga ucuguucuca guaaggcgaa
gaucuugcug cuugaugaac ccagugcuca uuuggaucca guaacauacc aaauaauuag
aagaacucua aaacaagcau uugcugauug cacaguaauu cucugugaac acaggauaga
agcaaugcug gaaugccaac aauuuuuggu cauagaagag aacaaagugc ggcaguacga
uuccauccag aaacugcuga acgagaggag ccucuuccgg caagccauca gccccuccga
cagggugaag cucuuucccc accggaacuc aagcaagugc aagucuaagc cccagauugc
ugcucugaaa gaggagacag aagaagaggu gcaagauaca aggcuuuaga gagcagcaua
aauguugaca ugggacauuu gcucauggaa uuggagcucg ugggacaguc accucaugga
auuggagcuc guggaacagu uaccucugcc ucagaaaaca aggaugaauu aaguuuuuuu
uuaaaaaaga aacauuuggu aaggggaauu gaggacacug auaugggucu ugauaaaugg
cuuccuggca auagucaaau ugugugaaag guacuucaaa uccuugaaga uuuaccacuu
guguuuugca agccagauuu uccugaaaac ccuugccaug ugcuaguaau uggaaaggca
gcucuaaaug ucaaucagcc uaguugauca gcuuauuguc uagugaaacu cguuaauuug
uaguguugga gaagaacuga aaucauacuu cuuaggguua ugauuaagua augauaacug
gaaacuucag cgguuuauau aagcuuguau uccuuuuucu cuccucuccc caugauguuu
agaaacacaa cuauauuguu ugcuaagcau uccaacuauc ucauuuccaa gcaaguauua
gaauaccaca ggaaccacaa gacugcacau caaaauaugc cccauucaac aucuagugag
cagucaggaa agagaacuuc cagauccugg aaaucagggu uaguauuguc caggucuacc
aaaaaucuca auauuucaga uaaucacaau acaucccuua ccugggaaag ggcuguuaua
aucuuucaca ggggacagga ugguucccuu gaugaagaag uugauaugcc uuuucccaac
uccagaaagu gacaagcuca cagaccuuug aacuagaguu uagcuggaaa aguauguuag
ugcaaauugu cacaggacag cccuucuuuc cacagaagcu ccagguagag gguguguaag
uagauaggcc augggcacug uggguagaca cacaugaagu ccaagcauuu agauguauag
guugauggug guauguuuuc aggcuagaug uauguacuuc augcugucua cacuaagaga
gaaugagaga
cacacugaag aagcaccaau caugaauuag uuuuauaugc uucuguuuua uaauuuugug
aagcaaaauu uuuucucuag gaaauauuua uuuuaauaau guuucaaaca uauauaacaa
ugcuguauuu uaaaagaaug auuaugaauu acauuuguau aaaauaauuu uuauauuuga
aauauugacu uuuuauggca cuaguauuuc uaugaaauau uauguuaaaa cugggacagg
ggagaaccua gggugauauu aaccaggggc caugaaucac cuuuuggucu ggagggaagc
cuuggggcug augcaguugu ugcccacagc uguaugauuc ccagccagca cagccucuua
gaugcaguuc ugaagaagau gguaccacca gucugacugu uuccaucaag gguacacugc
cuucucaacu ccaaacugac ucuuaagaag acugcauuau auuuauuacu guaagaaaau
aucacuuguc aauaaaaucc auacauuugu gugaaa Target sequence for 37
AAGCAGCATATTCTCAATACTATGTTTCATTAATAATTAAT ASOs * (Bold-cryptic
AGAGATATATGAACACATAAAAGATTCAATTATAATCACC exon)
TTGTGGATCTAAATTTCAGTTGACTTGTCATCTTGATTTC
TGGAGACCACAAGGTAATGAAAAATAATTACAAGAGTC
TTCCATCTGTTGCAGTATTAAAATGGTGAGTAAGACACC
CTGAAAGGAAATGTTCTATTCATGGTACAATGCAATTACAG
CTAGCACCAAATTCAACACTGTTTAACTTTCAACATATTAT TTTG Sequence motif no. 1
38 GAUGGAAGA Sequence motif no. 2 39 CAACAGAUGGAAGA
Corresponding to positions 17279930-117280013 in the genome
according to the Assemble version used as updated in 2013 (UCSC
Genome Browser on Human December 2013 (GRCh38/hg38)).
Materials and Methods
Fisher Rat Thyroid (FRT) Cellular System
[0179] In order to screen for antisense oligonucleotides (ASOs)
that modulate the splicing of the 3849 +10 kb C-to-T mutated CFTR a
cellular screening system is required. As epithelial cells derived
from patients (nasal or bronchial epithelial cells) are difficult
to grow and have limited proliferation capacity the inventors have
generated, fisher rat thyroid (FRT) cells stably expressing CFTR
cDNA carrying the 3849 +10 kb C-to-T mutation (FRT 3849 mut). The
cDNA is transfected into FRT cells using the Flp-In system
(Invitrogen) that allows integration at a specific genomic
location. It is important to note that FRT Flp-In cells are widely
used for analysis of CFTR and can be used for RNA as well as
functional analyses. In order to study the effect of ASOs on
splicing, the cDNA is cloned to include a mini gene containing
intronic sequences around the exon of interest. As a positive
control FRT cells containing the cDNA with the mini gene carrying
the wild type (WT) sequence (C in the mutation site) were also
generated (FRT 3849 WT).
Transfection
[0180] Each ASO was transfected into FRT 3849 mutant cells using
Lipofectamine 2000 transfection reagent (Invitrogen) according to
the lipofectamine 2000 reagent protocol using the following
lipofectamine amounts: 96 well-0.15 .mu.l, 6 well-3 .mu.l, 10 mm
plate-15 .mu.l. In each experiment the effect of different ASOs was
analyzed in comparison to cells treated with control ASOs.
RNA Extraction
[0181] Twenty-four (24) hours following transfection of FRT 3849
mutant cells, total RNA was extracted using RNeasy Mini Kit
(QIAGEN). RNA concentration was determined using nanodrop. RNA-less
and reverse transcriptase-less reactions were used as controls.
Complementary DNA (cDNA) synthesis was performed using the High
Capacity cDNA Reverse Transcription kit (Applied Biosystems). The
cDNA was analyzed by PCR and qPCR.
Quantitative Detection of Aberrantly Spliced CFTR Transcripts
(qPCR)
[0182] Real-time PCR was performed in QuantStudio 12K Flex
Real-Time PCR System using TaqMan.RTM. Fast Advanced Master Mix
(Applied Biosystems) with TaqMan probes specific for the 84 bp
cryptic exon. The expression level was normalized to the transcript
levels of HPRT. Technical duplicates were analyzed for each sample.
Analysis was perform using the double delta Ct analysis.
Determine the Ration Between these Two Transcripts (PCR)
[0183] PCR was performed using the Platinum.TM. SuperFi.TM. Green
PCR Master Mix 12359-10 (Invitrogen) on cDNA diluted 1:2.5, 2 .mu.l
of diluted cDNA for each reaction. PCR products were then run on an
agarose gel for detection of the correctly and aberrantly spliced
transcripts. The gels were exposed to UV light to visualize the PCR
products and images were produced. The quantification of the
fraction of correctly spliced transcripts was quantified using
image J.
Half Maximal Effective Concentration (EC50) Analysis
[0184] FRT cells were transfected with each lead ASO in six
concentrations (0.1, 1, 2.5, 10, 50 and 100 nM). For concentrations
lower than 100 nM, control ASO was added for completion to total
concentration of 100 nM. In each experiment the effect of 3
different ASOs was analyzed in comparison to cells treated with 100
nM control ASOs.
CFTR Protein Analysis by Western Blot
[0185] For protein analysis FRT 3849 mutant cells were transfected
with 10 nM lead ASOs or control ASO every 24 hr for 3 days.
Twenty-four (24) hr following the last transfection protein was
extracted using RIPA buffer and analyzed by immunoblotting with a
CFTR antibody. 6% polyacrylamide gels were used for protein
separation. The gel was transferred to a nitrocellulose membrane,
and antibody hybridization and chemiluminescence were performed
according to the standard procedures. The primary antibodies used
in this analysis were mouse anti CFTR M3A7 (Millipore) and rabbit
anti Calnexin (Sigma). HRP-conjugated anti-rabbit and anti-mouse
secondary antibodies were obtained from Jackson ImmunoResearch
Laboratories.
Studies of CFTR Function Using a Membrane Potential Assay
[0186] FRT 3849 mutant cells are grown in 96-well (black, flat
bottom; corning) plates. 24 h following transfection of the
different ASOs, CFTR channel function was analyzed using the FLIPR
membrane potential assay as previously described. Briefly, the
cells were loaded with blue membrane potential dye (Molecular
Devices), which can detect changes in transmembrane potential. The
plate was then read in a fluorescence plate reader (BioTek Synergy
H1) for baseline levels followed by CFTR stimulation using the cAMP
agonist forskolin (10 .mu.M; Sigma), DMSO vehicle was used as a
negative control. CFTR-mediated depolarization of the plasma
membrane was detected as an increase in fluorescence and
hyperpolarization (or repolarization) as a decrease. To terminate
the functional assay, the CFTR inhibitor CFTRinh-172 (10 .mu.M;
Cystic Fibrosis Foundation Therapeutics) was added to each well.
Changes in transmembrane potential were normalized to the values
prior to activation.
Primary Nasal Epithelial Cell Sampling and Culture
[0187] Human nasal endothelial cells (HNE) cells were sampled by
nasal brushing of both nostrils. To increase the number of filters
available to test the corrector efficacy, the inventors used
conditionally reprogrammed and re-differentiated HNE cells. HNE
cells were then seeded on porous filters (0.33 cm.sup.2, Transwell,
Corning) and supplemented with culture medium. After 2 days, cells
were cultured in an air-liquid interface for 2-4 weeks. ASOs
dissolved in DDW, were added to the basal side of HNEs during the
differentiation.
Ussing Chamber Studies
[0188] Following differentiation HNE cells were analyzed for CFTR
channel using the Ussing chamber system as previously described
(Pranke et al., 2017). In general, the short-circuit-current (Isc)
was measured under voltage clamp conditions with an EVC4000
Precision V/I Clamp (World Precision Instruments). For all
measurements, chloride concentration gradient across the epithelium
was applied by differential composition of basal and apical Ringer
solutions. Inhibitors and activators were added after stabilization
of baseline Isc:sodium (Na+)-channel blocker Amiloride (100 .mu.M)
to inhibit apical epithelial Na+ channel (ENaC); cAMP agonists
Forskolin (10 .mu.M) to activate the transepithelial cAMP-dependent
current (including Cl- transport through CFTR channels); VX-770 (10
.mu.M) to potentiate CFTR activity; CFTR inhibitor CFTRinh172 (5
.mu.M) to specifically inhibit CFTR; and ATP (100 .mu.M) to
challenge the purinergic calcium-dependent Cl- secretion. The
change after Forskolin served as an index of CFTR.
Example 1
Novel Antisense Oligonucleotides (ASOs)
[0189] Twenty-six (26) new antisense oligonucleotides (ASOs) were
analyzed for their effect on the splicing of CFTR pre-mRNA
including the mutation 3849 +10 Kb C-to-T. The ASOs were modified
with 2'-O-Methyl phosphorothioate (2'OMP). Briefly, Fischer rat
thyroid (FRT) cells were transfected with the ASOs, incubated for
24 hours, and then RNA was extracted and the levels of aberrantly
spliced CFTR transcripts were measured by qRT-PCR (FIG. 3A). In
addition, the percentage of correctly spliced CFTR transcripts
after incubation with each ASO was also determined (FIG. 3B). The
values shown are the average fold change (mean.+-.s.e.m.) relative
to cells treated with a control ASO from 2-8 independent
experiments. Values are normalized against transcripts of the HPRT
gene.
[0190] As shown in FIG. 3, even at concentrations as low as 10 nM,
all of the tested ASOs, except for one (SPL84-26) were surprisingly
found to significantly decrease the levels of aberrantly spliced
CFTR mRNA transcripts (to at least 0.6 of control ASO), and
increase the prevalence of correctly spliced CFTR mRNA transcripts
to at least 10 percent, compared to the negligible effect of the
control ASO.
Example 2
Comparison of Novel ASOs and Previously Disclosed ASOs
[0191] The new ASOs provided herein were compared to several of the
ASOs previously described (Friedman et al., J. Biol. Chem., 1999).
The same methods were used as in Example 1 above, with the
exception that in these experiments the FRT cells were transfected
with lower concentrations of the ASOs (2.5 nM vs. 10 nM).
[0192] From the data presented in FIGS. 4A and 4B, it becomes
evident that even at concentrations as low as 2.5 nM, all of the
ASOs provided herein were surprisingly found to significantly
decrease the levels of aberrantly spliced CFTR mRNA transcripts (to
at least 80 percent of control ASO level), and increase the
prevalence of correctly spliced CFTR mRNA transcripts to at least
15 percent, compared to the negligible effect of the control ASO.
It is further important to note that these results are far superior
to those achieved by previously described ASOs in a direct
head-to-head comparison.
[0193] These results become even more surprising and promising when
considering that the genetically-manipulated FRT cells used in the
experiments contained a single copy of a CFTR gene having the 3849
+10 Kb C-to-T mutation, which is a condition more extreme than the
most acute phenotype of CF in humans, where two CFTR gene alleles
display little to none CFTR activity.
Example 3
Comparison of Novel ASOs and Previously Disclosed ASOs
[0194] The new ASOs provided herein were compared to several of the
ASOs previously described in WO2014/045283. The same methods were
used as in Example 1 above.
[0195] From the data presented in FIGS. 5A and 5B, it becomes
evident that all of the ASOs provided herein were comparable in
efficacy to the ASOs described in WO2014/045283, i.e. in decreasing
the levels of aberrantly spliced CFTR mRNA transcripts, and/or
increasing the prevalence of correctly spliced CFTR mRNA
transcripts, compared to the negligible effect of the control
ASO.
[0196] It is surprising to note that the new ASOs provided herein,
despite being shorter than the ASOs previously described in
WO2014/045283 (18-21 compared to 25 consecutive bases (16%-28%),
present comparable efficacies in any one of the experiments. It is
especially surprising and unpredictable since the ASOs previously
described in Friedman et al., 1999, having 18-19 consecutive bases,
displayed inferior efficacy in all the experiments.
[0197] From the data presented in Examples 1, 2 and 3 above, it is
readily apparent that the ASOs provided by the present invention
are highly capable of manipulating the splicing of CFTR pre-mRNA
transcripts containing the 3849 +10 Kb C-to-T mutation such that
the ratio between correctly spliced CFTR mRNA transcripts and
aberrantly spliced CFTR mRNA transcripts becomes much more
favorable.
[0198] These results become even more surprising and promising when
considering that the genetically-manipulated FRT cells used in the
experiments contained a single copy of a CFTR gene having the 3849
+10 Kb C-to-T mutation, which is a condition more extreme than the
most acute phenotype of CF in humans, where two CFTR gene alleles
display little to none CFTR activity.
Example 4
EC50 Analysis of Novel ASOs
[0199] The new ASOs SPL84-2, SPL84-17, SPL8422, SPL84-23 and
SPL84-25 provided herein were tested in several concentrations to
generate an EC50 curve indicating on the affinity and efficacy of
each ASO. As illustrated in FIG. 6 and Table 3 (herein below), the
ASOs were found to have a high efficiency and potency.
TABLE-US-00003 TABLE 3 SPL84- SPL84- SPL84- SPL84- SPL84- 1 17 22
23 25 Maximal effect (Fold 0.316 0.2784 0.2157 0.1868 0.1339
decrease from control ASO) EC50 (nM) 1.16 0.6246 0.3073 0.5133
0.9246
Example 5
Western Blot Analysis for CFTR Mature Protein Levels
[0200] The CFTR protein in a western blot can have two forms:
immature--un-glycosylated protein (band B), and mature--full length
glycosylated (band C). In homozygote WT cells most of the CFTR
protein is found in band C--full length and mature. In FRT cells
having the 3849 +10 Kb C-to-T mutation ("FRT mut") and treated with
a control ASO all of the CFTR protein was visualized as truncated
CFTR protein as in band B.
[0201] The transfection protocol was as follows: 10 nM of each ASO
was transfected into FRT mut cells on three consecutive days. In
each experiment the effect of several ASOs was analyzed in
comparison to cells treated with 10 nM control ASOs. Each ASO was
tested in 2 biological experiments. CFTR was stained by an antibody
detecting only the full length CFTR protein (.alpha.CFTR--M3A7;
FIG. 7). In the cells treated with control, no band C was detected,
while following ASO transfection band C appeared, indicative of the
formation of a full-length mature protein.
[0202] As illustrated in FIGS. 7A and 7B, the level of mature
full-length protein was increased following transfection with 10 nM
ASOs. These data provide direct evidence that the ASOs provided by
the present invention have a pronounced and favorable effect not
only on the pre-mRNA/mRNA level, but more importantly, on the
protein level.
Example 6
Functional Analysis
[0203] FLIPR.TM. (Fluorescence Imaging Plate Reader) is a
functional system for measuring changes in membrane potential by a
fluorescent indicator. FLIPR can be used to test CFTR activation
levels when the activation of CFTR is achieved by the addition of
Forskolin (FSK) and the specificity for the CFTR channel is
verified by the addition of CFTR specific inhibitor (inh-172). The
experiments were performed 24 hours following transfection of 10 nM
of each ASO to FRT mut cells.
[0204] FIG. 8A is a representative FLIPR trace demonstrating CFTR
activation induced by ASO SPL84-17. FIG. 8B illustrates the average
effect of several ASOs on CFTR function. (*, p value.ltoreq.0.05;
**, p value.ltoreq.0.01; ***, p value.ltoreq.0.001; ****, p
value.ltoreq.0.0001).
[0205] The data presented in FIGS. 8A and 8B clearly illustrates
that the new ASOs provided by the present invention have a
pronounced and favorable effect not only on the RNA and protein
levels, but even more importantly, on the functional level.
Example 7
Efficacy in Primary Cells of a CF Patient
[0206] CFTR channel activity was analyzed in differentiated primary
respiratory epithelial cells from CF patients. The Us sing chamber
is employed to measure trans-epithelial currents indicative of the
CFTR channel activity. This measurement is a strong predictor of
patient response to the treatment (R.sup.2=0.95).
[0207] Human Nasal Epithelial (HNE) cells from CF patients carrying
at least one CFTR allele with the 3849 +10 Kb C-to-T mutation were
provided. In order to increase the number of filter assays,
conditionally reprogrammed and re-differentiated HNE cells were
used. In these experiments the HNE cells were exposed to the ASOs
for 15-23 days, while differentiating on filters, with no addition
of a transfection reagent.
[0208] Ussing chamber experiments were performed on HNE cells from
5 CF patients. Four of them are heterozygous for the 3849 10+Kb
C-to-T mutation and a minimal function mutation: Paris patient #2
and Israel patient #4 (3849 +10 kb C-to-T/F508del), Israel patient
#1 (3849 +10 kb C-to-T/W1282X) and Israel patient #2 (3849 +10 Kb
C-to-T/405+1G-to-A). In patients heterozygous for the 3849 +10 Kb
C-to-T mutation, the maximum expected activity is 50% of WT, which
is sufficient to confer full health conditions. An additional
tested patient is homozygous for the 3849 +10 kb C-to-T mutation
(Paris patient #3).
[0209] First, 5 ASOs (SPL84-17, SPL84-22, SPL84-23, SPL84-2, and
SPL84-25) were analyzed in cells from two patients (one
heterozygote and one homozygote). In FIG. 9 the effects of the 5
ASOs in a compound heterozygote patient (Paris patient #2-3849 +10
kb C-to-T/F508del), are presented. In order to measure CFTR
activity levels in the most accurate way, the change after the
addition of a specific CFTR inhibitor (inh-172) was used as an
index. As can be seen in the representative traces (FIG. 9A) and in
the median of CFTR inh-172 (FIG. 9B) all tested ASOs led to a
significant activation of CFTR compared to the control ASO. In this
patient, SPL84-23 had the strongest effect reaching 55% of WT when
cells were exposed to SPL84-23. This level indicates a full
restoration of the 3849 allele. The other 4 ASOs also led to a
significant induction of the activity ranging from 34%-42.5% of
WT.
[0210] The effect of each of the 5 ASOs was also analysed in HNEs
from a patient homozygous for the 3849 +10 kb C-to-T mutation
(Paris #3). Consistent with the first tested heterozygote patient,
ASO SPL84-23 showed full restoration of CFTR activity reaching
activity levels of WT individuals. In this patient, three
additional ASOs (SPL84-17, SPL84-22, and SPL84-2) also induced full
restoration, indicating that these 3 ASOs also have a potential for
high therapeutic efficacy. Nevertheless, the levels of CFTR
activity between these ASOs varied, SPL84-17 and SPL84-22 showed
higher levels of CFTR activity compared to SPL84-2 (231% and 186%
compared to 151%, respectively). SPL84-25 also induced a high level
of activation although lower than the effects of the other ASOs
(93%) (FIG. 10).
[0211] Based on the results from the cells of these patients, the
inventors chose to further analyse the effect of the 3 ASOs which
showed the highest CFTR activity levels: SPL84-17, SPL8422 and
SPL84-23 (FIGS. 9 and 10).
[0212] Next, these 3 ASOs were tested in two additional
heterozygote patients carrying different minimal function mutation
on the second allele (W1282X and 405+1G-to-A). It is worth noting
that the use of HNE cells enables access to patients with various
genotypes, but still carrying one 3849 +10 Kb C-to-T mutation.
Consistent with the results from the first heterozygote patient
(Paris #2), the 3 ASOs showed activation of CFTR in the additional
tested patients, with a variable effect between patients.
[0213] The effect of ASO SPL84-23, which showed the highest effect
in the tested heterozygotes, was tested in cells from an additional
3849 +10 kb C-to-T/F508del patient (Israel #4). In this patient the
exposure to the ASO led also to a significant activation of the
CFTR channel to 38% of WT levels.
[0214] Summarizing the functional results from all heterozygote
patients tested, SPL84-23 has the strongest effect across cells
from patients with different genotypes, reaching full restoration
of the CFTR activity (average of 42.5% of WT ranging from 23% to
73.5%). The other two ASOs, SPL84-17 and SPL84-22, also show a
significant restoration of the 3849 +10 Kb C-to-T allele, with a
more modest effect (average of 30% and 31% of WT, respectively)
(FIG. 11).
[0215] Various data indicate that avoiding CF disease symptoms
(e.g., lung function) requires only partial restoration of
protein/RNA levels. As an indication, splice site variants that
allow CFTR transcript levels to reach 10-25% of normal levels have
been found in individuals that do not have CF lung disease. Thus,
the results provide a solid indication for the potential of the
ASOs as drug candidates with a significant clinical benefit.
Example 8
ASOs Effect on Splicing
[0216] To test whether the significant activation of CFTR by ASO
SPL84-23 treatment results from reduction in the level of
aberrantly spliced CFTR transcripts, the effect of ASO SPL84-23 on
the splicing pattern was further analyzed by extracting RNA from
the same filters used for the Us sing chamber measurements. As can
be seen in FIG. 12A, the level of the aberrantly spliced CFTR
transcripts, carrying the 84 bp cryptic exon, was significantly
reduced in the HNE cells of both the heterozygotes and homozygote
patients (as measured by qRT-PCR and calculating the relative fold
change in CFTR aberrant transcript levels between control ASO and
SPL84-23). In the homozygote cells, since all transcripts originate
from the 3849 +10 kb C-to-T mutated alleles, the absolute level of
correctly and aberrantly spliced transcripts can be visualized on
agarose gel using RT-PCR. The results demonstrate that SPL84-23
treatment resulted in a dramatic effect on the splicing pattern. In
this patient, untreated cells or cells treated with the control ASO
generated mainly aberrantly spliced CFTR with very low levels of
correctly spliced transcripts. However, in SPL84-23 treated cells
most of the RNA transcripts were correctly spliced (FIG. 12B).
Example 9
Chemical Modifications of AOs Effect CFTR Function in HNE Cells
from a CF Patient
[0217] The effect of an ASO with the same sequence and different
chemical modifications was evaluated. 2'OMP is considered a
second-generation ASO chemistry and the effect of another
second-generation modification, 2'-Methoxy Ethyl (2'MOE), was
tested. Both modifications have similar characteristics of: (1)
slowing down degradation by protecting the ASOs from nucleases; and
(2) having higher affinity to the target.
[0218] The effect of SPL84-23 having 2'OMPs or 2'MOE modification
in primary human nasal epithelial cells from patients carrying the
3849 +10 kb C to T mutation (free uptake) was compared using the
Ussing chamber assay as described in Example 7.
[0219] CFTR function was measured in nasal epithelial cells from a
patient homozygous for the 3849 mutation, following treatment of
naked ASOs (free uptake, no transfection reagent) (FIG. 13). The
ASOs were tested in 2 concentrations as indicated. SPL84-23 2'MOE
showed a significant higher effect on the CFTR function compared to
SPL84-23 2'OMP, in the two concentrations. Importantly, while
SPL84-23 2'MOE completely rescued the CFTR function (to WT levels),
in both 200 nM and 50 nM ASO concentrations, SPL84-23 2'OMP
completely rescued the function only in the higher (200 nM)
concentration.
Example 10
Optimization of ASO Improves Efficacy and Penetration
[0220] The inventors further optimized leading synthetic
oligonucleotide candidates in order to improve their efficacy and
penetration. The effects of chemistry and shortening of the ASO
length were examined.
[0221] Changing the chemical modification from 2'OMe to IONIS
2.sup.nd generation 2'MOE and also ASO length from 17-20 bases were
attempted.
[0222] Short versions of AS084-23 were screened for their effect on
splicing by free uptake on primary patient HNE cells
(SPL84-23-1--SPL84-23-9). On average in the three 3849 heterozygote
patients analyzed, the effect of SPL84-23-1 (19 mer) was comparable
to SPL84-23 (FIG. 14).
[0223] Optimization of ASO length was further examined in primary
HNE cells and in primary HBE cells (human bronchial epithelial
cells) from a heterozygous a patient (3849/F508del).
[0224] SPL84-23-1 (19 mer) was shown to have a similar effect on
the CFTR function as the longer SPL84-23 ASO in primary HNE cells
(FIGS. 15A-15B), as well as in primary HBE cells (FIGS.
16A-16B).
[0225] While the present invention has been particularly described,
persons skilled in the art will appreciate that many variations and
modifications can be made. Therefore, the invention is not to be
construed as restricted to the particularly described embodiments,
and the scope and concept of the invention will be more readily
understood by reference to the claims, which follow.
Sequence CWU 1
1
71120RNAArtificial SequenceSynthetic 1cugcaacaga uggaagacuc
20219RNAArtificial SequenceSynthetic 2caacagaugg aagacucuu
19319RNAArtificial SequenceSynthetic 3cuccagaaau caagaugac
19419RNAArtificial SequenceSynthetic 4uacugcaaca gauggaaga
19520RNAArtificial SequenceSynthetic 5aucaagauga caagucaacu
20619RNAArtificial SequenceSynthetic 6guggucucca gaaaucaag
19719RNAArtificial SequenceSynthetic 7gauggaagac ucuuguaau
19821RNAArtificial SequenceSynthetic 8caagaugaca agucaacuga a
21922RNAArtificial SequenceSynthetic 9gaaaucaaga ugacaaguca ac
221019RNAArtificial SequenceSynthetic 10accuuguggu cuccagaaa
191120RNAArtificial SequenceSynthetic 11ccagaaauca agaugacaag
201221RNAArtificial SequenceSynthetic 12caccauuuua auacugcaac a
211321RNAArtificial SequenceSynthetic 13uggaagacuc uuguaauuau u
211421RNAArtificial SequenceSynthetic 14gaugacaagu caacugaaau u
211519RNAArtificial SequenceSynthetic 15cuuucagggu gucuuacuc
191619RNAArtificial SequenceSynthetic 16ucaggguguc uuacucacc
191720RNAArtificial SequenceSynthetic 17auuaccuugu ggucuccaga
201821RNAArtificial SequenceSynthetic 18gacaagucaa cugaaauuua g
211921RNAArtificial SequenceSynthetic 19caacugaaau uuagauccac a
212021RNAArtificial SequenceSynthetic 20agucaacuga aauuuagauc c
212121RNAArtificial SequenceSynthetic 21gugucuuacu caccauuuua a
212218RNAArtificial SequenceSynthetic 22gugucuuacu caccauuu
182319RNAArtificial SequenceSynthetic 23cucaccauuu uaauacugc
192418RNAArtificial SequenceSynthetic 24gacaagucaa cugaaauu
182520RNAArtificial SequenceSynthetic 25cuuacucacc auuuuaauac
202618RNAArtificial SequenceSynthetic 26gucuuacuca ccauuuua
182719RNAArtificial SequenceSynthetic 27caagucaacu gaaauuuag
192819RNAArtificial SequenceSynthetic 28cuuguaauua uuuuuacau
192925RNAArtificial SequenceSynthetic 29aaaucaagau gacaagucaa cugaa
253025RNAArtificial SequenceSynthetic 30cuuguggucu ccagaaauca agaug
253125RNAArtificial SequenceSynthetic 31aacagaugga agacucuugu aauua
253225RNAArtificial SequenceSynthetic 32ucaggguguc uuacucacca uuuua
253318RNAArtificial SequenceSynthetic 33gaccacuugc cacccauc
18346130RNAHomo sapiens 34aauuggaagc aaaugacauc acagcagguc
agagaaaaag gguugagcgg caggcaccca 60gaguaguagg ucuuuggcau uaggagcuug
agcccagacg gcccuagcag ggaccccagc 120gcccgagaga ccaugcagag
gucgccucug gaaaaggcca gcguugucuc caaacuuuuu 180uucagcugga
ccagaccaau uuugaggaaa ggauacagac agcgccugga auugucagac
240auauaccaaa ucccuucugu ugauucugcu gacaaucuau cugaaaaauu
gaaagagaau 300gggauagaga gcuggcuuca aagaaaaauc cuaaacucau
uaaugcccuu cggcgauguu 360uuuucuggag auuuauguuc uauggaaucu
uuuuauauuu aggggaaguc accaaagcag 420uacagccucu cuuacuggga
agaaucauag cuuccuauga cccggauaac aaggaggaac 480gcucuaucgc
gauuuaucua ggcauaggcu uaugccuucu cuuuauugug aggacacugc
540uccuacaccc agccauuuuu ggccuucauc acauuggaau gcagaugaga
auagcuaugu 600uuaguuugau uuauaagaag acuuuaaagc ugucaagccg
uguucuagau aaaauaagua 660uuggacaacu uguuagucuc cuuuccaaca
accugaacaa auuugaugaa ggacuugcau 720uggcacauuu cguguggauc
gcuccuuugc aaguggcacu ccucaugggg cuaaucuggg 780aguuguuaca
ggcgucugcc uucuguggac uugguuuccu gauaguccuu gcccuuuuuc
840aggcugggcu agggagaaug augaugaagu acagagauca gagagcuggg
aagaucagug 900aaagacuugu gauuaccuca gaaaugauug aaaauaucca
aucuguuaag gcauacugcu 960gggaagaagc aauggaaaaa augauugaaa
acuuaagaca aacagaacug aaacugacuc 1020ggaaggcagc cuaugugaga
uacuucaaua gcucagccuu cuucuucuca ggguucuuug 1080ugguguuuuu
aucugugcuu cccuaugcac uaaucaaagg aaucauccuc cggaaaauau
1140ucaccaccau cucauucugc auuguucugc gcauggcggu cacucggcaa
uuucccuggg 1200cuguacaaac augguaugac ucucuuggag caauaaacaa
aauacaggau uucuuacaaa 1260agcaagaaua uaagacauug gaauauaacu
uaacgacuac agaaguagug auggagaaug 1320uaacagccuu cugggaggag
ggauuugggg aauuauuuga gaaagcaaaa caaaacaaua 1380acaauagaaa
aacuucuaau ggugaugaca gccucuucuu caguaauuuc ucacuucuug
1440guacuccugu ccugaaagau auuaauuuca agauagaaag aggacaguug
uuggcgguug 1500cuggauccac uggagcaggc aagacuucac uucuaauggu
gauuauggga gaacuggagc 1560cuucagaggg uaaaauuaag cacaguggaa
gaauuucauu cuguucucag uuuuccugga 1620uuaugccugg caccauuaaa
gaaaauauca ucuuuggugu uuccuaugau gaauauagau 1680acagaagcgu
caucaaagca ugccaacuag aagaggacau cuccaaguuu gcagagaaag
1740acaauauagu ucuuggagaa gguggaauca cacugagugg aggucaacga
gcaagaauuu 1800cuuuagcaag agcaguauac aaagaugcug auuuguauuu
auuagacucu ccuuuuggau 1860accuagaugu uuuaacagaa aaagaaauau
uugaaagcug ugucuguaaa cugauggcua 1920acaaaacuag gauuuugguc
acuucuaaaa uggaacauuu aaagaaagcu gacaaaauau 1980uaauuuugca
ugaagguagc agcuauuuuu augggacauu uucagaacuc caaaaucuac
2040agccagacuu uagcucaaaa cucaugggau gugauucuuu cgaccaauuu
agugcagaaa 2100gaagaaauuc aauccuaacu gagaccuuac accguuucuc
auuagaagga gaugcuccug 2160ucuccuggac agaaacaaaa aaacaaucuu
uuaaacagac uggagaguuu ggggaaaaaa 2220ggaagaauuc uauucucaau
ccaaucaacu cuauacgaaa auuuuccauu gugcaaaaga 2280cucccuuaca
aaugaauggc aucgaagagg auucugauga gccuuuagag agaaggcugu
2340ccuuaguacc agauucugag cagggagagg cgauacugcc ucgcaucagc
gugaucagca 2400cuggccccac gcuucaggca cgaaggaggc agucuguccu
gaaccugaug acacacucag 2460uuaaccaagg ucagaacauu caccgaaaga
caacagcauc cacacgaaaa gugucacugg 2520ccccucaggc aaacuugacu
gaacuggaua uauauucaag aagguuaucu caagaaacug 2580gcuuggaaau
aagugaagaa auuaacgaag aagacuuaaa ggagugcuuu uuugaugaua
2640uggagagcau accagcagug acuacaugga acacauaccu ucgauauauu
acuguccaca 2700agagcuuaau uuuugugcua auuuggugcu uaguaauuuu
ucuggcagag guggcugcuu 2760cuuugguugu gcuguggcuc cuuggaaaca
cuccucuuca agacaaaggg aauaguacuc 2820auaguagaaa uaacagcuau
gcagugauua ucaccagcac caguucguau uauguguuuu 2880acauuuacgu
gggaguagcc gacacuuugc uugcuauggg auucuucaga ggucuaccac
2940uggugcauac ucuaaucaca gugucgaaaa uuuuacacca caaaauguua
cauucuguuc 3000uucaagcacc uaugucaacc cucaacacgu ugaaagcagg
ugggauucuu aauagauucu 3060ccaaagauau agcaauuuug gaugaccuuc
ugccucuuac cauauuugac uucauccagu 3120uguuauuaau ugugauugga
gcuauagcag uugucgcagu uuuacaaccc uacaucuuug 3180uugcaacagu
gccagugaua guggcuuuua uuauguugag agcauauuuc cuccaaaccu
3240cacagcaacu caaacaacug gaaucugaag gcaggagucc aauuuucacu
caucuuguua 3300caagcuuaaa aggacuaugg acacuucgug ccuucggacg
gcagccuuac uuugaaacuc 3360uguuccacaa agcucugaau uuacauacug
ccaacugguu cuuguaccug ucaacacugc 3420gcugguucca aaugagaaua
gaaaugauuu uugucaucuu cuucauugcu guuaccuuca 3480uuuccauuuu
aacaacagga gaaggagaag gaagaguugg uauuauccug acuuuagcca
3540ugaauaucau gaguacauug cagugggcug uaaacuccag cauagaugug
gauagcuuga 3600ugcgaucugu gagccgaguc uuuaaguuca uugacaugcc
aacagaaggu aaaccuacca 3660agucaaccaa accauacaag aauggccaac
ucucgaaagu uaugauuauu gagaauucac 3720acgugaagaa agaugacauc
uggcccucag ggggccaaau gacugucaaa gaucucacag 3780caaaauacac
agaaggugga aaugccauau uagagaacau uuccuucuca auaaguccug
3840gccagagggu gggccucuug ggaagaacug gaucagggaa gaguacuuug
uuaucagcuu 3900uuuugagacu acugaacacu gaaggagaaa uccagaucga
uggugugucu ugggauucaa 3960uaacuuugca acaguggagg aaagccuuug
gagugauacc acagaaagua uuuauuuuuu 4020cuggaacauu uagaaaaaac
uuggaucccu augaacagug gagugaucaa gaaauaugga 4080aaguugcaga
ugagguuggg cucagaucug ugauagaaca guuuccuggg aagcuugacu
4140uuguccuugu ggaugggggc uguguccuaa gccauggcca caagcaguug
augugcuugg 4200cuagaucugu ucucaguaag gcgaagaucu ugcugcuuga
ugaacccagu gcucauuugg 4260auccaguaac auaccaaaua auuagaagaa
cucuaaaaca agcauuugcu gauugcacag 4320uaauucucug ugaacacagg
auagaagcaa ugcuggaaug ccaacaauuu uuggucauag 4380aagagaacaa
agugcggcag uacgauucca uccagaaacu gcugaacgag aggagccucu
4440uccggcaagc caucagcccc uccgacaggg ugaagcucuu uccccaccgg
aacucaagca 4500agugcaaguc uaagccccag auugcugcuc ugaaagagga
gacagaagaa gaggugcaag 4560auacaaggcu uuagagagca gcauaaaugu
ugacauggga cauuugcuca uggaauugga 4620gcucguggga cagucaccuc
auggaauugg agcucgugga acaguuaccu cugccucaga 4680aaacaaggau
gaauuaaguu uuuuuuuaaa aaagaaacau uugguaaggg aauugaggac
4740acugauaugg gucuugauaa auggcuuccu ggcaauaguc aaauugugug
aaagguacuu 4800caaauccuug aagauuuacc acuuguguuu ugcaagccag
auuuuccuga aaacccuugc 4860caugugcuag uaauuggaaa ggcagcucua
aaugucaauc agccuaguug aucagcuuau 4920ugucuaguga aacucguuaa
uuuguagugu uggagaagaa cugaaaucau acuucuuagg 4980guuaugauua
aguaaugaua acuggaaacu ucagcgguuu auauaagcuu guauuccuuu
5040uucucuccuc uccccaugau guuuagaaac acaacuauau uguuugcuaa
gcauuccaac 5100uaucucauuu ccaagcaagu auuagaauac cacaggaacc
acaagacugc acaucaaaau 5160augccccauu caacaucuag ugagcaguca
ggaaagagaa cuuccagauc cuggaaauca 5220ggguuaguau uguccagguc
uaccaaaaau cucaauauuu cagauaauca caauacaucc 5280cuuaccuggg
aaagggcugu uauaaucuuu cacaggggac aggaugguuc ccuugaugaa
5340gaaguugaua ugccuuuucc caacuccaga aagugacaag cucacagacc
uuugaacuag 5400aguuuagcug gaaaaguaug uuagugcaaa uugucacagg
acagcccuuc uuuccacaga 5460agcuccaggu agagggugug uaaguagaua
ggccaugggc acugugggua gacacacaug 5520aaguccaagc auuuagaugu
auagguugau ggugguaugu uuucaggcua gauguaugua 5580cuucaugcug
ucuacacuaa gagagaauga gagacacacu gaagaagcac caaucaugaa
5640uuaguuuuau augcuucugu uuuauaauuu ugugaagcaa aauuuuuucu
cuaggaaaua 5700uuuauuuuaa uaauguuuca aacauauaua acaaugcugu
auuuuaaaag aaugauuaug 5760aauuacauuu guauaaaaua auuuuuauau
uugaaauauu gacuuuuuau ggcacuagua 5820uuucuaugaa auauuauguu
aaaacuggga caggggagaa ccuaggguga uauuaaccag 5880gggccaugaa
ucaccuuuug gucuggaggg aagccuuggg gcugaugcag uuguugccca
5940cagcuguaug auucccagcc agcacagccu cuuagaugca guucugaaga
agaugguacc 6000accagucuga cuguuuccau caaggguaca cugccuucuc
aacuccaaac ugacucuuaa 6060gaagacugca uuauauuuau uacuguaaga
aaauaucacu ugucaauaaa auccauacau 6120uugugugaaa 61303584DNAHomo
sapiens 35ttgacttgtc atcttgattt ctggagacca caaggtaatg aaaaataatt
acaagagtct 60tccatctgtt gcagtattaa aatg 84366216RNAHomo sapiens
36aauuggaagc aaaugacauc acagcagguc agagaaaaag gguugagcgg caggcaccca
60gaguaguagg ucuuuggcau uaggagcuug agcccagacg gcccuagcag ggaccccagc
120gcccgagaga ccaugcagag gucgccucug gaaaaggcca gcguugucuc
caaacuuuuu 180uucagcugga ccagaccaau uuugaggaaa ggauacagac
agcgccugga auugucagac 240auauaccaaa ucccuucugu ugauucugcu
gacaaucuau cugaaaaauu ggaaagagaa 300ugggauagag agcuggcuuc
aaagaaaaau ccuaaacuca uuaaugcccu ucggcgaugu 360uuuuucugga
gauuuauguu cuauggaauc uuuuuauauu uaggggaagu caccaaagca
420guacagccuc ucuuacuggg aagaaucaua gcuuccuaug acccggauaa
caaggaggaa 480cgcucuaucg cgauuuaucu aggcauaggc uuaugccuuc
ucuuuauugu gaggacacug 540cuccuacacc cagccauuuu uggccuucau
cacauuggaa ugcagaugag aauagcuaug 600uuuaguuuga uuuauaagaa
gacuuuaaag cugucaagcc guguucuaga uaaaauaagu 660auuggacaac
uuguuagucu ccuuuccaac aaccugaaca aauuugauga aggacuugca
720uuggcacauu ucguguggau cgcuccuuug caaguggcac uccucauggg
gcuaaucugg 780gaguuguuac aggcgucugc cuucugugga cuugguuucc
ugauaguccu ugcccuuuuu 840caggcugggc uagggagaau gaugaugaag
uacagagauc agagagcugg gaagaucagu 900gaaagacuug ugauuaccuc
agaaaugauu gaaaauaucc aaucuguuaa ggcauacugc 960ugggaagaag
caauggaaaa aaugauugaa aacuuaagac aaacagaacu gaaacugacu
1020cggaaggcag ccuaugugag auacuucaau agcucagccu ucuucuucuc
aggguucuuu 1080gugguguuuu uaucugugcu ucccuaugca cuaaucaaag
gaaucauccu ccggaaaaua 1140uucaccacca ucucauucug cauuguucug
cgcauggcgg ucacucggca auuucccugg 1200gcuguacaaa caugguauga
cucucuugga gcaauaaaca aaauacagga uuucuuacaa 1260aagcaagaau
auaagacauu ggaauauaac uuaacgacua cagaaguagu gauggagaau
1320guaacagccu ucugggagga gggauuuggg gaauuauuug agaaagcaaa
acaaaacaau 1380aacaauagaa aaacuucuaa uggugaugac agccucuucu
ucaguaauuu cucacuucuu 1440gguacuccug uccugaaaga uauuaauuuc
aagauagaaa gaggacaguu guuggcgguu 1500gcuggaucca cuggagcagg
caagacuuca cuucuaaugg ugauuauggg agaacuggag 1560ccuucagagg
guaaaauuaa gcacagugga agaauuucau ucuguucuca guuuuccugg
1620auuaugccug gcaccauuaa agaaaauauc aucuuuggug uuuccuauga
ugaauauaga 1680uacagaagcg ucaucaaagc augccaacua gaagaggaca
ucuccaaguu ugcagagaaa 1740gacaauauag uucuuggaga agguggaauc
acacugagug gaggucaacg agcaagaauu 1800ucuuuagcaa gagcaguaua
caaagaugcu gauuuguauu uauuagacuc uccuuuugga 1860uaccuagaug
uuuuaacaga aaaagaaaua uuugaaagcu gugucuguaa acugauggcu
1920aacaaaacua ggauuuuggu cacuucuaaa auggaacauu uaaagaaagc
ugacaaaaua 1980uuaauuuugc augaagguag cagcuauuuu uaugggacau
uuucagaacu ccaaaaucua 2040cagccagacu uuagcucaaa acucauggga
ugugauucuu ucgaccaauu uagugcagaa 2100agaagaaauu caauccuaac
ugagaccuua caccguuucu cauuagaagg agaugcuccu 2160gucuccugga
cagaaacaaa aaaacaaucu uuuaaacaga cuggagaguu uggggaaaaa
2220aggaagaauu cuauucucaa uccaaucaac ucuauacgaa aauuuuccau
ugugcaaaag 2280acucccuuac aaaugaaugg caucgaagag gauucugaug
agccuuuaga gagaaggcug 2340uccuuaguac cagauucuga gcagggagag
gcgauacugc cucgcaucag cgugaucagc 2400acuggcccca cgcuucaggc
acgaaggagg cagucugucc ugaaccugau gacacacuca 2460guuaaccaag
gucagaacau ucaccgaaag acaacagcau ccacacgaaa agugucacug
2520gccccucagg caaacuugac ugaacuggau auauauucaa gaagguuauc
ucaagaaacu 2580ggcuuggaaa uaagugaaga aauuaacgaa gaagacuuaa
aggagugcuu uuuugaugau 2640auggagagca uaccagcagu gacuacaugg
aacacauacc uucgauauau uacuguccac 2700aagagcuuaa uuuuugugcu
aauuuggugc uuaguaauuu uucuggcaga gguggcugcu 2760ucuuugguug
ugcuguggcu ccuuggaaac acuccucuuc aagacaaagg gaauaguacu
2820cauaguagaa auaacagcua ugcagugauu aucaccagca ccaguucgua
uuauguguuu 2880uacauuuacg ugggaguagc cgacacuuug cuugcuaugg
gauucuucag aggucuacca 2940cuggugcaua cucuaaucac agugucgaaa
auuuuacacc acaaaauguu acauucuguu 3000cuucaagcac cuaugucaac
ccucaacacg uugaaagcag gugggauucu uaauagauuc 3060uccaaagaua
uagcaauuuu ggaugaccuu cugccucuua ccauauuuga cuucauccag
3120uuguuauuaa uugugauugg agcuauagca guugucgcag uuuuacaacc
cuacaucuuu 3180guugcaacag ugccagugau aguggcuuuu auuauguuga
gagcauauuu ccuccaaacc 3240ucacagcaac ucaaacaacu ggaaucugaa
ggcaggaguc caauuuucac ucaucuuguu 3300acaagcuuaa aaggacuaug
gacacuucgu gccuucggac ggcagccuua cuuugaaacu 3360cuguuccaca
aagcucugaa uuuacauacu gccaacuggu ucuuguaccu gucaacacug
3420cgcugguucc aaaugagaau agaaaugauu uuugucaucu ucuucauugc
uguuaccuuc 3480auuuccauuu uaacaacagg agaaggagaa ggaagaguug
guauuauccu gacuuuagcc 3540augaauauca ugaguacauu gcagugggcu
guaaacucca gcauagaugu ggauagcuug 3600augcgaucug ugagccgagu
cuuuaaguuc auugacaugc caacagaagg uaaaccuacc 3660aagucaacca
aaccauacaa gaauggccaa cucucgaaag uuaugauuau ugagaauuca
3720cacgugaaga aagaugacau cuggcccuca gggggccaaa ugacugucaa
agaucucaca 3780gcaaaauaca cagaaggugg aaaugccaua uuagagaaca
uuuccuucuc aauaaguccu 3840ggccagaggu ugacuuguca ucuugauuuc
uggagaccac aagguaauga aaaauaauua 3900caagagucuu ccaucuguug
caguauuaaa auggugggcc ucuugggaag aacuggauca 3960gggaagagua
cuuuguuauc agcuuuuuug agacuacuga acacugaagg agaaauccag
4020aucgauggug ugucuuggga uucaauaacu uugcaacagu ggaggaaagc
cuuuggagug 4080auaccacaga aaguauuuau uuuuucugga acauuuagaa
aaaacuugga ucccuaugaa 4140caguggagug aucaagaaau auggaaaguu
gcagaugagg uugggcucag aucugugaua 4200gaacaguuuc cugggaagcu
ugacuuuguc cuuguggaug ggggcugugu ccuaagccau 4260ggccacaagc
aguugaugug cuuggcuaga ucuguucuca guaaggcgaa gaucuugcug
4320cuugaugaac ccagugcuca uuuggaucca guaacauacc aaauaauuag
aagaacucua 4380aaacaagcau uugcugauug cacaguaauu cucugugaac
acaggauaga agcaaugcug 4440gaaugccaac aauuuuuggu cauagaagag
aacaaagugc ggcaguacga uuccauccag 4500aaacugcuga acgagaggag
ccucuuccgg caagccauca gccccuccga cagggugaag 4560cucuuucccc
accggaacuc aagcaagugc aagucuaagc cccagauugc ugcucugaaa
4620gaggagacag aagaagaggu gcaagauaca aggcuuuaga gagcagcaua
aauguugaca 4680ugggacauuu gcucauggaa uuggagcucg ugggacaguc
accucaugga auuggagcuc 4740guggaacagu uaccucugcc ucagaaaaca
aggaugaauu aaguuuuuuu uuaaaaaaga 4800aacauuuggu aaggggaauu
gaggacacug auaugggucu ugauaaaugg cuuccuggca 4860auagucaaau
ugugugaaag guacuucaaa uccuugaaga uuuaccacuu guguuuugca
4920agccagauuu uccugaaaac ccuugccaug ugcuaguaau uggaaaggca
gcucuaaaug 4980ucaaucagcc uaguugauca gcuuauuguc uagugaaacu
cguuaauuug uaguguugga 5040gaagaacuga aaucauacuu cuuaggguua
ugauuaagua augauaacug gaaacuucag 5100cgguuuauau aagcuuguau
uccuuuuucu cuccucuccc caugauguuu agaaacacaa 5160cuauauuguu
ugcuaagcau uccaacuauc ucauuuccaa gcaaguauua gaauaccaca
5220ggaaccacaa gacugcacau caaaauaugc cccauucaac aucuagugag
cagucaggaa 5280agagaacuuc cagauccugg aaaucagggu uaguauuguc
caggucuacc aaaaaucuca 5340auauuucaga uaaucacaau acaucccuua
ccugggaaag ggcuguuaua aucuuucaca 5400ggggacagga ugguucccuu
gaugaagaag uugauaugcc uuuucccaac uccagaaagu 5460gacaagcuca
cagaccuuug aacuagaguu uagcuggaaa aguauguuag ugcaaauugu
5520cacaggacag cccuucuuuc cacagaagcu ccagguagag gguguguaag
uagauaggcc 5580augggcacug uggguagaca cacaugaagu ccaagcauuu
agauguauag guugauggug 5640guauguuuuc aggcuagaug uauguacuuc
augcugucua cacuaagaga gaaugagaga 5700cacacugaag aagcaccaau
caugaauuag
uuuuauaugc uucuguuuua uaauuuugug 5760aagcaaaauu uuuucucuag
gaaauauuua uuuuaauaau guuucaaaca uauauaacaa 5820ugcuguauuu
uaaaagaaug auuaugaauu acauuuguau aaaauaauuu uuauauuuga
5880aauauugacu uuuuauggca cuaguauuuc uaugaaauau uauguuaaaa
cugggacagg 5940ggagaaccua gggugauauu aaccaggggc caugaaucac
cuuuuggucu ggagggaagc 6000cuuggggcug augcaguugu ugcccacagc
uguaugauuc ccagccagca cagccucuua 6060gaugcaguuc ugaagaagau
gguaccacca gucugacugu uuccaucaag gguacacugc 6120cuucucaacu
ccaaacugac ucuuaagaag acugcauuau auuuauuacu guaagaaaau
6180aucacuuguc aauaaaaucc auacauuugu gugaaa 621637284DNAHomo
sapiens 37aagcagcata ttctcaatac tatgtttcat taataattaa tagagatata
tgaacacata 60aaagattcaa ttataatcac cttgtggatc taaatttcag ttgacttgtc
atcttgattt 120ctggagacca caaggtaatg aaaaataatt acaagagtct
tccatctgtt gcagtattaa 180aatggtgagt aagacaccct gaaaggaaat
gttctattca tggtacaatg caattacagc 240tagcaccaaa ttcaacactg
tttaactttc aacatattat tttg 284389RNAArtificial SequenceSynthetic
38gauggaaga 93914RNAArtificial SequenceSynthetic 39caacagaugg aaga
144019RNAArtificial SequenceSynthetic 40cugcaacaga uggaagacu
194119RNAArtificial SequenceSynthetic 41ugcaacagau ggaagacuc
194218RNAArtificial SequenceSynthetic 42cugcaacaga uggaagac
184318RNAArtificial SequenceSynthetic 43gcaacagaug gaagacuc
184418RNAArtificial SequenceSynthetic 44ugcaacagau ggaagacu
184517RNAArtificial SequenceSynthetic 45cugcaacaga uggaaga
174617RNAArtificial SequenceSynthetic 46caacagaugg aagacuc
174717RNAArtificial SequenceSynthetic 47ugcaacagau ggaagac
174817RNAArtificial SequenceSynthetic 48gcaacagaug gaagacu
174918RNAArtificial SequenceSynthetic 49cuccagaaau caagauga
185018RNAArtificial SequenceSynthetic 50uccagaaauc aagaugac
185117RNAArtificial SequenceSynthetic 51cuccagaaau caagaug
175217RNAArtificial SequenceSynthetic 52ccagaaauca agaugac
175317RNAArtificial SequenceSynthetic 53uccagaaauc aagauga
175418RNAArtificial SequenceSynthetic 54caacagaugg aagacucu
185518RNAArtificial SequenceSynthetic 55aacagaugga agacucuu
185617RNAArtificial SequenceSynthetic 56acagauggaa gacucuu
175717RNAArtificial SequenceSynthetic 57aacagaugga agacucu
175818RNAArtificial SequenceSynthetic 58uacugcaaca gauggaag
185918RNAArtificial SequenceSynthetic 59acugcaacag auggaaga
186017RNAArtificial SequenceSynthetic 60uacugcaaca gauggaa
176117RNAArtificial SequenceSynthetic 61acugcaacag auggaag
176219RNAArtificial SequenceSynthetic 62aucaagauga caagucaac
196319RNAArtificial SequenceSynthetic 63ucaagaugac aagucaacu
196418RNAArtificial SequenceSynthetic 64aucaagauga caagucaa
186518RNAArtificial SequenceSynthetic 65caagaugaca agucaacu
186618RNAArtificial SequenceSynthetic 66ucaagaugac aagucaac
186717RNAArtificial SequenceSynthetic 67aucaagauga caaguca
176817RNAArtificial SequenceSynthetic 68aagaugacaa gucaacu
176917RNAArtificial SequenceSynthetic 69ucaagaugac aagucaa
177017RNAArtificial SequenceSynthetic 70caagaugaca agucaac
177120RNAArtificial SequenceSynthetic 71aauuauuuuu cauuaccuug
20
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