U.S. patent application number 16/302339 was filed with the patent office on 2020-03-19 for polynucleotides encoding porphobilinogen deaminase for the treatment of acute intermittent porphyria.
The applicant listed for this patent is Fundacion Para La Investigacion Medica Aplicada, ModernaTX, Inc.. Invention is credited to Matias Antonio Avila Zaragoza, Kerry Benenato, Pedro Berraondo Lopez, Antonio Fontanellas Roma, Lin Tung Guey, Stephen Hoge, Lei Jiang, Ellalahewage Sathyajith Kumarasinghe, Paolo Martini, Iain McFadyen, Vladimir Presnyak, Staci Sabnis.
Application Number | 20200085916 16/302339 |
Document ID | / |
Family ID | 67223874 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200085916 |
Kind Code |
A1 |
Martini; Paolo ; et
al. |
March 19, 2020 |
POLYNUCLEOTIDES ENCODING PORPHOBILINOGEN DEAMINASE FOR THE
TREATMENT OF ACUTE INTERMITTENT PORPHYRIA
Abstract
The invention relates to mRNA therapy for the treatment of Acute
Intermittent Porphyria (AIP). mRNAs for use in the invention, when
administered in vivo, encode human porphobilinogen deaminase
(PBGD), isoforms thereof, functional fragments thereof, and fusion
proteins comprising PBGD. mRNAs of the invention are preferably
encapsulated in lipid nanoparticles (LNPs) to affect efficient
delivery to cells and/or tissues in subjects, when administered
thereto. mRNA therapies of the invention increase and/or restore
deficient levels of PBGD expression and/or activity in subjects.
mRNA therapies of the invention further decrease levels of toxic
metabolites associated with deficient PBGD activity in subjects,
namely porphobilinogen and aminolevulinate (PBG and ALA).
Inventors: |
Martini; Paolo; (Boston,
MA) ; Hoge; Stephen; (Cambridge, MA) ;
Benenato; Kerry; (Cambridge, MA) ; Presnyak;
Vladimir; (Manchester, NH) ; Jiang; Lei;
(Cambridge, MA) ; McFadyen; Iain; (Medford,
MA) ; Kumarasinghe; Ellalahewage Sathyajith;
(Cambridge, MA) ; Fontanellas Roma; Antonio;
(Pamplona, ES) ; Berraondo Lopez; Pedro;
(Pamplona, ES) ; Avila Zaragoza; Matias Antonio;
(Pamplona, ES) ; Guey; Lin Tung; (Lexington,
MA) ; Sabnis; Staci; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc.
Fundacion Para La Investigacion Medica Aplicada |
Cambridge
Pamplona |
MA |
US
ES |
|
|
Family ID: |
67223874 |
Appl. No.: |
16/302339 |
Filed: |
May 18, 2017 |
PCT Filed: |
May 18, 2017 |
PCT NO: |
PCT/US2017/033418 |
371 Date: |
November 16, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62338161 |
May 18, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/08 20180101; C12N
9/1085 20130101; A61K 48/005 20130101; C12N 15/88 20130101; C12Y
205/01061 20130101; A61K 48/0033 20130101; A61K 38/45 20130101 |
International
Class: |
A61K 38/45 20060101
A61K038/45; C12N 9/10 20060101 C12N009/10; C12N 15/88 20060101
C12N015/88; A61K 48/00 20060101 A61K048/00; A61P 7/08 20060101
A61P007/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 9, 2017 |
EP |
17382259.4 |
Claims
1.-28. (canceled)
29. A pharmaceutical composition comprising a lipid nanoparticle,
wherein the lipid nanoparticle comprises a compound having the
Formula (I) ##STR00179## or a salt or stereoisomer thereof, wherein
R.sub.1 is selected from the group consisting of C.sub.5-30 alkyl,
C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R'; R.sub.2 and
R.sub.3 are independently selected from the group consisting of H,
C.sub.1-14 alkyl, C.sub.2-14 alkenyl, --R*YR'', --YR'', and
--R*OR'', or R.sub.2 and R.sub.3, together with the atom to which
they are attached, form a heterocycle or carbocycle; R.sub.4 is
selected from the group consisting of a C.sub.3-6 carbocycle,
--(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR, --CQ(R).sub.2,
and unsubstituted C.sub.1-6 alkyl, where Q is selected from a
carbocycle, heterocycle, --OR, --O(CH.sub.2).sub.nN(R).sub.2,
--C(O)OR, --OC(O)R, --CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN,
--N(R).sub.2, --C(O)N(R).sub.2, --N(R)C(O)R, --N(R)S(O).sub.2R,
--N(R)C(O)N(R).sub.2, --N(R)C(S)N(R).sub.2, --N(R)R.sub.8,
--O(CH.sub.2).sub.nOR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and --C(R)N(R).sub.2C(O)OR, and
each n is independently selected from 1, 2, 3, 4, and 5; each
R.sub.5 is independently selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each R.sub.6 is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; M and M' are independently
selected from --C(O)O--, --OC(O)--, --C(O)N(R')--, --N(R')C(O)--,
--C(O)--, --C(S)--, --C(S)S--, --SC(S)--, --CH(OH)--,
--P(O)(OR')O--, --S(O).sub.2--, --S--S--, an aryl group, and a
heteroaryl group; R.sub.7 is selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; R.sub.8 is selected from
the group consisting of C.sub.3-6 carbocycle and heterocycle;
R.sub.9 is selected from the group consisting of H, CN, NO.sub.2,
C.sub.1-6 alkyl, --OR, --S(O).sub.2R, --S(O).sub.2N(R).sub.2,
C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and heterocycle; each R is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; each R' is independently selected
from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl,
--R*YR'', --YR'', and H; each R'' is independently selected from
the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
each R* is independently selected from the group consisting of
C.sub.1-12 alkyl and C.sub.2-12 alkenyl; each Y is independently a
C.sub.3-6 carbocycle; each X is independently selected from the
group consisting of F, Cl, Br, and I; and m is selected from 5, 6,
7, 8, 9, 10, 11, 12, and 13; and provided that when R.sub.4 is
--(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR, or
--CQ(R).sub.2, then (i) Q is not --N(R).sub.2 when n is 1, 2, 3, 4
or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n
is 1 or 2, wherein the lipid nanoparticle comprises an mRNA that
comprises an open reading frame (ORF) encoding an porphobilinogen
deaminase (PBGD) polypeptide, wherein the composition is suitable
for administration to a human subject in need of treatment for
acute intermittent porphyria (AIP).
30. The pharmaceutical composition of claim 29, wherein the lipid
nanoparticle comprises the compound is of Formula (IA):
##STR00180## or a salt or stereoisomer thereof, wherein l is
selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and
9; M.sub.1 is a bond or M'; R.sub.4 is unsubstituted C.sub.1-3
alkyl, or --(CH.sub.2).sub.nQ, in which Q is OH,
--NHC(S)N(R).sub.2, --NHC(O)N(R).sub.2, --N(R)C(O)R,
--N(R)S(O).sub.2R, --N(R)R.sub.8, --NHC(.dbd.NR.sub.9)N(R).sub.2,
--NHC(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
heteroaryl or heterocycloalkyl; M and M' are independently selected
from --C(O)O--, --OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, --S--S--
an aryl group, and a heteroaryl group; and R.sub.2 and R.sub.3 are
independently selected from the group consisting of H, C.sub.1-14
alkyl, and C.sub.2-14 alkenyl.
31. The pharmaceutical composition of claim 29, wherein m is 5, 7,
or 9.
32. The pharmaceutical composition of claim 29, wherein the
compound is of Formula (II) ##STR00181## or a salt or stereoisomer
thereof, wherein l is selected from 1, 2, 3, 4, and 5; M.sub.1 is a
bond or M'; R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 2, 3, or 4, and Q is OH,
--NHC(S)N(R).sub.2, --NHC(O)N(R).sub.2, --N(R)C(O)R,
--N(R)S(O).sub.2R, --N(R)R.sub.8, --NHC(.dbd.NR.sub.9)N(R).sub.2,
--NHC(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
heteroaryl, or heterocycloalkyl; M and M' are independently
selected from --C(O)O--, --OC(O)--, --C(O)N(R')--, --P(O)(OR')O--,
--S--S--, an aryl group, and a heteroaryl group; and R.sub.2 and
R.sub.3 are independently selected from the group consisting of H,
C.sub.1-14 alkyl, and C.sub.2-14 alkenyl.
33. The pharmaceutical composition of claim 30, wherein M.sub.1 is
M'.
34. The pharmaceutical composition of claim 33, wherein M and M'
are independently --C(O)O-- or --OC(O)--.
35. The pharmaceutical composition of claim 30, wherein l is 1, 3,
or 5.
36. The pharmaceutical composition of claim 29, wherein the
compound is selected from the group consisting of Compound 1 to
Compound 232, salts and stereoisomers thereof, and any combination
thereof.
37. (canceled)
38. The pharmaceutical composition of claim 29, wherein the
compound is Compound 18, a salt or a stereoisomer thereof, or any
combination thereof.
39.-143. (canceled)
144. A method of expressing a porphobilinogen deaminase (PBGD)
polypeptide in a human subject in need thereof comprising
administering to the subject an effective amount of the
pharmaceutical composition of claim 29, wherein the pharmaceutical
composition is suitable for administrating as a single dose or as a
plurality of single unit doses to the subject.
145. A method of treating, preventing or delaying the onset of
acute intermittent porphyria (AIP) signs or symptoms in a human
subject in need thereof comprising administering to the subject an
effective amount of the pharmaceutical composition of claim 29,
wherein the administration treats, prevents or delays the onset of
one or more of the signs or symptoms of AIP in the subject.
146. (canceled)
147. A method of reducing an aminolevulinate acid (ALA), a
porphobilinogen (PBG) and/or a porphyrin urinary excretion level in
a human subject comprising administering to the subject an
effective amount of the pharmaceutical composition of claim 29,
wherein the administration reduces the ALA, PBG and/or porphyrin
urinary excretion level in the subject.
148. The method of claim 147, wherein (i) the ALA urinary
excretions level is reduced by at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, at least 98%, at
least 99%, or at least 100% as compared to the subject's baseline
ALA excretion level or a reference ALA excretion level during an
acute porphyria attack, for at least 24 hours, at least 48 hours,
at least 72 hours, at least 96 hours, or at least 120 hours
post-administration, (ii) the PBG urinary excretions level is
reduced by at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 95%, at least 98%, at least 99%, or at least
100% as compared to the subject's PBG excretion baseline level or a
reference PBG excretion level during an acute porphyria attack, for
at least 24 hours, at least 48 hours, at least 72 hours, at least
96 hours, or at least 120 hours post-administration, and/or (iii)
the porphyrin urinary excretions level is reduced by at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, at least 99%, or at least 100% as compared to
the subject's baseline porphyrin excretion level or a reference
porphyrin excretion level during an acute porphyria attack, for at
least 24 hours, at least 48 hours, at least 72 hours, at least 96
hours, or at least 120 hours post-administration.
149. A method of reducing an alanine transaminase (ALT), a
aspartate transaminase (AST) and/or a bilirubin serum level in a
human subject comprising administering to the subject an effective
amount of the pharmaceutical composition of claim 29, wherein the
administration reduces the ALT, AST and/or bilirubin serum level in
the subject.
150. The method of claim 149, wherein (i) the ALT serum level is
reduced by at least 90%, at least 80%, at least 70%, at least 60%,
at least 50%, at least 40%, or at least 30% as compared to the
subject's baseline ALT serum level or a reference ALT serum level
within at least 24 hours, at least 48 hours, at least 72 hours, at
least 96 hours, or at least 120 hours post-administration, (ii) the
AST serum level is reduced by at least 90%, at least 80%, at least
70%, at least 60%, at least 50%, at least 40%, or at least 30% as
compared to the subject's baseline AST serum level or a reference
AST serum level, for at least 24 hours, at least 48 hours, at least
72 hours, at least 96 hours, or at least 120 hours
post-administration, and/or (iii) the bilirubin serum level is
reduced by at least 90%, at least 80%, at least 70%, at least 60%,
at least 50%, at least 40%, or at least 30% as compared to the
subject's baseline bilirubin serum level or a reference bilirubin
serum level, for at least 24 hours, at least 48 hours, at least 72
hours, at least 96 hours, or at least 120 hours
post-administration.
151. The method of claim 145, wherein 12 hours after the
pharmaceutical composition or polynucleotide is administered to the
subject, the PBGD activity in the subject is increased at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 100%, at least
150%, at least 200%, at least 300%, at least 400%, at least 500%,
or at least 600% compared to the subject's baseline PBGD
activity.
152.-154. (canceled)
155. The method of claim 145, wherein 24 hours after the
pharmaceutical composition or polynucleotide is administered to the
subject: (a) the level of ALA in the subject is reduced by at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, or 100% compared to the subject's baseline ALA;
(b) the level of PBG in the subject is reduced by at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, or 100% compared to the subject's baseline PBG; (c) the
level of porphyrin in the subject is reduced by at least about 20%,
at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, or 100% compared to the subject's baseline porphyrin;
(d) the level of ALT in the subject is reduced by at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, or 100% compared to the subject's baseline ALT; (e) the
level of AST in the subject is reduced by at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, or 100% compared to the subject's baseline AST; or (f) the
level of AST in the subject is reduced by at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, or 100% compared to the subject's baseline AST.
156.-178. (canceled)
179. The pharmaceutical composition of claim 29, wherein the lipid
nanoparticle comprises from about 45 mol % to about 55 mol % of
ionizable lipid.
180. The pharmaceutical composition of claim 29, wherein the lipid
nanoparticle comprises from about 35 mol % to about 40 mol % of
structural lipid.
181. The pharmaceutical composition of claim 29, wherein the lipid
nanoparticle comprises from amount 2 mol % to about 5 mol % PEG
lipid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is the National Stage of International
Application No. PCT/US2017/033418, filed on May 18, 2017, which
claims the priority benefit of U.S. Provisional Application No.
62/338,161, filed May 18, 2016 and EP Application No. EP17382259.4,
filed May 9, 2017, each of which is hereby incorporated by
reference herein in its entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The content of the electronically submitted sequence listing
(Name: 3529.069PC02 Patent-In_ST25.txt, Size: 222,958 bytes; and
Date of Creation: May 16, 2017) is herein incorporated by reference
in its entirety.
BACKGROUND
[0003] Acute intermittent porphyria (AIP) is an autosomal dominant
metabolic disorder associated with impaired production of heme, the
oxygen-binding prosthetic group of hemoglobin. The causative gene
for AIP is porphobilinogen deaminase (PBGD) (NM_000190; NP_000181;
also referred to as hydroxymethylbilane synthase (HMBS or
HM-synthase) and uroporphyrinogen I synthase). Song G et al., FASEB
J. 23: 396-404 (2009). PBGD (E.C. 2.5.1.61) is one of the eight
enzymes involved in the porphyrin-heme biosynthetic pathway. PBGD's
biological function is to catalyze the head to tail condensation of
four porphobilinogen molecules into the linear hydroxymethylbilane.
There are two primary isoforms of PBGD: the 44-kDa housekeeping
enzyme (isoform 1), which is expressed in all tissues, and the
42-kDa erythrocyte-specific enzyme (isoform 2). The ubiquitous PBGD
isoform 1 is 361 amino acid residues, while the
erythrocyte-specific variant (PBGD isoform 2) is 344 amino acids.
Id.
[0004] Mutations within the PBGD gene can result in the complete or
partial loss of PBGD function, resulting in impaired heme
production and the abnormal accumulation of aminolevulinic acid
(ALA) and porphobilinogen (PBG) in cytoplasm of cells, plasma and
urine. Id.
[0005] While signs and symptoms can be variable, patients suffering
from AIP often exhibit neurological (e.g., agitation, delirium,
seizures, loss of motor function, respiratory paralysis) and
gastrointestinal (e.g., extreme abdominal pain, vomiting, painful
urination) issues. These signs and symptoms can be triggered by
various factors (e.g., drugs, hormones, and alcohol) and usually
occur as episodes or attacks that develop over course of several
hours or few days. Hrdinka M et al., Physiol Res. 2: S119-36
(2006). In between these episodes or attacks, AIP patients can
otherwise appear healthy. However, if left untreated, AIP can
potentially cause life-threatening complications, including death.
AIP has an estimate prevalence of about 5.9 per million people
worldwide (Elder G et al., J. Inherit. Metab. Dis. 36:849-57
(2012)). While AIP patients from all ethnic groups have been
reported, the disorder is much more prevalent in both the Dutch and
Swedish populations (1 in 10,000 to 8 in 10,000). Tjensvoll K et
al., Dis Markers. 19: 41-6 (2003-2004).
[0006] Historic treatment for AIP was primarily via lifestyle
modification (e.g., avoiding alcohol, smoking and known
porphyrogenic drugs and diet) and management of individual sign and
symptoms. Badminton M N et al., Int J Clin Pract. 56: 272-8 (2002).
In extreme cases, regular hematin infusions and/or liver
transplantation had been recommended. Seth A K et al., Liver
Transpl. 13: 1219-27 (2007).
[0007] Today, the current Standard of Care (SOC) for AIP is
Panhematin therapy, also known as "hemin" therapy. The SOC therapy
is based on a down-regulation of hepatic heme synthesis using heme
administration. Notably however, heme therapy is indicated only if
an acute attack of porphyria is proven by a marked increase in
urine PBG. Moreover, there remain several unmet medical needs
including ineffectiveness in chronic AIP, and short-acting efficacy
(lasts only 1-2 days). Moreover, recurrent hyper-activation of the
hepatic heme synthesis pathway can be associated with neurological
and metabolic manifestations and long-term complications including
chronic kidney disease and increased risk of hepatocellular
carcinoma in certain AIP patients. Prophylactic heme infusion can
be an effective strategy in some of these patients, but it induces
tolerance and its frequent application may be associated with
thromboembolic disease and hepatic siderosis.
[0008] Emerging therapies including enzyme replacement therapy
(ERT) or gene therapy (e.g., HMBS-gene transfer), as well as a
potential therapy based on ALASI-gene expression inhibition, are
being developed. Zymenex (also known as "Porphozym"), a recombinant
human PBGD, failed in Phase 3 clinical trials in 2009. While the
therapy had an excellent safety profile, the treatment failed to
sufficiently reduce the levels of the surrogate markers of PBGD
activity (i.e., PBG and ALA). In the trial, PBG declined, but ALA
did not. Moreover, the PBG enzyme wasn't targeted to hepatocytes.
In a Phase I open label liver-directed gene therapy clinical trial
for acute intermittent porphyria, D'Avola and colleagues reported
the results of the first gene therapy trial for AIP and the
first-in-human use of AAV5 in which they showed evidence of safety,
although there was no clear efficacy signal. It is possible that
the biology of the disease and the lack of reliable read-outs might
account for the lack of efficacy. To date, orthotopic liver
transplantation is the only curative treatment in patients with
recurrent acute attacks.
[0009] Thus, with the exception of liver transplants, most
treatments often fail to completely and reliably treat the
disorder. Therefore, there is an ongoing need for improved
therapeutics to treat AIP.
BRIEF SUMMARY
[0010] The present invention provides mRNA therapeutics for the
treatment of acute intermittent porphyria (AIP). The mRNA
therapeutics of the invention are particularly well-suited for the
treatment of AIP as the technology provides for the intracellular
delivery of mRNA encoding PBGD followed by de novo synthesis of
functional PBGD protein within target cells. The instant invention
features the incorporation of modified nucleotides within
therapeutic mRNAs to (1) minimize unwanted immune activation (e.g.,
the innate immune response associated with the in vivo introduction
of foreign nucleic acids) and (2) optimize the translation
efficiency of mRNA to protein. Exemplary aspects of the invention
feature a combination of nucleotide modifications to reduce the
innate immune response and sequence optimization, in particular,
within the open reading frame (ORF) of therapeutic mRNAs encoding
PBGD to enhance protein expression.
[0011] In further embodiments, the mRNA therapeutic technology of
the instant invention also features delivery of mRNA encoding PBGD
via a lipid nanoparticle (LNP) delivery system. The instant
invention features novel ionizable lipid-based LNPs which have
improved properties when combined with mRNA encoding PBGD and
administered in vivo, for example, cellular uptake, intracellular
transport and/or endosomal release or endosomal escape. The LNP
formulations of the invention also demonstrate reduced
immunogenicity associated with the in vivo administration of
LNPs.
[0012] In certain aspects, the invention relates to compositions
and delivery formulations comprising a polynucleotide, e.g., a
ribonucleic acid (RNA), e.g., a messenger RNA (mRNA), encoding
porphobilinogen deaminase and methods for treating acute
intermittent porphyria (AIP) in a subject in need thereof by
administering the same.
[0013] The present disclosure provides a pharmaceutical composition
comprising a lipid nanoparticle encapsulated mRNA that comprises an
open reading frame (ORF) encoding an porphobilinogen deaminase
(PBGD) polypeptide, wherein the composition is suitable for
administration to a human subject in need of treatment for acute
intermittent porphyria (AIP).
[0014] The present disclosure further provides a pharmaceutical
composition comprising: (a) a mRNA that comprises (i) an open
reading frame (ORF) encoding an porphobilinogen deaminase (PBGD)
polypeptide, wherein the ORF comprises at least one chemically
modified nucleobase, sugar, backbone, or any combination thereof,
(ii) an untranslated region (UTR) comprising a microRNA (miRNA)
binding site; and (b) a delivery agent, wherein the pharmaceutical
composition is suitable for administration to a human subject in
need of treatment for acute intermittent porphyria (AIP).
[0015] The present disclosure further provides a pharmaceutical
composition comprising an mRNA comprising an open reading frame
(ORF) encoding a human porphobilinogen deaminase (PBGD)
polypeptide, wherein the composition when administered to a subject
in need thereof as a single intravenous dose is sufficient to
reduce urinary excretion of: (i) aminolevulinate acid (ALA) at
least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold
or at least 50-fold as compared to a reference ALA excretion level
(e.g., during an acute porphyria attack), for at least 24 hours, at
least 48 hours, at least 72 hours, at least 96 hours, or at least
120 hours post-administration, (ii) porphobilinogen (PBG) at least
2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at
least 50-fold as compared to a reference PBG excretion level (e.g.,
during an acute porphyria attack), for at least 24 hours, at least
48 hours, at least 72 hours, at least 96 hours, or at least 120
hours post-administration, and/or (iii) porphyrin at least at least
2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at
least 50-fold as compared to a reference porphyrin excretion level
(e.g., during an acute porphyria attack), for at least 24 hours, at
least 48 hours, at least 72 hours, at least 96 hours, or at least
120 hours post-administration.
[0016] The present disclosure further provides a pharmaceutical
composition comprising an mRNA comprising an open reading frame
(ORF) encoding a human porphobilinogen deaminase (PBGD)
polypeptide, wherein the composition when administered to a subject
in need thereof as a single intravenous dose is sufficient to
reduce urinary excretion of: (i) aminolevulinate acid (ALA) by at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95% or at least 98%, at least 99%, or 100% as compared to
the subject's baseline level or a reference ALA excretion level
(e.g., in a subject with AIP or during an acute porphyria attack),
for at least 24 hours, at least 48 hours, at least 72 hours, at
least 96 hours, or at least 120 hours post-administration, (ii)
porphobilinogen (PBG) by at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95% or at least 98%, at least
99%, or 100% as compared to the subject's baseline level or a
reference PBG excretion level (e.g., in a subject with AIP or
during an acute porphyria attack), for at least 24 hours, at least
48 hours, at least 72 hours, at least 96 hours, or at least 120
hours post-administration, and/or (iii) porphyrin by at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95% or at least 98%, at least 99%, or 100% as compared to the
subject's baseline level or a reference porphyrin excretion level
(e.g., in a subject with AIP or during an acute porphyria attack),
for at least 24 hours, at least 48 hours, at least 72 hours, at
least 96 hours, or at least 120 hours post-administration.
[0017] The present disclosure further provides a pharmaceutical
composition comprising an mRNA comprising an open reading frame
(ORF) encoding a human porphobilinogen deaminase (PBGD)
polypeptide, wherein the composition when administered to a subject
in need thereof as a single intravenous dose is sufficient to
reduce serum levels of: (i) alanine transaminase (ALT) to at least
within 10-fold, at least within 5-fold, at least within 2-fold, or
at least within 1.5-fold or to within at least 50%, at least 40%,
at least 30%, at least 20%, or at least 10% of a reference ALT
serum level within at least 24 hours, at least 48 hours, at least
72 hours, at least 96 hours, or at least 120 hours
post-administration, (ii) aspartate transaminase (AST) to at least
within 10-fold, at least within 5-fold, at least within 2-fold, or
at least within 1.5-fold or within at least 50%, at least 40%, at
least 30%, at least 20%, or at least 10% of a reference AST serum
level, for at least 24 hours, at least 48 hours, at least 72 hours,
at least 96 hours, or at least 120 hours post-administration,
and/or (iii) bilirubin to at least within 10-fold, at least within
5-fold, at least within 2-fold, or at least within 1.5 fold or
within at least 50%, at least 40%, at least 30%, at least 20%, or
at least 10% of a reference bilirubin serum level, for at least 24
hours, at least 48 hours, at least 72 hours, at least 96 hours, or
at least 120 hours post-administration.
[0018] The present disclosure further provides a pharmaceutical
composition comprising an mRNA comprising an open reading frame
(ORF) encoding a human porphobilinogen deaminase (PBGD)
polypeptide, wherein the composition when administered to a subject
in need thereof as a single intravenous dose is sufficient to
reduce serum levels of: (i) alanine transaminase (ALT) by at least
90%, at least 80%, at least 70%, at least 60%, at least 50%, at
least 40%, or at least 30% as compared to the subject's baseline
level or a reference ALT serum level (e.g., in a subject with AIP
or during an acute porphyria attack), within at least 24 hours, at
least 48 hours, at least 72 hours, at least 96 hours, or at least
120 hours post-administration, (ii) aspartate transaminase (AST) by
at least 90%, at least 80%, at least 70%, at least 60%, at least
50%, at least 40%, or at least 30% as compared to the subject's
baseline level or a reference AST serum level (e.g., in a subject
with AIP or during an acute porphyria attack), for at least 24
hours, at least 48 hours, at least 72 hours, at least 96 hours, or
at least 120 hours post-administration, and/or (iii) bilirubin by
at least 90%, at least 80%, at least 70%, at least 60%, at least
50%, at least 40%, or at least 30% as compared to the subject's
baseline level or a reference bilirubin serum level (e.g., in a
subject with AIP or during an acute porphyria attack), for at least
24 hours, at least 48 hours, at least 72 hours, at least 96 hours,
or at least 120 hours post-administration.
[0019] The present disclosure further provides a pharmaceutical
composition comprising an mRNA comprising an open reading frame
(ORF) encoding a human porphobilinogen deaminase (PBGD)
polypeptide, wherein the composition when administered to a subject
in need thereof as a single intravenous dose is sufficient to: (i)
maintain hepatic PBGD activity levels at or above a reference
physiological level or at a supraphysiological level for at least
24 hours, at least 48 hours, at least 72 hours, at least 96 hours,
or at least 120 hours post-administration, and/or (ii) maintain
hepatic PBGD activity levels at 50% or more of a reference hepatic
PBGD activity level for at least 24 hours, at least 48 hours, at
least 72 hours, or at least 96 hours post-administration.
[0020] In some embodiments, the pharmaceutical compositions
disclosed herein further comprise a delivery agent.
[0021] The present disclosure provides a polynucleotide comprising
an open reading frame (ORF) encoding a porphobilinogen deaminase
(PBGD) polypeptide, wherein the uracil or thymine content of the
ORF relative to the theoretical minimum uracil or thymine content
of a nucleotide sequence encoding the PBGD polypeptide (% U.sub.TM
or % T.sub.TM), is between about 100% and about 150%. In some
embodiments, the % U.sub.TM or % T.sub.TM is between about 105% and
about 145%, between about 105% and about 140%, between about 110%
and about 140%, between about 110% and about 145%, between about
115% and about 135%, between about 105% and about 135%, between
about 110% and about 135%, between about 115% and about 145%, or
between about 115% and about 140%. In some embodiments, the uracil
or thymine content of the ORF relative to the uracil or thymine
content of the corresponding wild-type ORF (% U.sub.WT or %
T.sub.WT) is less than 100%. In some embodiments, the % U.sub.WT or
% T.sub.WT is less than about 95%, less than about 90%, less than
about 85%, less than 80%, less than 79%, less than 78%, less than
77%, less than 76%, less than 75%, less than 74%, or less than 73%.
In some embodiments, the % U.sub.WT or % T.sub.WT is between 65%
and 73%. In some embodiments, the uracil or thymine content in the
ORF relative to the total nucleotide content in the ORF (% U.sub.TL
or % TTL) is less than about 50%, less than about 40%, less than
about 30%, or less than about 19%. In some embodiments, the %
U.sub.TL or % T.sub.TL is less than about 19%. In some embodiments,
the % U.sub.TL or % T.sub.TL is between about 13% and about 15%. In
some embodiments, the guanine content of the ORF with respect to
the theoretical maximum guanine content of a nucleotide sequence
encoding the PBGD polypeptide (% G.sub.TMX) is at least 69%, at
least 70%, at least 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, or about 100%. In some
embodiments, the % G.sub.TMX is between about 70% and about 80%,
between about 71% and about 79%, between about 71% and about 78%,
or between about 71% and about 77%.
[0022] In some embodiments, the cytosine content of the ORF
relative to the theoretical maximum cytosine content of a
nucleotide sequence encoding the PBGD polypeptide (% C.sub.TMX) is
at least 59%, at least 60%, at least about 65%, at least about 70%,
at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, or about 100%. In some
embodiments, the % C.sub.TMX is between about 60% and about 80%,
between about 62% and about 80%, between about 63% and about 79%,
or between about 68% and about 76%. In some embodiments, the
guanine and cytosine content (G/C) of the ORF relative to the
theoretical maximum G/C content in a nucleotide sequence encoding
the PBGD polypeptide (% G/C.sub.TMX) is at least about 81%, at
least about 85%, at least about 90%, at least about 95%, or about
100%. In some embodiments, the % G/C.sub.TMX is between about 80%
and about 100%, between about 85% and about 99%, between about 90%
and about 97%, or between about 91% and about 96%. In some
embodiments, the G/C content in the ORF relative to the G/C content
in the corresponding wild-type ORF (% G/C.sub.WT) is at least 102%,
at least 103%, at least 104%, at least 105%, at least 106%, at
least 107%, at least 110%, at least 115%, or at least 120%. In some
embodiments, the average G/C content in the 3.sup.rd codon position
in the ORF is at least 20%, at least 21%, at least 22%, at least
23%, at least 24%, at least 25%, at least 26%, at least 27%, at
least 28%, at least 29%, or at least 30% higher than the average
G/C content in the 3.sup.rd codon position in the corresponding
wild-type ORF.
[0023] In some embodiments, the ORF has at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100% sequence identity to a sequence
selected from the group consisting of SEQ ID NOs: 9 to 33, and 89
to 117. In some embodiments, the ORF has at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% sequence identity to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 9 to 33, and 89 to 117. In some
embodiments, the ORF has at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% sequence
identity to the nucleic acid sequence of SEQ ID NO: 104, 112, or
114. In some embodiments, the ORF has at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100% sequence identity
to the nucleic acid sequence of SEQ ID NO: 104, 112, or 114. In
some embodiments, the ORF comprises the nucleic acid sequence of
SEQ ID NO: 104, 112, or 114.
[0024] In some embodiments, the PBGD polypeptide comprises an amino
acid sequence at least about 95%, at least about 96%, at least
about 97%, at least about 98%, at least about 99%, or about 100%
identical to (i) the polypeptide sequence of wild type PBGD,
isoform 1 (SEQ ID NO: 1), (ii) the polypeptide sequence of wild
type PBGD, isoform 2 (SEQ ID NO: 3), the polypeptide sequence of
wild type PBGD, isoform 3 (SEQ ID NO: 5), or the polypeptide
sequence of wild type PBGD, isoform 4 (SEQ ID NO: 7), and wherein
the PBGD polypeptide has porphobilinogen deaminase activity. In
some embodiments, the PBGD polypeptide is a variant, derivative, or
mutant having a porphobilinogen deaminase activity (e.g., the SM
gain of function variant, SEQ ID NO: 152). In some embodiments, the
gain-of-function mutant PBGD comprises an 1291M mutation, an N340S
mutation, or a combination thereof. In some embodiments, the
gain-of-function mutant PBGD comprises the polypeptide sequence of
SEQ ID NO: 152. In some embodiments, the PBGD polypeptide is a PBGD
fusion protein. In some embodiments, the PBGD fusion protein
comprises heterologous protein moiety. In some embodiments, the
heterologous protein moiety is an apolipoprotein. In some
embodiments, the apolipoprotein is human apolipoprotein A1. In some
embodiments, the human apolipoprotein A1 is mature human
apolipoprotein A1. In some embodiments, a fusion protein comprising
PBGD and mature human apolipoprotein A1 comprises the polypeptide
sequence of SEQ ID NO: 154.
[0025] In some embodiments, the polynucleotide sequence further
comprises a nucleotide sequence encoding a transit peptide.
[0026] In some embodiments, the polynucleotide is single stranded.
In some embodiments, the polynucleotide is double stranded. In some
embodiments, the polynucleotide is DNA. In some embodiments, the
polynucleotide is RNA. In some embodiments, the polynucleotide is
mRNA. In some embodiments, the polynucleotide comprises at least
one chemically modified nucleobase, sugar, backbone, or any
combination thereof. In some embodiments, the at least one
chemically modified nucleobase is selected from the group
consisting of pseudouracil (.psi.), N1-methylpseudouracil
(m1.psi.), 2-thiouracil (s2U), 4'-thiouracil, 5-methylcytosine,
5-methyluracil, and any combination thereof. In some embodiments,
the at least one chemically modified nucleobase is selected from
the group consisting of pseudouracil (W), N1-methylpseudouracil
(m1.psi.), 1-ethylpseudouracil, 2-thiouracil (s2U), 4'-thiouracil,
5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any
combination thereof. In some embodiments, the at least one
chemically modified nucleobase is 5-methoxyuracil. In some
embodiments, at least about 25%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 95%, at least
about 99%, or 100% of the uracils are 5-methoxyuracils. In some
embodiments, at least about 25%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 95%, at least
about 99%, or 100% of the uracils or thymines are chemically
modified. In some embodiments, at least about 25%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 95%, at least about 99%, or 100% of the guanines are
chemically modified. In some embodiments, at least about 25%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about 99%, or 100% of the
cytosines are chemically modified. In some embodiments, at least
about 25%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 99%, or 100% of
the adenines are chemically modified.
[0027] In some embodiments, the polynucleotide further comprises a
miRNA binding site.
[0028] In some embodiments, the polynucleotide comprises at least
two different microRNA (miR) binding sites.
[0029] In some embodiments, the microRNA is expressed in an immune
cell of hematopoietic lineage or a cell that expresses TLR7 and/or
TLR8 and secretes pro-inflammatory cytokines and/or chemokines, and
wherein the polynucleotide (e.g., mRNA) comprises one or more
modified nucleobases.
[0030] In some embodiments, the mRNA comprises at least one first
microRNA binding site of a microRNA abundant in an immune cell of
hematopoietic lineage and at least one second microRNA binding site
is of a microRNA abundant in endothelial cells.
[0031] In some embodiments, the mRNA comprises multiple copies of a
first microRNA binding site and at least one copy of a second
microRNA binding site.
[0032] In some embodiments, the mRNA comprises first and second
microRNA binding sites of the same microRNA.
[0033] In some embodiments, the microRNA binding sites are of the
3p and 5p arms of the same microRNA.
[0034] In some embodiments, the microRNA binding site comprises one
or more nucleotide sequences selected from Table 3 or Table 4.
[0035] In some embodiments, the microRNA binding site binds to
miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16,
miR-21, miR-223, miR-24, miR-27 or miR-26a, or any combination
thereof.
[0036] In some embodiments, the microRNA binding site binds to
miR126-3p, miR-142-3p, miR-142-5p, or miR-155, or any combination
thereof.
[0037] In some embodiments, the microRNA binding site is a miR-126
binding site. In some embodiments, at least one microRNA binding
site is a miR-142 binding site. In some embodiments, one microRNA
binding site is a miR-126 binding site and the second microRNA
binding site is for a microRNA selected from the group consisting
of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16,
miR-21, miR-223, miR-24 and miR-27.
[0038] In some embodiments, the mRNA comprises at least one
miR-126-3p binding site and at least one miR-142-3p binding site.
In some embodiments, the mRNA comprises at least one miR-142-3p
binding site and at least one 142-5p binding site.
[0039] In some embodiments, the microRNA binding sites are located
in the 5' UTR, 3' UTR, or both the 5' UTR and 3' UTR of the mRNA.
In some embodiments, the microRNA binding sites are located in the
3' UTR of the mRNA. In some embodiments, the microRNA binding sites
are located in the 5' UTR of the mRNA. In some embodiments, the
microRNA binding sites are located in both the 5' UTR and 3' UTR of
the mRNA. In some embodiments, at least one microRNA binding site
is located in the 3' UTR immediately adjacent to the stop codon of
the coding region of the mRNA. In some embodiments, at least one
microRNA binding site is located in the 3' UTR 70-80 bases
downstream of the stop codon of the coding region of the mRNA. In
some embodiments, at least one microRNA binding site is located in
the 5' UTR immediately preceding the start codon of the coding
region of the mRNA. In some embodiments, at least one microRNA
binding site is located in the 5' UTR 15-20 nucleotides preceding
the start codon of the coding region of the mRNA. In some
embodiments, at least one microRNA binding site is located in the
5' UTR 70-80 nucleotides preceding the start codon of the coding
region of the mRNA.
[0040] In some embodiments, the mRNA comprises multiple copies of
the same microRNA binding site positioned immediately adjacent to
each other or with a spacer of less than 5, 5-10, 10-15, or 15-20
nucleotides.
[0041] In some embodiments, the mRNA comprises multiple copies of
the same microRNA binding site located in the 3' UTR, wherein the
first microRNA binding site is positioned immediately adjacent to
the stop codon and the second and third microRNA binding sites are
positioned 30-40 bases downstream of the 3' most residue of the
first microRNA binding site.
[0042] In some embodiments, the microRNA binding site comprises one
or more nucleotide sequences selected from SEQ ID NO:36 and SEQ ID
NO:38. In some embodiments, the miRNA binding site binds to
miR-142. In some embodiments, the miRNA binding site binds to
miR-142-3p or miR-142-5p. In some embodiments, the miR-142
comprises SEQ ID NO: 34.
[0043] In some embodiments, the microRNA binding site comprises one
or more nucleotide sequences selected from SEQ ID NO:158 and SEQ ID
NO:160. In some embodiments, the miRNA binding site binds to
miR-126. In some embodiments, the miRNA binding site binds to
miR-126-3p or miR-126-5p. In some embodiments, the miR-126
comprises SEQ ID NO: 156.
[0044] In some embodiments, the mRNA comprises a 3' UTR comprising
a microRNA binding site that binds to miR-142, miR-126, or a
combination thereof.
[0045] In some embodiments, the polynucleotide, e.g., mRNA, further
comprises a 3' UTR. In some embodiments, the 3' UTR comprises a
nucleic acid sequence at least about 90%, at least about 95%, at
least about 96%, at least about 97%, at least about 98%, at least
about 99%, or 100% identical to a 3'UTR sequence selected from the
group consisting of SEQ ID NOs: 57 to 81, 84, 149 to 151, 161 to
172, 192 to 199, or any combination thereof. In some embodiments,
the miRNA binding site is located within the 3' UTR.
[0046] In some embodiments, the 3' UTR comprises a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 57 to
81, 84, 149 to 151, 161 to 172, 192 to 199, and any combination
thereof. In some embodiments, the mRNA comprises a 3' UTR
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 149 to 151, or any combination thereof.
In some embodiments, the mRNA comprises a 3' UTR comprising a
nucleic acid sequence of SEQ ID NO: 150. In some embodiments, the
mRNA comprises a 3' UTR comprising a nucleic acid sequence of SEQ
ID NO: 151.
[0047] In some embodiments, the polynucleotide, e.g., mRNA, further
comprises a 5' UTR. In some embodiments, the 5' UTR comprises a
nucleic acid sequence at least 90%, at least about 95%, at least
about 96%, at least about 97%, at least about 98%, at least about
99%, or about 100% identical to a 5'UTR sequence selected from the
group consisting of SEQ ID NO: 39 to 56, 83, 189 to 191, or any
combination thereof. In some embodiments, the 5' UTR comprises a
sequence selected from the group consisting of SEQ ID NO: 39 to 56,
83, 189 to 191, and any combination thereof. In some embodiments,
the mRNA comprises a 5' UTR comprising the nucleic acid sequence of
SEQ ID NO: 39.
[0048] In some embodiments, the polynucleotide, e.g., mRNA, further
comprises a 5' terminal cap. In some embodiments, the 5' terminal
cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,
2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,
2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5'
methylG cap, or an analog thereof. In some embodiments, the 5'
terminal cap comprises a Cap1.
[0049] In some embodiments, the polynucleotide, e.g., mRNA, further
comprises a poly-A region. In some embodiments, the poly-A region
is at least about 10, at least about 20, at least about 30, at
least about 40, at least about 50, at least about 60, at least
about 70, at least about 80, or at least about 90 nucleotides in
length. In some embodiments, the poly-A region has about 10 to
about 200, about 20 to about 180, about 50 to about 160, about 70
to about 140, about 80 to about 120 nucleotides in length.
[0050] In some embodiments, the polynucleotide, e.g., mRNA, encodes
a PBGD polypeptide that is fused to one or more heterologous
polypeptides. In some embodiments, the one or more heterologous
polypeptides increase a pharmacokinetic property of the PBGD
polypeptide. In some embodiments, upon administration to a subject,
the polynucleotide has (i) a longer plasma half-life; (ii)
increased expression of a PBGD polypeptide encoded by the ORF;
(iii) a lower frequency of arrested translation resulting in an
expression fragment; (iv) greater structural stability; or (v) any
combination thereof, relative to a corresponding polynucleotide
comprising SEQ ID NO: 2, 4, 6, 8, or 153. In some embodiments, the
polynucleotide encodes a PBGD polypeptide that is fused to human
apolipoprotein A1 (e.g., SEQ ID NO: 155).
[0051] In some embodiments, the polynucleotide, e.g., mRNA,
comprises (i) a 5'-terminal cap; (ii) a 5'-UTR; (iii) an ORF
encoding a PBGD polypeptide; (iv) a 3'-UTR; and (v) a poly-A
region. In some embodiments, the 3'-UTR comprises a miRNA binding
site. In some embodiments, the polynucleotide comprises a nucleic
acid sequence selected from the group consisting of SEQ ID NO:
118-148, for example, SEQ ID NO: 133, 141, 144, or 145. In some
embodiments the polynucleotide further comprises a 5'-terminal cap
(e.g., Cap1) and a poly-A-tail region (e.g., about 100 nucleotides
in length).
[0052] The present disclosure also provides a method of producing a
polynucleotide, e.g., mRNA, of the present invention, the method
comprising modifying an ORF encoding a PBGD polypeptide by
substituting at least one uracil nucleobase with an adenine,
guanine, or cytosine nucleobase, or by substituting at least one
adenine, guanine, or cytosine nucleobase with a uracil nucleobase,
wherein all the substitutions are synonymous substitutions. In some
embodiments, the method further comprises replacing at least about
90%, at least about 95%, at least about 99%, or about 100% of
uracils with 5-methoxyuracils.
[0053] The present disclosure also provides a composition
comprising (a) a polynucleotide, e.g., mRNA, of the invention; and
(b) a delivery agent. In some embodiments, the delivery agent
comprises a lipidoid, a liposome, a lipoplex, a lipid nanoparticle,
a polymeric compound, a peptide, a protein, a cell, a nanoparticle
mimic, a nanotube, or a conjugate. In some embodiments, the
delivery agent comprises a lipid nanoparticle. In some embodiments,
the lipid nanoparticle comprises a lipid selected from the group
consisting of
3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine
(KL10),
N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanami-
ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane
(KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),
heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate
(DLin-MC3-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),
(13Z,165Z)--N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608),
2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-
-octadeca-9,12-di en-1-yloxy]propan-1-amine (Octyl-CLinDMA),
(2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-
,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA
(2R)),
(2S)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-
,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA
(2S)), and any combinations thereof. In some embodiments, the lipid
nanoparticle comprises DLin-MC3-DMA.
[0054] In some embodiments, the delivery agent comprises a compound
having the Formula (I)
##STR00001##
or a salt or stereoisomer thereof, wherein
[0055] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0056] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0057] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR,
[0058] --CHQR, --CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl,
where Q is selected from a carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --N(R)R.sub.8, --O(CH.sub.2).sub.nOR,
--N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and --C(R)N(R).sub.2C(O)OR, and
each n is independently selected from 1, 2, 3, 4, and 5;
[0059] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0060] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0061] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0062] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0063] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0064] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0065] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0066] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0067] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0068] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0069] each Y is independently a C.sub.3-6 carbocycle;
[0070] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0071] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
and
[0072] provided when R.sub.4 is --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, or --CQ(R).sub.2, then (i) Q is not
--N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or
7-membered heterocycloalkyl when n is 1 or 2.
[0073] The present disclosure also provides a composition
comprising a nucleotide sequence encoding a PBGD polypeptide and a
delivery agent, wherein the delivery agent comprises a compound
having the Formula (I)
##STR00002##
[0074] or a salt or stereoisomer thereof, wherein
[0075] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0076] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0077] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --N(R)R.sub.8, --O(CH.sub.2).sub.nOR,
--N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and --C(R)N(R).sub.2C(O)OR, and
each n is independently selected from 1, 2, 3, 4, and 5;
[0078] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0079] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0080] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0081] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0082] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0083] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0084] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0085] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0086] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0087] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0088] each Y is independently a C.sub.3-6 carbocycle;
[0089] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0090] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
and
[0091] provided when R.sub.4 is --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, or --CQ(R).sub.2, then (i) Q is not
--N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or
7-membered heterocycloalkyl when n is 1 or 2.
[0092] In some embodiments, the delivery agent comprises a compound
having the Formula (I), or a salt or stereoisomer thereof,
wherein
[0093] R.sub.1 is selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0094] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0095] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR,
[0096] --CHQR, --CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl,
where Q is selected from a carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, and --C(R)N(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5;
[0097] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0098] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0099] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[0100] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0101] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0102] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0103] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0104] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0105] each Y is independently a C.sub.3-6 carbocycle;
[0106] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0107] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
and
[0108] provided when R.sub.4 is --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, or --CQ(R).sub.2, then (i) Q is not
--N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or
7-membered heterocycloalkyl when n is 1 or 2.
[0109] In some embodiments, the compound is of Formula (IA):
##STR00003##
or a salt or stereoisomer thereof, wherein
[0110] l is selected from 1, 2, 3, 4, and 5;
[0111] m is selected from 5, 6, 7, 8, and 9;
[0112] M.sub.1 is a bond or M';
[0113] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 1, 2, 3, 4, or 5 and Q is OH,
--NHC(S)N(R).sub.2, --NHC(O)N(R).sub.2, --N(R)C(O)R,
--N(R)S(O).sub.2R, --N(R)R.sub.8, --NHC(.dbd.NR.sub.9)N(R).sub.2,
--NHC(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
heteroaryl, or heterocycloalkyl;
[0114] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, --S--S--, an aryl group,
and a heteroaryl group; and
[0115] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[0116] In some embodiments, m is 5, 7, or 9.
[0117] In some embodiments, the compound is of Formula (IA), or a
salt or stereoisomer thereof, wherein
[0118] l is selected from 1, 2, 3, 4, and 5;
[0119] m is selected from 5, 6, 7, 8, and 9;
[0120] M.sub.1 is a bond or M';
[0121] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 1, 2, 3, 4, or 5 and Q is OH,
--NHC(S)N(R).sub.2, or --NHC(O)N(R).sub.2;
[0122] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, an aryl group, and a
heteroaryl group; and
[0123] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[0124] In some embodiments, m is 5, 7, or 9.
[0125] In some embodiments, the compound is of Formula (II):
##STR00004##
or a salt or stereoisomer thereof, wherein
[0126] l is selected from 1, 2, 3, 4, and 5;
[0127] M.sub.1 is a bond or M';
[0128] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 2, 3, or 4 and Q is OH,
--NHC(S)N(R).sub.2, --NHC(O)N(R).sub.2, --N(R)C(O)R,
--N(R)S(O).sub.2R, --N(R)R.sub.8, --NHC(.dbd.NR.sub.9)N(R).sub.2,
--NHC(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
heteroaryl, or heterocycloalkyl;
[0129] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, --S--S--, an aryl group,
and a heteroaryl group; and
[0130] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[0131] In some embodiments, the compound is of Formula (II), or a
salt or stereoisomer thereof, wherein
[0132] l is selected from 1, 2, 3, 4, and 5;
[0133] M.sub.1 is a bond or M';
[0134] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 2, 3, or 4 and Q is OH,
--NHC(S)N(R).sub.2, or --NHC(O)N(R).sub.2;
[0135] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, an aryl group, and a
heteroaryl group; and
[0136] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[0137] In some embodiments, M.sub.1 is M'.
[0138] In some embodiments, M and M' are independently --C(O)O-- or
--OC(O)--.
[0139] In some embodiments, l is 1, 3, or 5.
[0140] In some embodiments, the compound is selected from the group
consisting of Compound 1 to Compound 232, salts and stereoisomers
thereof, and any combination thereof.
[0141] In some embodiments, the compound is selected from the group
consisting of Compound 1 to Compound 147, salts and stereoisomers
thereof, and any combination thereof.
[0142] In some embodiments, the compound is of the Formula
(IIa),
##STR00005##
or a salt or stereoisomer thereof.
[0143] In some embodiments, the compound is of the Formula
(IIb),
##STR00006##
or a salt or stereoisomer thereof.
[0144] In some embodiments, the compound is of the Formula (IIc) or
(IIe),
##STR00007##
or a salt or stereoisomer thereof.
[0145] In some embodiments, R.sub.4 is as described herein. In some
embodiments, R.sub.4 is selected from --(CH.sub.2).sub.nQ and
--(CH.sub.2).sub.nCHQR.
[0146] In some embodiments, the compound is of the Formula
(IId),
##STR00008##
or a salt or stereoisomer thereof,
[0147] wherein n is selected from 2, 3, and 4, and m, R', R'', and
R.sub.2 through R.sub.6 are as described herein. For example, each
of R.sub.2 and R.sub.3 may be independently selected from the group
consisting of C.sub.5-14 alkyl and C.sub.5-14 alkenyl.
[0148] In some embodiments, the compound is of the Formula (IId),
or a salt or stereoisomer thereof,
[0149] wherein R.sub.2 and R.sub.3 are independently selected from
the group consisting of C.sub.5-14 alkyl and C.sub.5-14 alkenyl, n
is selected from 2, 3, and 4, and R', R'', R.sub.5, R.sub.6 and m
are as defined herein.
[0150] In some embodiments, R.sub.2 is C.sub.8 alkyl.
[0151] In some embodiments, R.sub.3 is C.sub.5 alkyl, C.sub.6
alkyl, C.sub.7 alkyl, C.sub.8 alkyl, or C.sub.9 alkyl.
[0152] In some embodiments, m is 5, 7, or 9.
[0153] In some embodiments, each R.sub.5 is H.
[0154] In some embodiments, each R.sub.6 is H. In some embodiments,
the delivery agent comprises a compound having the Formula
(III)
##STR00009##
or salts or stereoisomers thereof, wherein
[0155] ring A is
##STR00010##
[0156] t is 1 or 2;
[0157] A.sub.1 and A.sub.2 are each independently selected from CH
or N;
[0158] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[0159] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
independently selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R''MR', --R*YR'', --YR'', and
--R*OR'';
[0160] each M is independently selected from the group consisting
of --C(O)O--, --OC(O)--, --OC(O)O--, --C(O)N(R')--, --N(R')C(O)--,
--C(O)--, --C(S)--, --C(S)S--, --SC(S)--, --CH(OH)--,
--P(O)(OR')O--, --S(O).sub.2--, an aryl group, and a heteroaryl
group;
[0161] X.sup.1, X.sup.2, and X.sup.3 are independently selected
from the group consisting of a bond, --CH.sub.2--,
--(CH.sub.2).sub.2--, --CHR--, --CHY--, --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--,
--CH.sub.2--OC(O)--, --CH(OH)--, --C(S)--, and --CH(SH)--;
[0162] each Y is independently a C.sub.3-6 carbocycle;
[0163] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0164] each R is independently selected from the group consisting
of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle;
[0165] each R' is independently selected from the group consisting
of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H; and each R'' is
independently selected from the group consisting of C.sub.3-12
alkyl and C.sub.3-12 alkenyl,
[0166] wherein when ring A is
##STR00011##
then
[0167] i) at least one of X.sup.1, X.sup.2, and X.sup.3 is not
--CH.sub.2--; and/or
[0168] ii) at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 is --R''MR'.
[0169] In some embodiments, the compound is of any of Formulae
(IIIa1)-(IIIa6):
##STR00012##
[0170] The compounds of Formula (III) or any of (IIIa1)-(IIIa6)
include one or more of the following features when applicable.
[0171] In some embodiments, ring A is
##STR00013##
[0172] In some embodiments, ring A is
##STR00014##
[0173] In some embodiments, ring A is
##STR00015##
[0174] In some embodiments, ring A is
##STR00016##
[0175] In some embodiments, ring A is
##STR00017##
[0176] In some embodiments, ring A is
##STR00018##
wherein ring, in which the N atom is connected with X.sup.2.
[0177] In some embodiments, Z is CH.sub.2.
[0178] In some embodiments, Z is absent.
[0179] In some embodiments, at least one of A.sub.1 and A.sub.2 is
N.
[0180] In some embodiments, each of A.sub.1 and A.sub.2 is N.
[0181] In some embodiments, each of A.sub.1 and A.sub.2 is CH.
[0182] In some embodiments, A.sub.1 is N and A.sub.2 is CH.
[0183] In some embodiments, A.sub.1 is CH and A.sub.2 is N.
[0184] In some embodiments, at least one of X.sup.1, X.sup.2, and
X.sup.3 is not --CH.sub.2--. For example, in certain embodiments,
X.sup.1 is not --CH.sub.2--. In some embodiments, at least one of
X.sup.1, X.sup.2, and X.sup.3 is --C(O)--.
[0185] In some embodiments, X.sup.2 is --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--, or
--CH.sub.2--OC(O)--.
[0186] In some embodiments, X.sup.3 is --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--, or
--CH.sub.2--OC(O)--. In other embodiments, X.sup.3 is
--CH.sub.2--.
[0187] In some embodiments, X.sup.3 is a bond or
--(CH.sub.2).sub.2--.
[0188] In some embodiments, R.sub.1 and R.sub.2 are the same. In
certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are the same. In
some embodiments, R.sub.4 and R.sub.5 are the same. In certain
embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
the same.
[0189] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is --R''MR'. In some embodiments, at
most one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
--R''MR'. For example, at least one of R.sub.1, R.sub.2, and
R.sub.3 may be --R''MR', and/or at least one of R.sub.4 and R.sub.5
is --R''MR'. In certain embodiments, at least one M is --C(O)O--.
In some embodiments, each M is --C(O)O--. In some embodiments, at
least one M is --OC(O)--. In some embodiments, each M is --OC(O)--.
In some embodiments, at least one M is --OC(O)O--. In some
embodiments, each M is --OC(O)O--. In some embodiments, at least
one R'' is C.sub.3 alkyl. In certain embodiments, each R'' is
C.sub.3 alkyl. In some embodiments, at least one R'' is C.sub.5
alkyl. In certain embodiments, each R'' is C.sub.5 alkyl. In some
embodiments, at least one R'' is C.sub.6 alkyl. In certain
embodiments, each R'' is C.sub.6 alkyl. In some embodiments, at
least one R'' is C.sub.7 alkyl. In certain embodiments, each R'' is
C.sub.7 alkyl. In some embodiments, at least one R' is C.sub.5
alkyl. In certain embodiments, each R' is C.sub.5 alkyl. In other
embodiments, at least one R' is C.sub.1 alkyl. In certain
embodiments, each R' is C.sub.1 alkyl. In some embodiments, at
least one R' is C.sub.2 alkyl. In certain embodiments, each R' is
C.sub.2 alkyl.
[0190] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is C.sub.12 alkyl. In certain
embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 are C.sub.12 alkyl.
[0191] In some embodiments, the delivery agent comprises a compound
having the Formula (IV)
##STR00019##
or salts or stereoisomer thereof, wherein
[0192] A.sub.1 and A.sub.2 are each independently selected from CH
or N and at least one of A.sub.1 and A.sub.2 is N;
[0193] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[0194] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
independently selected from the group consisting of C.sub.6-20
alkyl and C.sub.6-20 alkenyl;
[0195] wherein when ring A is
##STR00020##
then
[0196] i) R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are the
same, wherein R.sub.1 is not C.sub.12 alkyl, C.sub.18 alkyl, or
C.sub.18 alkenyl;
[0197] ii) only one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 is selected from C.sub.6-20 alkenyl;
[0198] iii) at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 have a different number of carbon atoms than at least one
other of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5;
[0199] iv) R.sub.1, R.sub.2, and R.sub.3 are selected from
C.sub.6-20 alkenyl, and R.sub.4 and R.sub.5 are selected from
C.sub.6-20 alkyl; or
[0200] v) R.sub.1, R.sub.2, and R.sub.3 are selected from
C.sub.6-20 alkyl, and R.sub.4 and R.sub.5 are selected from
C.sub.6-20 alkenyl.
[0201] In some embodiments, the compound is of Formula (IVa):
##STR00021##
[0202] The compounds of Formula (IV) or (IVa) include one or more
of the following features when applicable.
[0203] In some embodiments, Z is CH.sub.2.
[0204] In some embodiments, Z is absent.
[0205] In some embodiments, at least one of A.sub.1 and A.sub.2 is
N.
[0206] In some embodiments, each of A.sub.1 and A.sub.2 is N.
[0207] In some embodiments, each of A.sub.1 and A.sub.2 is CH.
[0208] In some embodiments, A.sub.1 is N and A.sub.2 is CH.
[0209] In some embodiments, A.sub.1 is CH and A.sub.2 is N.
[0210] In some embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 are the same, and are not C.sub.12 alkyl, C.sub.18 alkyl,
or C.sub.18 alkenyl. In some embodiments, R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same and are C.sub.9 alkyl or
C.sub.14 alkyl.
[0211] In some embodiments, only one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is selected from C.sub.6-20 alkenyl. In
certain such embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 have the same number of carbon atoms. In some embodiments,
R.sub.4 is selected from C.sub.5-20 alkenyl. For example, R.sub.4
may be C.sub.12 alkenyl or C.sub.18 alkenyl.
[0212] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 have a different number of carbon
atoms than at least one other of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5.
[0213] In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are
selected from C.sub.6-20 alkenyl, and R.sub.4 and R.sub.5 are
selected from C.sub.6-20 alkyl. In other embodiments, R.sub.1,
R.sub.2, and R.sub.3 are selected from C.sub.6-20 alkyl, and
R.sub.4 and R.sub.5 are selected from C.sub.6-20 alkenyl. In some
embodiments, R.sub.1, R.sub.2, and R.sub.3 have the same number of
carbon atoms, and/or R.sub.4 and R.sub.5 have the same number of
carbon atoms. For example, R.sub.1, R.sub.2, and R.sub.3, or
R.sub.4 and R.sub.5, may have 6, 8, 9, 12, 14, or 18 carbon atoms.
In some embodiments, R.sub.1, R.sub.2, and R.sub.3, or R.sub.4 and
R.sub.5, are C.sub.18 alkenyl (e.g., linoleyl). In some
embodiments, R.sub.1, R.sub.2, and R.sub.3, or R.sub.4 and R.sub.5,
are alkyl groups including 6, 8, 9, 12, or 14 carbon atoms.
[0214] In some embodiments, R.sub.1 has a different number of
carbon atoms than R.sub.2, R.sub.3, R.sub.4, and R.sub.5. In other
embodiments, R.sub.3 has a different number of carbon atoms than
R.sub.1, R.sub.2, R.sub.4, and R.sub.5. In further embodiments,
R.sub.4 has a different number of carbon atoms than R.sub.1,
R.sub.2, R.sub.3, and R.sub.5.
[0215] In other embodiments, the delivery agent comprises a
compound having the Formula (V)
##STR00022##
or salts or stereoisomers thereof, in which
[0216] A.sub.3 is CH or N;
[0217] A.sub.4 is CH.sub.2 or NH; and at least one of A.sub.3 and
A.sub.4 is N or NH;
[0218] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[0219] R.sub.1, R.sub.2, and R.sub.3 are independently selected
from the group consisting of C.sub.5-20 alkyl, C.sub.5-20 alkenyl,
--R''MR', --R*YR'', --YR'', and --R*OR'';
[0220] each M is independently selected from --C(O)O--, --OC(O)--,
--C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--, --C(S)S--,
--SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--, an aryl
group, and a heteroaryl group;
[0221] X.sup.1 and X.sup.2 are independently selected from the
group consisting of --CH.sub.2--, --(CH.sub.2).sub.2--, --CHR--,
--CHY--, --C(O)--, --C(O)O--, --OC(O)--, --C(O)--CH.sub.2--,
--CH.sub.2--C(O)--, --C(O)O--CH.sub.2--, --OC(O) --CH.sub.2--,
--CH.sub.2--C(O)O--, --CH.sub.2--OC(O)--, --CH(OH)--, --C(S)--, and
--CH(SH)--;
[0222] each Y is independently a C.sub.3-6 carbocycle;
[0223] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0224] each R is independently selected from the group consisting
of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle;
[0225] each R' is independently selected from the group consisting
of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H; and
[0226] each R'' is independently selected from the group consisting
of C.sub.3-12 alkyl and C.sub.3-12 alkenyl.
[0227] In some embodiments, the compound is of Formula (Va):
##STR00023##
[0228] The compounds of Formula (V) or (Va) include one or more of
the following features when applicable.
[0229] In some embodiments, Z is CH.sub.2.
[0230] In some embodiments, Z is absent.
[0231] In some embodiments, at least one of A.sub.3 and A.sub.4 is
N or NH.
[0232] In some embodiments, A.sub.3 is N and A.sub.4 is NH.
[0233] In some embodiments, A.sub.3 is N and A.sub.4 is
CH.sub.2.
[0234] In some embodiments, A.sub.3 is CH and A.sub.4 is NH.
[0235] In some embodiments, at least one of X.sup.1 and X.sup.2 is
not --CH.sub.2--. For example, in certain embodiments, X.sup.1 is
not --CH.sub.2--. In some embodiments, at least one of X.sup.1 and
X.sup.2 is --C(O)--.
[0236] In some embodiments, X.sup.2 is --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--, or
--CH.sub.2--OC(O)--.
[0237] In some embodiments, R.sub.1, R.sub.2, and R.sub.3 are
independently selected from the group consisting of C.sub.5-20
alkyl and C.sub.5-20 alkenyl. In some embodiments, R.sub.1,
R.sub.2, and R.sub.3 are the same. In certain embodiments, R.sub.1,
R.sub.2, and R.sub.3 are C.sub.6, C.sub.9, C.sub.12, or C.sub.14
alkyl. In other embodiments, R.sub.1, R.sub.2, and R.sub.3 are
C.sub.18 alkenyl. For example, R.sub.1, R.sub.2, and R.sub.3 may be
linoleyl.
[0238] In other embodiments, the delivery agent comprises a
compound having the Formula (VI):
##STR00024##
or salts or stereoisomers thereof, in which
[0239] A.sub.6 and A.sub.7 are each independently selected from CH
or N, wherein at least one of A.sub.6 and A.sub.7 is N;
[0240] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[0241] X.sup.4 and X.sup.5 are independently selected from the
group consisting of --CH.sub.2--, --(CH.sub.2).sub.2--, --CHR--,
--CHY--, --C(O)--, --C(O)O--, --OC(O)--, --C(O)--CH.sub.2--,
--CH.sub.2--C(O)--, --C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--,
--CH.sub.2--C(O)O--, --CH.sub.2--OC(O)--, --CH(OH)--, --C(S)--, and
--CH(SH)--;
[0242] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 each are
independently selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R''MR', --R*YR'', --YR'', and
--R*OR'';
[0243] each M is independently selected from the group consisting
of --C(O)O--, --OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--,
--C(S)--, --C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--,
--S(O).sub.2--, an aryl group, and a heteroaryl group;
[0244] each Y is independently a C.sub.3-6 carbocycle;
[0245] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0246] each R is independently selected from the group consisting
of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle;
[0247] each R' is independently selected from the group consisting
of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H; and
[0248] each R'' is independently selected from the group consisting
of C.sub.3-12 alkyl and C.sub.3-12 alkenyl.
[0249] In some embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 each are independently selected from the group consisting
of C.sub.6-20 alkyl and C.sub.6-20 alkenyl.
[0250] In some embodiments, R.sub.1 and R.sub.2 are the same. In
certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are the same. In
some embodiments, R.sub.4 and R.sub.5 are the same. In certain
embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
the same.
[0251] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is C.sub.9-12 alkyl. In certain
embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 independently is C.sub.9, C.sub.12 or C.sub.14 alkyl. In
certain embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
and R.sub.5 is C.sub.9 alkyl.
[0252] In some embodiments, A.sub.6 is N and A.sub.7 is N. In some
embodiments, A.sub.6 is CH and A.sub.7 is N.
[0253] In some embodiments, X.sup.4 is --CH.sub.2-- and X.sup.5 is
--C(O)--. In some embodiments, X.sup.4 and X.sup.5 are
--C(O)--.
[0254] In some embodiments, when A.sub.6 is N and A.sub.7 is N, at
least one of X.sup.4 and X.sup.5 is not --CH.sub.2--, e.g., at
least one of X.sup.4 and X.sup.5 is --C(O)--. In some embodiments,
when A.sub.6 is N and A.sub.7 is N, at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is --R''MR'.
[0255] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is not --R''MR'.
[0256] In some embodiments, the composition disclosed herein is a
nanoparticle composition. In some embodiments, the delivery agent
further comprises a phospholipid. In some embodiments, the
phospholipid is selected from the group consisting of [0257]
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), [0258]
1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), [0259]
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), [0260]
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), [0261]
1,2-distearoyl-sn-glycero-3-phosphocholine (DSpC), [0262]
1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), [0263]
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), [0264]
1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),
[0265]
1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
(OChemsPC), [0266] 1-hexadecyl-sn-glycero-3-phosphocholine (C16
Lyso PC), [0267] 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,
[0268] 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, [0269]
1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, [0270]
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), [0271]
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE),
[0272] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, [0273]
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, [0274]
1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, [0275]
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, [0276]
1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, [0277]
1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt
(DOPG), sphingomyelin, and any mixtures thereof.
[0278] In some embodiments, the delivery agent further comprises a
structural lipid. In some embodiments, the structural lipid is
selected from the group consisting of cholesterol, fecosterol,
sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,
tomatidine, ursolic acid, alpha-tocopherol, and any mixtures
thereof.
[0279] In some embodiments, the delivery agent further comprises a
PEG lipid. In some embodiments, the PEG lipid is selected from the
group consisting of a PEG-modified phosphatidylethanolamnine, a
PEG-modified phosphatidic acid, a PEG-modified ceramide, a
PEG-modified dialkylamine, a PEG-modified diacylglycerol, a
PEG-modified dialkylglycerol, and any mixtures thereof. In some
embodiments, the PEG lipid has the Formula:
##STR00025##
wherein r is an integer between 1 and 100. In some embodiments, the
PEG lipid is Compound 428.
[0280] In some embodiments, the delivery agent further comprises an
ionizable lipid selected from the group consisting of [0281]
3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine
(KL10), [0282]
N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinedie-
thanamine (KL22), [0283]
14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),
[0284] 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),
[0285] 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA), [0286] heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (DLin-MC3-DMA), [0287]
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), [0288] 1,2-dioleyloxy-N,N-dimethylaminopropane
(DODMA), [0289]
2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-
-octadeca-9,12-di en-1-yloxy]propan-1-amine (Octyl-CLinDMA), [0290]
(2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-
,12Z)-octadeca-9, 12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA
(2R)), and [0291]
(2S)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-
-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine
(Octyl-CLinDMA (2S)).
[0292] In some embodiments, the delivery agent further comprises a
phospholipid, a structural lipid, a PEG lipid, or any combination
thereof. In some embodiments, the delivery agent comprises Compound
18, DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of
about 50:10:38.5:1.5.
[0293] In some embodiments, the composition is formulated for in
vivo delivery. In some embodiments, the composition is formulated
for intramuscular, subcutaneous, or intradermal delivery.
[0294] The present disclosure further provides a polynucleotide
comprising an mRNA comprising: (i) a 5' UTR, (ii) an open reading
frame (ORF) encoding a human porphobilinogen deaminase (PBGD)
polypeptide, wherein the ORF comprises a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 9 to 33 and 89 to
117, and (iii) a 3' UTR comprising a microRNA binding site selected
from miR-142, miR-126, or a combination thereof, wherein the mRNA
comprises at least one chemically modified nucleobase.
[0295] The present disclosure further provides a polynucleotide
comprising an mRNA comprising: (i) a 5'-terminal cap; (ii) a 5' UTR
comprising a sequence selected from the group consisting of SEQ ID
NO: 39 to 56, 83, 189 to 191, and any combination thereof; (iii) an
open reading frame (ORF) encoding a human porphobilinogen deaminase
(PBGD) polypeptide, wherein the ORF comprises a sequence selected
from the group consisting of SEQ ID NOs: 9 to 33 and 89 to 117,
wherein the mRNA comprises at least one chemically modified
nucleobase selected from the group consisting of pseudouracil
(.psi.), N1-methylpseudouracil (m1.psi.), 1-ethylpseudouracil,
2-thiouracil (s2U), 4'-thiouracil, 5-methylcytosine,
5-methyluracil, 5-methoxyuracil, and any combination thereof; and
(iv) a 3' UTR comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 57 to 81, 84, 149 to 151, 161 to
172, 192 to 199, and any combination thereof; and (v) a
poly-A-region.
[0296] In some embodiments, the polynucleotide comprises a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 133,
141, 144, and 145.
[0297] The present disclosure further provides a pharmaceutical
composition comprising the polynucleotide, e.g., an mRNA, and a
delivery agent. In some embodiments, the delivery agent is a lipid
nanoparticle comprising Compound 18, Compound 236, a salt or a
stereoisomer thereof, or any combination thereof. In some
embodiments, the polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide disclosed herein is formulated with a
delivery agent comprising, e.g., a compound having the Formula (I),
e.g., any of Compounds 1-232, e.g., Compound 18; a compound having
the Formula (III), (IV), (V), or (VI), e.g., any of Compounds
233-342, e.g., Compound 236; or a compound having the Formula
(VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or any
combination thereof. In some embodiments, the delivery agent
comprises Compound 18, DSPC, Cholesterol, and Compound 428, e.g.,
with a mole ratio of about 50:10:38.5:1.5.
[0298] In one aspect of the embodiments disclosed herein, the
subject is a human subject in need of treatment or prophylaxis for
acute intermittent porphyria (AIP) and/or an acute porphyria
attack.
[0299] In one aspect of the embodiments disclosed herein, upon
administration to the subject, the mRNA has: (i) a longer plasma
half-life; (ii) increased expression of a PBGD polypeptide encoded
by the ORF; (iii) a lower frequency of arrested translation
resulting in an expression fragment; (iv) greater structural
stability; or (v) any combination thereof, relative to a
corresponding mRNA having the nucleic acid sequence of SEQ ID NO:
2, 4, 6, or 8 and/or administered as naked mRNA.
[0300] In some embodiments, a pharmaceutical composition or
polynucleotide disclosed herein is suitable for administration as a
single unit dose or a plurality of single unit doses.
[0301] In some embodiments, a pharmaceutical composition or
polynucleotide disclosed herein is suitable for reducing the level
of one or more biomarkers of AIP in the subject.
[0302] In some embodiments, a pharmaceutical composition or
polynucleotide disclosed herein is for use in treating, preventing
or delaying the onset of AIP signs or symptoms in the subject. In
some embodiments, the signs or symptoms include pain, seizures,
paralysis, neuropathy, death, or a combination thereof.
[0303] The present disclosure also provides a host cell comprising
a polynucleotide of the invention. In some embodiments, the host
cell is a eukaryotic cell. The present disclosure also provides a
vector comprising a polynucleotide of the invention. Also provided
is a method of making a polynucleotide of the invention comprising
synthesizing the polynucleotide enzymatically or chemically. The
present disclosure also provides a polypeptide encoded by a
polynucleotide of the invention, a composition comprising a
polynucleotide of the invention, a host cell comprising a
polynucleotide of the invention, a vector comprising a
polynucleotide of the invention, or produced by the method of
making disclosed herein.
[0304] The present disclosure also provides a method of expressing
in vivo an active PBGD polypeptide in a subject in need thereof
comprising administering to the subject an effective amount of the
polynucleotide of the invention, a composition comprising a
polynucleotide of the invention, a host cell comprising a
polynucleotide of the invention, a vector comprising a
polynucleotide of the invention. Also provided is a method of
treating acute intermittent porphyria (AIP) in a subject in need
thereof comprising administering to the subject a therapeutically
effective amount of the polynucleotide of the invention, a
composition comprising a polynucleotide of the invention, a host
cell comprising a polynucleotide of the invention, a vector
comprising a polynucleotide of the invention, wherein the
administration alleviates the signs or symptoms of AIP in the
subject.
[0305] The present disclosure also provides a method to prevent or
delay the onset of AIP signs or symptoms in a subject in need
thereof comprising administering to the subject a prophylactically
effective amount of the polynucleotide of the invention, a
composition comprising a polynucleotide of the invention, a host
cell comprising a polynucleotide of the invention, a vector
comprising a polynucleotide of the invention before AIP signs or
symptoms manifest, wherein the administration prevents or delays
the onset of AIP signs or symptoms in the subject. Also provided is
a method to ameliorate the signs or symptoms of AIP in a subject in
need thereof comprising administering to the subject a
therapeutically effective amount of the polynucleotide of the
invention, a composition comprising a polynucleotide of the
invention, a host cell comprising a polynucleotide of the
invention, a vector comprising a polynucleotide of the invention
before AIP signs or symptoms manifest, wherein the administration
ameliorates AIP signs or symptoms in the subject.
[0306] The present disclosure further provides a method of
expressing a porphobilinogen deaminase (PBGD) polypeptide in a
human subject in need thereof comprising administering to the
subject an effective amount of a pharmaceutical composition or a
polynucleotide, e.g., an mRNA, described herein, wherein the
pharmaceutical composition or polynucleotide is suitable for
administrating as a single dose or as a plurality of single unit
doses to the subject.
[0307] The present disclosure further provides a method of
treating, preventing or delaying the onset of acute intermittent
porphyria (AIP) signs or symptoms in a human subject in need
thereof comprising administering to the subject an effective amount
of a pharmaceutical composition or a polynucleotide, e.g., an mRNA,
described herein, wherein the administration treats, prevents or
delays the onset of one or more of the signs or symptoms of AIP in
the subject.
[0308] The present disclosure further provides a method for the
treatment of acute intermittent porphyria (AIP), comprising
administering to a human subject suffering from AIP a single
intravenous dose of a pharmaceutical composition or a
polynucleotide, e.g., an mRNA, described herein.
[0309] The present disclosure further provides a method of reducing
an aminolevulinate acid (ALA), a porphobilinogen (PBG) and/or a
porphyrin urinary excretion level in a human subject comprising
administering to the subject an effective amount of a
pharmaceutical composition or a polynucleotide, e.g., an mRNA,
described herein, wherein the administration reduces the ALA, PBG
and/or porphyrin urinary excretion level in the subject. In some
embodiments,
[0310] (i) ALA urinary excretion level is reduced at least 2-fold,
at least 5-fold, at least 10-fold, at least 20-fold or at least
50-fold as compared to a reference ALA excretion level during an
acute porphyria attack, for at least 24 hours, at least 48 hours,
at least 72 hours, at least 96 hours, or at least 120 hours
post-administration,
[0311] (ii) PBG urinary excretion level is reduced at least 2-fold,
at least 5-fold, at least 10-fold, at least 20-fold or at least
50-fold as compared to a reference PBG excretion level during an
acute porphyria attack, for at least 24 hours, at least 48 hours,
at least 72 hours, at least 96 hours, or at least 120 hours
post-administration, and/or
[0312] (iii) porphyrin urinary excretion level is reduced at least
at least 2-fold, at least 5-fold, at least 10-fold, at least
20-fold or at least 50-fold as compared to a reference porphyrin
excretion level during an acute porphyria attack, for at least 24
hours, at least 48 hours, at least 72 hours, at least 96 hours, or
at least 120 hours post-administration.
[0313] The present disclosure further provides a method of reducing
an aminolevulinate acid (ALA), a porphobilinogen (PBG) and/or a
porphyrin urinary excretion level in a human subject comprising
administering to the subject an effective amount of a
pharmaceutical composition or a polynucleotide described herein,
wherein the administration reduces the ALA, PBG and/or porphyrin
urinary excretion level in the subject. In some embodiments,
[0314] (i) ALA urinary excretion level is reduced by at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, at least 99%, or at least 100% as compared to
the subject's baseline level or a reference ALA excretion level
(e.g., in a subject with AIP or during an acute porphyria attack),
for at least 24 hours, at least 48 hours, at least 72 hours, at
least 96 hours, or at least 120 hours post-administration,
[0315] (ii) PBG urinary excretion level is reduced by at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, at least 99%, or at least 100% as compared to
the subject's baseline level or a reference PBG excretion level
(e.g., in a subject with AIP or during an acute porphyria attack),
for at least 24 hours, at least 48 hours, at least 72 hours, at
least 96 hours, or at least 120 hours post-administration,
and/or
[0316] (iii) porphyrin urinary excretion level is reduced by at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%, at least 98%, at least 99%, or at least 100% as
compared to the subject's baseline level or a reference porphyrin
excretion level (e.g., in a subject with AIP or during an acute
porphyria attack), for at least 24 hours, at least 48 hours, at
least 72 hours, at least 96 hours, or at least 120 hours
post-administration.
[0317] The present disclosure further provides a method of reducing
an alanine transaminase (ALT), a aspartate transaminase (AST)
and/or a bilirubin serum level in a human subject comprising
administering to the subject an effective amount of a
pharmaceutical composition or a polynucleotide, e.g., an mRNA,
described herein, wherein the administration reduces the ALT, AST
and/or bilirubin serum level in the subject. In some
embodiments,
[0318] (i) ALT serum level is reduced to at least within 10-fold,
at least within 5-fold, at least within 2-fold, or at least within
1.5-fold as compared to a reference ALT serum level within at least
24 hours, at least 48 hours, at least 72 hours, at least 96 hours,
or at least 120 hours post-administration,
[0319] (ii) AST serum level is reduced to at least within 10-fold,
at least within 5-fold, at least within 2-fold, or at least within
1.5-fold, as compared to a reference AST serum level, for at least
24 hours, at least 48 hours, at least 72 hours, at least 96 hours,
or at least 120 hours post-administration, and/or
[0320] (iii) bilirubin serum level is reduced to at least within
10-fold, at least within 5-fold, at least within 2-fold, or at
least within 1.5 fold as compared to a reference bilirubin serum
level, for at least 24 hours, at least 48 hours, at least 72 hours,
at least 96 hours, or at least 120 hours post-administration.
[0321] The present disclosure further provides a method of reducing
an alanine transaminase (ALT), a aspartate transaminase (AST)
and/or a bilirubin serum level in a human subject comprising
administering to the subject an effective amount of a
pharmaceutical composition or a polynucleotide described herein,
wherein the administration reduces the ALT, AST and/or bilirubin
serum level in the subject. In some embodiments,
[0322] (i) ALT serum level is reduced by at least 90%, at least
80%, at least 70%, at least 60% at least 50%, at least 40%, or at
least 30% as compared to the subject's baseline level or a
reference ALT serum level (e.g., in a subject with AIP or during an
acute porphyria attack) within at least 24 hours, at least 48
hours, at least 72 hours, at least 96 hours, or at least 120 hours
post-administration,
[0323] (ii) AST serum level is reduced by at least 90%, at least
80%, at least 70%, at least 60%, at least 50%, at least 40%, or at
least 30% as compared to the subject's baseline level or a
reference AST serum level (e.g., in a subject with AIP or during an
acute porphyria attack), for at least 24 hours, at least 48 hours,
at least 72 hours, at least 96 hours, or at least 120 hours
post-administration, and/or
[0324] (iii) bilirubin serum level is reduced by at least 90%, at
least 80%, at least 70%, at least 60%, at least 50%, at least 40%,
or at least 30% as compared to the subject's baseline level or a
reference bilirubin serum level (e.g., in a subject with AIP or
during an acute porphyria attack), for at least 24 hours, at least
48 hours, at least 72 hours, at least 96 hours, or at least 120
hours post-administration.
[0325] In some embodiments, 12 hours after the pharmaceutical
composition or polynucleotide is administered to the subject, the
PBGD activity in the subject is increased at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 100%, at least 150%, at least
200%, at least 300%, at least.sup.400%, at least 500%, or at least
600% compared to the subject's baseline PBGD activity.
[0326] In some embodiments, the PBGD activity is increased in the
liver of the subject.
[0327] In some embodiments, the increased PBGD activity persists
for greater than 24, 36, 48, 60, 72, or 96 hours.
[0328] In some embodiments, the pharmaceutical composition or
polynucleotide is administered to the subject during an acute
porphyria attack.
[0329] In some embodiments, after administration of the
pharmaceutical composition or polynucleotide to the subject, e.g.,
within 24 hours, the level of ALA in the subject is reduced by at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, or 100% compared to the subject's baseline
ALA.
[0330] In some embodiments, the level of ALA is reduced in one or
more of the urine, plasma, serum, and/or liver of the subject.
[0331] In some embodiments, after administration to the subject the
level of ALA in the subject is reduced compared to the baseline
level in the subject for at least one day, at least two days, at
least three days, at least four days, at least five days, at least
one week, at least two weeks, at least three weeks, or at least one
month.
[0332] In some embodiments, after administration of the
pharmaceutical composition or polynucleotide to the subject, e.g.,
within 24 hours, the level of PBG in the subject is reduced by at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, or 100% compared to the subject's baseline
ALA.
[0333] In some embodiments, the level of PBG is reduced in one or
more of the urine, plasma, serum, and/or liver of the subject.
[0334] In some embodiments, after administration to the subject the
level of PBG in the subject is reduced compared to the baseline
level in the subject for at least one day, at least two days, at
least three days, at least four days, at least five days, at least
one week, at least two weeks, at least three weeks, or at least one
month.
[0335] In some embodiments, after administration of the
pharmaceutical composition or polynucleotide to the subject, e.g.,
within 24 hours, the level of porphyrin in the subject is reduced
by at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, or 100% compared to the subject's
baseline ALA.
[0336] In some embodiments, the level of porphyrin is reduced in
one or more of the urine, plasma, serum, and/or liver of the
subject.
[0337] In some embodiments, after administration to the subject the
level of porphyrin in the subject is reduced compared to the
baseline level in the subject for at least one day, at least two
days, at least three days, at least four days, at least five days,
at least one week, at least two weeks, at least three weeks, or at
least one month.
[0338] In some embodiments, after administration of the
pharmaceutical composition or polynucleotide to the subject, e.g.,
within 24 hours, the level of ALT in the subject is reduced by at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, or 100% compared to the subject's baseline
ALT.
[0339] In some embodiments, the level of ALT is reduced in one or
more of the urine, plasma, serum, and/or liver of the subject.
[0340] In some embodiments, after administration to the subject the
level of ALT in the subject is reduced compared to the baseline
level in the subject for at least one day, at least two days, at
least three days, at least four days, at least five days, at least
one week, at least two weeks, at least three weeks, or at least one
month.
[0341] In some embodiments, after administration of the
pharmaceutical composition or polynucleotide to the subject, e.g.,
within 24 hours, the level of AST in the subject is reduced by at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, or 100% compared to the subject's baseline
AST.
[0342] In some embodiments, the level of AST is reduced in one or
more of the urine, plasma, serum, and/or liver of the subject.
[0343] In some embodiments, after administration to the subject the
level of AST in the subject is reduced compared to the baseline
level in the subject for at least one day, at least two days, at
least three days, at least four days, at least five days, at least
one week, at least two weeks, at least three weeks, or at least one
month.
[0344] In some embodiments, after administration of the
pharmaceutical composition or polynucleotide to the subject, e.g.,
within 24 hours, the level of bilirubin in the subject is reduced
by at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, or 100% compared to the subject's
baseline bilirubin.
[0345] In some embodiments, the level of bilirubin is reduced in
one or more of the urine, plasma, serum, and/or liver of the
subject.
[0346] In some embodiments, after administration to the subject the
level of bilirubin in the subject is reduced compared to the
baseline level in the subject for at least one day, at least two
days, at least three days, at least four days, at least five days,
at least one week, at least two weeks, at least three weeks, or at
least one month.
[0347] In some embodiments, the AIP is clinically manifest (overt)
AIP.
[0348] In some embodiments, the AIP is clinically presymptomatic
(latent) AIP.
[0349] In some embodiments, the level the PBGD polypeptide activity
level is sufficient to reduce the risk of or prevent the onset of
an acute attack and/or sufficient to treat an acute attack.
[0350] In some embodiments, the pharmaceutical composition or
polynucleotide is administered as a single dose of less than 1.5
mg/kg, less than 1.25 mg/kg, less than 1 mg/kg, or less than 0.75
mg/kg.
[0351] In some embodiments, the administration to the subject is
about once a week, about once every two weeks, or about once a
month.
[0352] In some embodiments, the pharmaceutical composition or
polynucleotide is administered intravenously.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0353] FIG. 1A-D shows the protein sequence (FIG. 1A), table with
domain features (FIG. 1B), graphic representation of domain
structure (FIG. 1C), and nucleic acid sequence (FIG. 1D) of isoform
1 of PBGD.
[0354] FIG. 2A-D shows the protein sequence (FIG. 2A), table with
domain features (FIG. 2B), graphic representation of domain
structure (FIG. 2C), and nucleic acid sequence (FIG. 2D) of isoform
2 of PBGD.
[0355] FIG. 3A-D shows the protein sequence (FIG. 3A), table with
domain features (FIG. 3B), graphic representation of domain
structure (FIG. 3C), and nucleic acid sequence (FIG. 3D) of isoform
3 of PBGD.
[0356] FIG. 4A-D shows the protein sequence (FIG. 4A), table with
domain features (FIG. 4B), graphic representation of domain
structure (FIG. 4C), and nucleic acid sequence (FIG. 4D) of isoform
4 of PBGD.
[0357] FIG. 5 shows uracil (U) metrics corresponding to wild type
isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
The column labeled "U content (%)" corresponds to the % U.sub.TL
parameter. The column labeled "U Content v. WT (%)" corresponds to
% U.sub.WT. The column labeled "U Content v. Theoretical Minimum
(%)" corresponds to % U.sub.TM. The column labeled "UU pairs v. WT
(%)" corresponds to % UU.sub.WT.
[0358] FIG. 6 shows guanine (G) metrics corresponding to wild type
isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
The column labeled "G Content (%)" corresponds to % G.sub.TL. The
column labeled "G Content v. WT (%)" corresponds to % G.sub.WT. The
column labeled "G Content v. Theoretical Maximum (%)" corresponds
to % G.sub.TMX.
[0359] FIG. 7 shows cytosine (C) metrics corresponding to wild type
isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
The column labeled "C Content (%)" corresponds to % C.sub.TL. The
column labeled "C Content v. WT (%)" corresponds to % C.sub.WT. The
column labeled "C Content v. Theoretical Maximum (%)" corresponds
to % C.sub.TMX.
[0360] FIG. 8 shows guanine plus cytosine (G/C) metrics
corresponding to wild type isoform 1 of PBGD and 25 sequence
optimized PBGD polynucleotides. The column labeled "G/C Content
(%)" corresponds to % G/C.sub.TL. The column labeled "G/C Content
v. WT (%)" corresponds to % G/C.sub.WT. The column labeled "G/C
Content v. Theoretical Maximum (%)" corresponds to %
G/C.sub.TMX.
[0361] FIG. 9 shows a comparison between the G/C compositional bias
for codon positions 1, 2, 3 corresponding to the wild type isoform
1 of PBGD and 25 sequence optimized PBGD polynucleotides.
[0362] FIG. 10A-C shows the protein sequence (FIG. 10A), table with
domain features (FIG. 10B), graphic representation of domain
structure (FIG. 10C) of the I291M/N340S gain of function mutant of
isoform 1 of PBGD.
[0363] FIG. 11 shows the nucleic acid sequence of the I291M/N340S
gain of function mutant of isoform 1 of PBGD.
[0364] FIG. 12A-D shows the protein sequence (FIG. 12A), table with
domain features (FIG. 12B), graphic representation of domain
structure (FIG. 12C), and nucleic acid sequence (FIG. 12D) of a
fusion construct comprising the mature form of apolipoprotein A1
(sequence without signal peptide and propeptide) and isoform 1 of
PBGD.
[0365] FIG. 13 shows the levels of hepatic PBGD activity after IV
administration of chemically modified mRNAs encoding wild type PBGD
(COV1 and COV2), PBGD-SM protein variant, and ApoAI-PBGD-SM
conjugate to AIP mice. A single IV dose of mRNA construct at 1
nmol/kg was administered. PBGD activity in liver was quantitated as
pmol of uroporphyrin produced per mg of protein per hour. Levels of
hepatic PBGD activity observed in wild type mice and AIP mice are
also shown for comparison. Data are expressed as mean.+-.SD.
[0366] FIG. 14 shows the design of a pharmacodynamics study to
evaluate the effects of the administration of a single IV dose of
modified mRNA encoding wild type or SM variant of PBGD to AIP mice
subjected to three acute porphyria attacks triggered by
phenobarbital challenges. Intraperitoneal injections of
phenobarbital and intravenous administration of PBGD mRNA are
indicated by arrows above the time line of the study.
[0367] FIG. 15 shows urinary ALA excretion levels (micrograms of
ALA per mg of creatinine) in AIP mice subjected to porphyria
attacks caused by intraperitoneal phenobarbital challenges. In
accordance with the study design shown in FIG. 14, AIP mice were
administered a single intravenous injection of a chemically
modified mRNA encoding PBGD, SM variant of PBGD, or luciferase, all
formulated in MC3 and at 0.5 mg/kg. Data are expressed as
mean.+-.SD.
[0368] FIG. 16 shows urinary PBG excretion levels (micrograms of
PBG per mg of creatinine) in AIP mice subjected to porphyria
attacks caused by intraperitoneal phenobarbital challenges. In
accordance with the study design shown in FIG. 14, AIP mice were
administered a single intravenous injection of a chemically
modified mRNA encoding PBGD, SM variant of PBGD, or luciferase, all
formulated in MC3 and at 0.5 mg/kg. Data are expressed as
mean.+-.SD.
[0369] FIG. 17 shows pain measurements in AIP mice subjected to
porphyria attacks caused by intraperitoneal phenobarbital
challenges. In accordance with the study design shown in FIG. 14,
AIP mice were administered a single intravenous injection of a
chemically modified mRNA encoding PBGD, SM variant of PBGD, or
luciferase, all formulated in MC3 and at 0.5 mg/kg. Pain levels
were measured after the first, second, and third phenobarbital
challenge (pain scale was measured using the method reported in
Langford et al., Nat. Methods. 7(6): 447-9 (2010); Matsumiya et
al., J. Am. Assoc. Lab. Anim. Sci. 51(1): 42-9 (2012)). Data are
expressed as mean.+-.SD.
[0370] FIGS. 18A and 18B present assessments of peripheral
neuropathy as determined by rotarod (FIG. 18A) and footprint
measurements (gait patterns) (FIG. 18B) following a single IV
administration of PBGD, SM variant of PBGD, or luciferase mRNA (0.5
mg/kg) according to the study design shown in FIG. 14. Baseline
measurements and measurements after each phenobarbital challenge
are shown. Data are expressed as mean.+-.SD.
[0371] FIGS. 19A, 19B, 19C, 19D and 19E show correction of sciatic
nerve dysfunction following a single IV administration of PBGD or
SM variant of PBGD mRNA (0.5 mg/kg) compared to luciferase mRNA
(0.5 mg/kg) in AIP mice induced by three consecutive phenobarbital
challenges according to the study design shown in FIG. 14. FIG. 19A
shows sciatic nerve conduction data corresponding to AIP mice
administered mRNA encoding luciferase. FIG. 19B and FIG. 19C shows
sciatic nerve conduction data corresponding to AIP mice
administered modified mRNA encoding wild type PBGD (FIG. 19B) and
modified mRNA encoding PBGD-SM protein variant (FIG. 19C). FIG. 19D
and FIG. 19E, respectively, show latency and amplitude values from
sciatic nerve conduction data corresponding to AIP mice
administered mRNA encoding luciferase or modified mRNA encoding
wild type PBGD or modified mRNA encoding PBGD-SM variant.
[0372] FIGS. 20A, 20B, 20C, 20D, and 20E show hepatic expression of
PBGD protein observed after IV administration of modified mRNA
encoding wild type PBGD, or an AAV-PBGD vector. FIG. 20A shows
hepatic PBGD protein expression after administration of AAV-PBGD.
FIG. 20B shows basal PBGD protein expression levels. FIG. 20C, FIG.
20D and FIG. 20E shows hepatic PBGD protein levels on day 1, day 2
and day 4, respectively, after administration of modified mRNA
encoding wild type PBGD.
[0373] FIG. 21A and FIG. 21B show urinary ALA excretion levels
(micrograms of ALA per mg of creatinine) (FIG. 21A) and urinary PBG
excretion levels (micrograms of PBG per mg of creatinine) (FIG.
21B) in AIP mice subjected to porphyria attacks caused by
intraperitoneal phenobarbital challenges. AIP mice were
administered a single intravenous injection of a chemically
modified mRNA encoding PBGD or mRNA encoding luciferase. mRNAs were
formulated in MC3 or Compound 18 lipid nanoparticles. The mRNA
encoding luciferase was administered at 0.5 mg/kg. The mRNA
encoding PBGD was administered at 0.5 mg/kg and 0.1 mg/kg. Data are
expressed as mean.+-.SD.
[0374] FIG. 22A shows pain measurements in AIP mice subjected to
porphyria attacks caused by intraperitoneal phenobarbital
challenges. Pain levels were measured using a method reported in
Langford et al., Nature Methods. 7(6): 447-9 (2010) after the
first, second and third phenobarbital challenges. AIP mice were
administered a single intravenous injection of a chemically
modified mRNA encoding PBGD or mRNA encoding luciferase. mRNAs were
formulated in MC3 or Compound 18 lipid nanoparticles. The mRNA
encoding luciferase was administered at 0.5 mg/kg. The mRNA
encoding PBGD was administered at 0.5 mg/kg and 0.1 mg/kg. Data are
expressed as mean.+-.SD.
[0375] FIG. 22B shows rotarod measurements in AIP mice subjected to
porphyria attacks caused by intraperitoneal phenobarbital
challenges. Time spent on rotarod was measured before the
phenobarbital challenge as baseline, and after the first, second
and third phenobarbital challenges. AIP mice were administered a
single intravenous injection of a chemically modified mRNA encoding
PBGD or an mRNA encoding luciferase at the beginning of the study
(day 1). mRNAs were formulated in MC3 or Compound 18 lipid
nanoparticles. The mRNA encoding luciferase was administered at 0.5
mg/kg. The mRNA encoding PBGD was administered at 0.5 mg/kg and 0.1
mg/kg. Data are expressed as mean.+-.SD.
[0376] FIGS. 23A-B shows in vivo hepatic PBGD activity (expressed
as nM uroporphyrin 1 concentration) (FIG. 23A) and in vivo hepatic
PBGD protein expression (FIG. 23B) in wild type CD-1 mice
administered modified mRNA constructs encoding wild type PBGD or
luciferase as a control.
[0377] FIG. 24 shows the design of a multi-dose
pharmacodynamics/dose-response study to evaluate the effects of the
administration of a multiple doses of modified mRNA encoding human
wild type PBGD to AIP mice subjected to three acute porphyria
attacks triggered by phenobarbital challenges. Intraperitoneal
injections of phenobarbital and intravenous administration of PBGD
mRNA are indicated by arrows above the time line of the study.
During each induced porphyria attack, the AIP mice were
administered intravenous injections of a chemically modified mRNA
encoding PBGD (Construct #14) formulated in Compound 18 lipid
nanoparticles, which was administered at a dose of 0.5 mg/kg, 0.2
mg/kg, or 0.05 mg/kg every other week during each attack. PBS and
an mRNA encoding luciferase were used as controls.
[0378] FIG. 25 shows urinary ALA excretion levels (micrograms of
ALA per mg of creatinine) in AIP mice subjected to multiple
porphyria attacks induced by intraperitoneal phenobarbital
challenges according to the study design shown in FIG. 24. Data are
expressed as mean.+-.SD.
[0379] FIG. 26 shows urinary PBG excretion levels (micrograms of
PBG per mg of creatinine) in AIP mice subjected to multiple
porphyria attacks induced by intraperitoneal phenobarbital
challenges according to the study design shown in FIG. 24. Data are
expressed as mean.+-.SD.
[0380] FIG. 27 shows urinary porphyrin excretion levels (micrograms
of porphyrin per mg of creatinine) in AIP mice subjected to
multiple porphyria attacks induced by intraperitoneal phenobarbital
challenges according to the study design shown in FIG. 24. Data are
expressed as mean.+-.SD.
[0381] FIG. 28 shows pain measurements in AIP mice subjected to
multiple porphyria attacks induced by intraperitoneal phenobarbital
challenges according to the study design shown in FIG. 24. Pain
levels were measured using a method reported in Langford et al.,
Nature Methods, 7(6): 447-9 (2010) after the first, second and
third phenobarbital challenges. Data are expressed as mean.+-.SD.
P-values obtained from repeated measures ANOVA. **p<0.01,
***p<0.001
[0382] FIG. 29 present assessments of peripheral neuropathy as
determined by rotarod. Measurement at baseline and measurements
after each phenobarbital challenge are shown. AIP mice were
subjected to three porphyria attacks induced by intraperitoneal
phenobarbital challenges according to the study design shown in
FIG. 24. The figure also shows data corresponding to untreated wild
type animals (WT). Data are expressed as mean with SD. In case of
the statistical analysis (see asterisks), data were log transformed
prior to repeated measures ANOVA analysis to equalize variances and
comparisons between baseline and marks obtained after each
induction were made using Bonferroni's multiple comparisons.
*p<0.05, **p<0.01, ***p<0.001
[0383] FIG. 30 present assessments of peripheral neuropathy as
determined by gait pattern analysis. Measurements at baseline and
measurements after each phenobarbital challenge are shown. AIP mice
were subjected to three porphyria attacks induced by
intraperitoneal phenobarbital challenges according to the study
design shown in FIG. 24. The figure also shows data corresponding
to untreated wild type animals (WT). The stride length was measured
in the two hind legs of each of the animals. The bars represent
mean with S.D. Data were log transformed prior to repeated measures
ANOVA analysis to equalize variances and comparisons between
baseline and footprint marks obtained after each induction were
made using Bonferroni's multiple comparisons. *p<0.05,
**p<0.01, ***p<0.001
[0384] FIG. 31 present assessments of sciatic nerve dysfunction due
to recurrent acute porphyria attacks in AIP mice induced by
phenobarbital challenge according to the study design shown in FIG.
24. Amplitude values from sciatic nerve conduction data correspond
to mice under control conditions or after administration of a
modified mRNA encoding wild type human PBGD (at doses, e.g., 0.05
mg/kg, 0.2 mg/kg, and 0.5 mg/kg). Control measurements correspond
to: AIP mice treated with buffer, AIP mice administered mRNA
encoding luciferase, untreated wild type mice, and wild type mice
treated with phenobarbital. The bars represent mean with S.D.
P-values were obtained from a one-way ANOVA. *p<0.05,
***p<0.001
[0385] FIGS. 32A, 32B, and 32C present assessments of serum
transaminases and bilirubin levels in AIP mice according to the
study design shown in FIG. 24. Each drawing presents measurements
from mice under control conditions or after 3 IV administrations of
modified mRNA encoding human wild type PBGD (at doses of 0.05
mg/kg, 0.2 mg/kg, and 0.5 mg/kg). Control measurements correspond
to: AIP mice treated with buffer, AIP mice treated with mRNA
encoding luciferase, untreated wild type mice, and wild type mice
treated with phenobarbital. The bars represent mean with S.D. FIG.
32A shows levels of serum ALT transaminase (I.U./L) at sacrifice.
FIG. 32B shows levels of serum AST transaminase (I.U./L) at
sacrifice. FIG. 32C shows serum bilirubin levels (mg/dL).
[0386] FIGS. 33A, 33B, and 33C show the effect of the
administration of a modified mRNA encoding wild type PBGD on AIP
urine biomarker levels (ALA, PBG, and porphyrin, respectively)
during a porphyria attack triggered by phenobarbital challenges.
Each drawing shows results corresponding to AIP mice treated with
PBS without phenobarbital, mice treated with mRNA encoding
luciferase, and a modified mRNA encoding wild type human PBGD. FIG.
33A shows the effect on urine ALA levels, FIG. 33B shows the effect
on urine PBG levels, and FIG. 33C shows the effect on urine
porphyrin levels.
[0387] FIG. 34 shows the pharmacokinetic profile of hepatic PBGD
activity in WT CD-1 mice after IV administration of either a
control mRNA encoding luciferase, or a modified mRNA encoding human
wild type PBGD (0.5 mg/kg). The error bars represent S.D. Hepatic
PBGD activity was quantitated based on uroporphyrin levels (nM).
Splenic PBGD activity did not differ between mice administered
luciferase (vehicle control) mRNA and hPBGD mRNA (data not
shown).
[0388] FIG. 35A shows the decay in hepatic PBGD activity in WT CD-1
mice after IV administration of a modified mRNA encoding human wild
type PBGD (0.5 mg/kg). FIG. 35B shows the decay in hepatic PBGD
activity in AIP mice after IV administration of a modified mRNA
encoding human wild type PBGD. In both cases, the terminal t1/2 of
the PBGD activity, determined by noncompartmental analysis, was 8
days.
[0389] FIG. 36 shows the pharmacokinetic of hepatic human PBGD
protein concentration in WT CD-1 mice after IV administration of
either a control mRNA encoding luciferase, or a modified mRNA
encoding human wild type PBGD (0.5 mg/kg). The error bars represent
S.D. Human PBGD protein levels in liver were quantified by LC-MS/MS
using a human specific peptide (ASYPGLQFEIIAMSTTGDK; SEQ ID NO:
85).
[0390] FIG. 37 shows hepatic PBGD activity (measured as
uroporphyrin production, nM; left axis) and hPBGD mRNA levels in
liver (pg/uL; right axis) in WT CD1 mice administered a single IV
bolus of human PBGD mRNA (0.5 mg/kg).
[0391] FIGS. 38A, 38B, 38C, 38D, 38E, and 38F show hepatic
expression of PBGD protein observed in WT CD1 mice after IV
administration of modified mRNA encoding human wild type PBGD (0.5
mg/kg). FIGS. 38A, 38B, 38C, 38D, and 38E show hepatic PBGD protein
levels at 2 hours, 6 hours, 10 hours, 16 hours, and 24 hours,
respectively, after administration of modified mRNA encoding human
wild type PBGD. FIG. 38F shows basal PBGD protein expression levels
detected by Novus anti-PBGD antibody which doesn't cross-react with
endogenous mouse PBGD protein.
[0392] FIG. 39 shows hepatic PBGD activity levels after a single IV
administration of 5-methoxyuracil comprising chemically modified
mRNAs encoding wild type PBGD to AIP mice. A single IV dose of mRNA
construct at 0.2 mg/kg or 0.5 mg/kg was administered. Levels of
hepatic PBGD activity observed in wild type mice and AIP mice are
also shown for comparison. Data are expressed as mean.+-.SD.
[0393] FIGS. 40A and 40B shows a decrease in systolic (FIG. 40A)
and diastolic (FIG. 40B) blood pressure in AIP mice administered a
single IV injection of human PBGD mRNA (0.5 mg/kg). A porphyric
attack was induced in AIP mice by daily intraperitoneal
phenobarbital injections. Blood pressure in WT and AIP mice that
were not administered phenobarbital are shown as control
groups.
[0394] FIG. 41 shows the hepatic PBGD activity 1 and 2 days after a
single IV administration of human PBGD mRNA or luciferase vehicle
control mRNA (0.5 mg/kg) to Sprague Dawley rats. Data are presented
as mean.+-.SD. In contrast, PBGD activity levels in spleen did not
differ between treatment arms 1-2 days post-injection in these
rats.
[0395] FIG. 42A shows hepatic expression of PBGD protein observed
in Sprague Dawley rats at 24 hours after IV administration of
modified mRNA encoding human wild type PBGD (1 mg/kg). FIG. 42B
shows basal PBGD protein expression levels detected by Novus
anti-PBGD antibody which doesn't cross-react with endogenous rat
PBGD protein significantly.
[0396] FIGS. 43A-B shows in vivo hepatic PBGD activity (expressed
as expressed as pmol uroporphyrinogen/mg protein/hour) (FIG. 43A)
and in vivo hepatic PBGD protein expression (FIG. 43B) in
Cynomolgus macaque liver following administration of modified mRNA
constructs encoding wild type PBGD.
DETAILED DESCRIPTION
[0397] The present invention provides mRNA therapeutics for the
treatment of acute intermittent porphyria (AIP). Acute intermittent
porphyria (AIP) is a genetic metabolic disorder affecting the
production of heme, the oxygen-binding prosthetic group of
hemoglobin. AIP is caused by mutations in the HMBS gene, which
codes for the enzyme porphobilinogen deaminase (PBGD). Without
porphobilinogen deaminase (PBGD), a necessary cytoplasmic enzyme,
heme synthesis cannot finish, and the metabolite porphobilinogen
accumulates in the cytoplasm. mRNA therapeutics are particularly
well-suited for the treatment of AIP as the technology provides for
the intracellular delivery of mRNA encoding PBGD followed by de
novo synthesis of functional PBGD protein within target cells.
After delivery of mRNA to the target cells, the desired PBGD
protein is expressed by the cells' own translational machinery, and
hence, fully functional PBGD protein replaces the defective or
missing protein.
[0398] One challenge associated with delivering nucleic acid-based
therapeutics (e.g., mRNA therapeutics) in vivo stems from the
innate immune response which can occur when the body's immune
system encounters foreign nucleic acids. Foreign mRNAs can activate
the immune system via recognition through toll-like receptors
(TLRs), in particular TLR7/8, which is activated by single-stranded
RNA (ssRNA). In nonimmune cells, the recognition of foreign mRNA
can occur through the retinoic acid-inducible gene 1 (RIG-1).
Immune recognition of foreign mRNAs can result in unwanted cytokine
effects including interleukin-1.beta. (IL-1.beta.) production,
tumor necrosis factor-.alpha. (TNF-.alpha.) distribution and a
strong type I interferon (type I IFN) response. The instant
invention features the incorporation of different modified
nucleotides within therapeutic mRNAs to minimize the immune
activation and optimize the translation efficiency of mRNA to
protein. Particular aspects of the invention feature a combination
of nucleotide modification to reduce the innate immune response and
sequence optimization, in particular, within the open reading frame
(ORF) of therapeutic mRNAs encoding PBGD to enhance protein
expression.
[0399] Certain embodiments of the mRNA therapeutic technology of
the instant invention also feature delivery of mRNA encoding PBGD
via a lipid nanoparticle (LNP) delivery system. Lipid nanoparticles
(LNPs) are an ideal platform for the safe and effective delivery of
mRNAs to target cells. LNPs have the unique ability to deliver
nucleic acids by a mechanism involving cellular uptake,
intracellular transport and endosomal release or endosomal escape.
The instant invention features novel ionizable lipid-based LNPs
combined with mRNA encoding PBGD which have improved properties
when administered in vivo. Without being bound in theory, it is
believed that the novel ionizable lipid-based LNP formulations of
the invention have improved properties, for example, cellular
uptake, intracellular transport and/or endosomal release or
endosomal escape. LNPs administered by systemic route (e.g.,
intravenous (IV) administration), for example, in a first
administration, can accelerate the clearance of subsequently
injected LNPs, for example, in further administrations. This
phenomenon is known as accelerated blood clearance (ABC) and is a
key challenge, in particular, when replacing deficient enzymes
(e.g., PBGD) in a therapeutic context. This is because repeat
administration of mRNA therapeutics is in most instances essential
to maintain necessary levels of enzyme in target tissues in
subjects (e.g., subjects suffering from AIP.) Repeat dosing
challenges can be addressed on multiple levels. mRNA engineering
and/or efficient delivery by LNPs can result in increased levels
and or enhanced duration of protein (e.g., PBGD) being expressed
following a first dose of administration, which in turn, can
lengthen the time between first dose and subsequent dosing. It is
known that the ABC phenomenon is, at least in part, transient in
nature, with the immune responses underlying ABC resolving after
sufficient time following systemic administration. As such,
increasing the duration of protein expression and/or activity
following systemic delivery of an mRNA therapeutic of the invention
in one aspect, combats the ABC phenomenon. Moreover, LNPs can be
engineered to avoid immune sensing and/or recognition and can thus
further avoid ABC upon subsequent or repeat dosing. Exemplary
aspect of the invention feature novel LNPs which have been
engineered to have reduced ABC.
1. PORPHOBILINOGEN DEAMINASE (PBGD)
[0400] Porphobilinogen deaminase (PBGD; EC 4.3.1.8) is the third
enzyme of the biosynthetic pathway leading to the production of
heme. It catalyzes the synthesis of hydroxymethylbilane by stepwise
condensation of 4 porphobilinogen units. Hydroxymethylbilane is
then converted to uroporphyrinogen III by uroporphyrinogen III
synthetase. The structure of 40-42 kDa porphobilinogen deaminase,
which is highly conserved amongst organisms, consists of three
domains. Domains 1 and 2 are structurally very similar: each
consisting of five beta-sheets and three alpha helices in humans.
Domain 3 is positioned between the other two and has a flattened
beta-sheet geometry. A dipyrrole, a cofactor of this enzyme
consisting of two condensed porphobilinogen molecules, is
covalently attached to domain 3 and extends into the active site,
the cleft between domains 1 and 2. Several positively charged
arginine residues, positioned to face the active site from domains
1 and 2, have been shown to stabilize the carboxylate
functionalities on the incoming porphobilinogen as well as the
growing pyrrole chain. These structural features presumably favor
the formation of the final hydroxymethylbilane product.
Porphobilinogen deaminase usually exists in dimer units in the
cytoplasm of the cell.
[0401] The most well-known health issue involving porphobilinogen
deaminase is acute intermittent porphyria (AIP), an autosomal
dominant genetic disorder where insufficient hydroxymethylbilane is
produced, leading to a build-up of porphobilinogen in the cytoplasm
as well as elevation in ALA and PBG levels in plasma and urine.
This is caused by a gene mutation that, in 90% of cases, causes
decreased amounts of enzyme. However, mutations where less-active
enzymes and/or different isoforms have been described. See
Grandchamp et al. (1989) Nucleic Acids Res. 17:6637-49; and Astrin
et al. (1994) Hum. Mut. 4:243-52.
[0402] The coding sequence (CDS) for wild type PBGD canonical mRNA
sequence, corresponding to isoform 1, is described at the NCBI
Reference Sequence database (RefSeq) under accession number
NM_000109.3 ("Homo sapiens hydroxymethylbilane synthase (HBMS),
transcript variant 1, mRNA"). The wild type PBGD canonical protein
sequence, corresponding to isoform 1, is described at the RefSeq
database under accession number NP_000181.2 ("Porphobilinogen
deaminase isoform 1 [Homo sapiens]"). The PBGD isoform 1 protein is
361 amino acids long. It is noted that the specific nucleic acid
sequences encoding the reference protein sequence in the Ref Seq
sequences are the coding sequence (CDS) as indicated in the
respective RefSeq database entry.
[0403] Isoforms 2, 3, and 4 are produced by alternative
splicing.
[0404] The RefSeq protein and mRNA sequences for isoform 2 of PBGD
are NP_001019553.1 and NM_001024382.1, respectively. The RefSeq
protein and mRNA sequences for isoform 3 of PBGD are NP_001245137.1
and NM_001258208.1, respectively. The RefSeq protein and mRNA
sequences for isoform 4 of PBGD are NP_001245138.1 and
NM_001258209.1, respectively. Isoforms 2, 3, and 4 PBGD are encoded
by the CDS disclosed in each one of the above mentioned mRNA RefSeq
entries.
[0405] The isoform 2 polynucleotide contains an alternate, in-frame
exon in the 5' coding region and uses a downstream start codon,
compared to variant 1. It encodes a PBGD isoform 2 polypeptide,
which has a shorter N-terminus compared to isoform 1. The PBGD
isoform 2 protein is 344 amino acids long and lacks the amino acids
corresponding to positions 1-17 in isoform 1.
[0406] The isoform 3 polynucleotide lacks an alternate in-frame
exon compared to variant 1. The resulting PBGD isoform 3
polypeptide has the same N- and C-termini but is shorter compared
to isoform 1. The PBGD isoform 3 protein is 321 amino acids long
and lacks the amino acids corresponding to positions 218-257 in
isoform 1. The isoform 4 polynucleotide uses an alternate splice
junction at the 3' end of the first exon and lacks an alternate
in-frame exon compared to variant 1. The resulting PBGD isoform 4
polypeptide is shorter at the N-terminus and lacks an alternate
internal segment compared to isoform 1. The PBGD isoform 4 protein
is 304 amino acids long and lacks the amino acids corresponding to
positions 1-17 and positions 218-257 in isoform 1.
[0407] In certain aspects, the invention provides a polynucleotide
(e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA))
comprising a nucleotide sequence (e.g., an open reading frame
(ORF)) encoding a PBGD polypeptide. In some embodiments, the PBGD
polypeptide of the invention is a wild type PBGD isoform 1, 2, 3,
or 4 protein. In some embodiments, the PBGD polypeptide of the
invention is a variant, a peptide or a polypeptide containing a
substitution, and insertion and/or an addition, a deletion and/or a
covalent modification with respect to a wild-type PBGD isoform 1,
2, 3, or 4 sequence. In some embodiments, sequence tags or amino
acids, can be added to the sequences encoded by the polynucleotides
of the invention (e.g., at the N-terminal or C-terminal ends),
e.g., for localization. In some embodiments, amino acid residues
located at the carboxy, amino terminal, or internal regions of a
polypeptide of the invention can optionally be deleted providing
for fragments.
[0408] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) comprising a nucleotide sequence (e.g., an ORF) of the
invention encodes a substitutional variant of a PBGD isoform 1, 2,
3, or 4 sequence, which can comprise one, two, three or more than
three substitutions. In some embodiments, the substitutional
variant can comprise one or more conservative amino acids
substitutions. In other embodiments, the variant is an insertional
variant. In other embodiments, the variant is a deletional
variant.
[0409] As recognized by those skilled in the art, PBGD isoform 1,
2, 3, or 4 protein fragments, functional protein domains, variants,
and homologous proteins (orthologs) are also considered to be
within the scope of the PBGD polypeptides of the invention.
Nonlimiting examples of polypeptides encoded by the polynucleotides
of the invention are shown in FIGS. 1 to 4. For example, FIG. 1
shows the amino acid sequence of human PBGD wild type isoform
1.
[0410] Certain compositions and methods presented in this
disclosure refer to the protein or polynucleotide sequences of PBGD
isoform 1. A person skilled in the art will understand that such
disclosures are equally applicable to any other isoforms of PBGD
known in the art.
2. POLYNUCLEOTIDES AND OPEN READING FRAMES (ORFS)
[0411] The instant invention features mRNAs for use in treating
(i.e., prophylactically and/or therapeutically treating) AIP. The
mRNAs featured for use in the invention are administered to
subjects and encode human porphobilinogen deaminase (PBGD)
proteins(s) in vivo. Accordingly, the invention relates to
polynucleotides, e.g., mRNA, comprising an open reading frame of
linked nucleosides encoding human porphobilinogen deaminase (PBGD),
isoforms thereof, functional fragments thereof, and fusion proteins
comprising PBGD. In some embodiments, the open reading frame is
sequence-optimized. In particular embodiments, the invention
provides sequence-optimized polynucleotides comprising nucleotides
encoding the polypeptide sequence of isoforms 1, 2, 3 or 4 of human
PBGD, or sequence having high sequence identity with those sequence
optimized polynucleotides.
[0412] In certain aspects, the invention provides polynucleotides
(e.g., a RNA, e.g., an mRNA) that comprise a nucleotide sequence
(e.g., an ORF) encoding one or more PBGD polypeptides. In some
embodiments, the encoded PBGD polypeptide of the invention can be
selected from:
[0413] (i) a full length PBGD polypeptide (e.g., having the same or
essentially the same length as wild-type PBGD isoform 1, 2, 3 or
4);
[0414] (ii) a functional fragment of any of the PBGD isoforms
described herein (e.g., a truncated (e.g., deletion of carboxy,
amino terminal, or internal regions) sequence shorter than one of
wild-type isoforms 1, 2, 3 or 4; but still retaining PBGD enzymatic
activity);
[0415] (iii) a variant thereof, e.g., full length or truncated
isoform 1, 2, 3, or 4 protein in which one or more amino acids have
been replaced, e.g., variants that retain all or most of the PBGD
activity of the polypeptide with respect to a reference isoform
(such as, e.g., T59I, D178N, or any other natural or artificial
variants known in the art, or a variant comprising the I291M and
N340S mutations); or
[0416] (iv) a fusion protein comprising (i) a full length PBGD
isoform 1, 2, 3, or 4 protein, a functional fragment or a variant
thereof, and (ii) at least one heterologous protein (e.g.,
Apolipoprotein A1).
[0417] In certain embodiments, the encoded PBGD polypeptide is a
mammalian PBGD polypeptide, such as a human PBGD polypeptide, a
functional fragment or a variant thereof.
[0418] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention increases PBGD protein expression levels
and/or detectable PBGD enzymatic activity levels in cells when
introduced in those cells, e.g., by at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or at least 100%, compared to PBGD protein
expression levels and/or detectable PBGD enzymatic activity levels
in the cells prior to the administration of the polynucleotide of
the invention. PBGD protein expression levels and/or PBGD enzymatic
activity can be measured according to methods know in the art. In
some embodiments, the polynucleotide is introduced to the cells in
vitro. In some embodiments, the polynucleotide is introduced to the
cells in vivo.
[0419] In some embodiments, the polynucleotides (e.g., a RNA, e.g.,
an mRNA) of the invention comprise a nucleotide sequence (e.g., an
ORF) that encodes a wild-type human PBGD, e.g., wild-type isoform 1
of human PBGD (SEQ ID NO: 1, see FIG. 1), wild-type isoform 2 of
human PBGD (SEQ ID NO: 3, see FIG. 2), wild-type isoform 3 of human
PBGD (SEQ ID NO: 5, see FIG. 3), or wild-type isoform 4 of human
PBGD (SEQ ID NO: 7, see FIG. 4).
[0420] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a sequence optimized nucleic
acid sequence, wherein the open reading frame (ORF) of the sequence
optimized nucleic acid sequence is derived from a wild-type PBGD
sequence (e.g., wild-type isoforms 1, 2, 3 or 4). For example, for
polynucleotides of invention comprising a sequence optimized ORF
encoding PBGD isoform 2, the corresponding wild type sequence is
the native PBGD isoform 2. Similarly, for a sequence optimized mRNA
encoding a functional fragment of isoform 1, the corresponding wild
type sequence is the corresponding fragment from PBGD isoform
1.
[0421] In some embodiments, the polynucleotides (e.g., a RNA, e.g.,
an mRNA) of the invention comprise a nucleotide sequence encoding
PBGD isoform 1 having the full length sequence of human PBGD
isoform 1 (i.e., including the initiator methionine). In mature
human PBGD isoform 1, the initiator methionine can be removed to
yield a "mature PBGD" comprising amino acid residues of 2-361 of
the translated product. The teachings of the present disclosure
directed to the full sequence of human PBGD (amino acids 1-361) are
also applicable to the mature form of human PBGD lacking the
initiator methionine (amino acids 2-361). Thus, in some
embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) of
the invention comprise a nucleotide sequence encoding PBGD isoform
1 having the mature sequence of human PBGD isoform 1 (i.e., lacking
the initiator methionine). In some embodiments, the polynucleotide
(e.g., a RNA, e.g., an mRNA) of the invention comprising a
nucleotide sequence encoding PBGD isoform 1 having the full length
or mature sequence of human PBGD isoform 1 is sequence
optimized.
[0422] In some embodiments, the polynucleotides (e.g., a RNA, e.g.,
an mRNA) of the invention comprise a nucleotide sequence (e.g., an
ORF) encoding a mutant PBGD polypeptide, e.g., a double mutant
I291M/N340S PBGD. The protein (SEQ ID NO: 152) and polynucleotide
(SEQ ID NO: 153) of double mutant I291M/N340S human PBGD isoform 1
("SM PBGD") are shown in FIG. 10A and FIG. 11. In some embodiments,
the polynucleotides of the invention comprise an ORF encoding a
PBGD polypeptide that comprises at least one point mutation in the
PBGD sequence and retains PBGD enzymatic activity. In some
embodiments, the mutant PBGD polypeptide has a PBGD activity which
is at least 10%, at least 15%, at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
100% of the PBGD activity of the corresponding wild-type PBGD
(i.e., the same PBGD isoform but without the mutation(s)). In some
embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the
invention comprising an ORF encoding a mutant PBGD polypeptide is
sequence optimized.
[0423] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) that encodes a PBGD polypeptide with mutations that do not
alter PBGD enzymatic activity. Such mutant PBGD polypeptides can be
referred to as function-neutral. In some embodiments, the
polynucleotide comprises an ORF that encodes a mutant PBGD
polypeptide comprising one or more function-neutral point
mutations.
[0424] In some embodiments, the mutant PBGD polypeptide has higher
PBGD enzymatic activity than the corresponding wild-type PBGD. In
some embodiments, the mutant PBGD polypeptide has a PBGD activity
that is at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, or at
least 100% higher than the activity of the corresponding wild-type
PBGD (i.e., the same PBGD isoform but without the mutation(s)).
[0425] In some embodiments, the polynucleotides (e.g., a RNA, e.g.,
an mRNA) of the invention comprise a nucleotide sequence (e.g., an
ORF) encoding a functional PBGD fragment, e.g., where one or more
fragments correspond to a polypeptide subsequence of a wild type
PBGD polypeptide and retain PBGD enzymatic activity. In some
embodiments, the PBGD fragment has a PBGD activity which is at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or at least
100% of the PBGD activity of the corresponding full length PBGD. In
some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA)
of the invention comprising an ORF encoding a functional PBGD
fragment is sequence optimized.
[0426] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) encoding a PBGD fragment that has higher PBGD enzymatic
activity than the corresponding full length PBGD. Thus, in some
embodiments the PBGD fragment has a PBGD activity which is at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or at least 100%
higher than the PBGD activity of the corresponding full length
PBGD.
[0427] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) encoding a PBGD fragment that is 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% shorter than wild-type isoform 1, 2,
3, or 4 of PBGD.
[0428] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence,
functional fragment, or variant thereof), wherein the nucleotide
sequence is at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% identical to the sequence of SEQ ID
NO:2, 4, 6 or 8 (see, e.g., panel D in FIGS. 1, 2, 3 and 4,
respectively).
[0429] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence,
functional fragment, or variant thereof), wherein the nucleotide
sequence has at least 70%, at least 71%, at least 72%, at least
73%, at least 74%, at least 75%, at least 76%, at least 77%, at
least 78%, at least 79%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or 100% sequence identity
to a sequence selected from the group consisting of SEQ ID NOs: 9
to 33, and 89 to 117. See TABLE 2.
[0430] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence,
functional fragment, or variant thereof), wherein the nucleotide
sequence has 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%,
70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to
75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to
100%, sequence identity to a sequence selected from the group
consisting of SEQ ID NOs: 9 to 33, and 89 to 117. See TABLE 2.
[0431] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises an ORF encoding a PBGD
polypeptide (e.g., the wild-type sequence, functional fragment, or
variant thereof), wherein the polynucleotide comprises a nucleic
acid sequence having 70% to 100%, 75% to 100%, 80% to 100%, 85% to
100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%,
70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95%
to 100%, sequence identity to a sequence selected from the group
consisting of SEQ ID NOs: 118-148. See TABLE 5.
[0432] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence,
functional fragment, or variant thereof), wherein the nucleotide
sequence is at least 75%, at least 76%, at least 77%, at least 78%,
at least 79%, least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or 100% identical to the sequence of
SEQ ID NO:2, 4, 6 or 8 (see, e.g., panel D FIGS. 1, 2, 3 and 4,
respectively).
[0433] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises a nucleotide sequence (e.g., an
ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence,
functional fragment, or variant thereof), wherein the nucleotide
sequence is between 70% and 90% identical; between 75% and 85%
identical; between 76% and 84% identical; between 77% and 83%
identical, between 77% and 82% identical, or between 78% and 81%
identical to the sequence of SEQ ID NO:2, 4, 6 or 8 (see, e.g.,
panel D in FIGS. 1, 2, 3, and 4, respectively).
[0434] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention comprises from about 900 to about 100,000
nucleotides (e.g., from 900 to 1,000, from 900 to 1,100, from 900
to 1,200, from 900 to 1,300, from 900 to 1,400, from 900 to 1,500,
from 1,000 to 1,100, from 1,000 to 1,100, from 1,000 to 1,200, from
1,000 to 1,300, from 1,000 to 1,400, from 1,000 to 1,500, from
1,083 to 1,200, from 1,083 to 1,400, from 1,083 to 1,600, from
1,083 to 1,800, from 1,083 to 2,000, from 1,083 to 3,000, from
1,083 to 5,000, from 1,083 to 7,000, from 1,083 to 10,000, from
1,083 to 25,000, from 1,083 to 50,000, from 1,083 to 70,000, or
from 1,083 to 100,000).
[0435] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g.,
an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence,
functional fragment, or variant thereof), wherein the length of the
nucleotide sequence (e.g., an ORF) is at least 500 nucleotides in
length (e.g., at least or greater than about 500, 600, 700, 80,
900, 1,000, 1,050, 1,083, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,
1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500,
2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400,
3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300,
4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200,
5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 7,000,
8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,
70,000, 80,000, 90,000 or up to and including 100,000
nucleotides).
[0436] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g.,
an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence,
functional fragment, or variant thereof), and further comprises at
least one nucleic acid sequence that is noncoding, e.g., a miRNA
binding site. In some embodiments, the polynucleotide (e.g., a RNA,
e.g., an mRNA) of the invention further comprises a 5'-UTR (e.g.,
selected from the sequences of SEQ ID NOs: 39 to 56, 83, 189 to
191) and a 3'UTR (e.g., selected from the sequences of SEQ ID NOs:
57 to 81, 84, 149 to 151, 161 to 172, 192 to 199). In some
embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the
invention comprises a sequence selected from the group consisting
of SEQ ID NO: 118-148, e.g., SEQ ID NO: 133, 141, 144 or 145. In a
further embodiment, the polynucleotide (e.g., a RNA, e.g., an mRNA)
comprises a 5' terminal cap (e.g., Cap0, Cap1, ARCA, inosine,
N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine,
8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,
2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof)
and a poly-A-tail region (e.g., about 100 nucleotides in length).
In a further embodiment, the polynucleotide (e.g., a RNA, e.g., an
mRNA) a comprises a 3' UTR comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 149 to 151, or
any combination thereof. In a further embodiment, the
polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 3' UTR
comprising a nucleic acid sequence of SEQ ID NO: 150. In a further
embodiment, the polynucleotide (e.g., a RNA, e.g., an mRNA)
comprises a 3' UTR comprising a nucleic acid sequence of SEQ ID NO:
151.
[0437] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) comprising a nucleotide sequence
(e.g., an ORF) encoding a PBGD polypeptide is single stranded or
double stranded.
[0438] In some embodiments, the polynucleotide of the invention
comprising a nucleotide sequence (e.g., an ORF) encoding a PBGD
polypeptide (e.g., the wild-type sequence, functional fragment, or
variant thereof) is DNA or RNA. In some embodiments, the
polynucleotide of the invention is RNA. In some embodiments, the
polynucleotide of the invention is, or functions as, a messenger
RNA (mRNA). In some embodiments, the mRNA comprises a nucleotide
sequence (e.g., an ORF) that encodes at least one PBGD polypeptide,
and is capable of being translated to produce the encoded PBGD
polypeptide in vitro, in vivo, in situ, or ex vivo.
[0439] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) comprises a sequence-optimized
nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide
(e.g., the wild-type sequence, functional fragment, or variant
thereof), wherein the polynucleotide comprises at least one
chemically modified nucleobase, e.g., 5-methoxyuracil. In some
embodiments, the polynucleotide further comprises a miRNA binding
site, e.g., a miRNA binding site that binds to miR-142 and/or a
miRNA binding site that binds to miR-126. In some embodiments, the
polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein is
formulated with a delivery agent comprising, e.g., a compound
having the Formula (I), e.g., any of Compounds 1-232, e.g.,
Compound 18; a compound having the Formula (III), (IV), (V), or
(VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a
compound having the Formula (VIII), e.g., any of Compounds 419-428,
e.g., Compound 428, or any combination thereof. In some
embodiments, the delivery agent comprises Compound 18, DSPC,
Cholesterol, and Compound 428, e.g., with a mole ratio of about
50:10:38.5:1.5.
3. SIGNAL SEQUENCES
[0440] The polynucleotides (e.g., a RNA, e.g., an mRNA) of the
invention can also comprise nucleotide sequences that encode
additional features that facilitate trafficking of the encoded
polypeptides to therapeutically relevant sites. One such feature
that aids in protein trafficking is the signal sequence, or
targeting sequence. The peptides encoded by these signal sequences
are known by a variety of names, including targeting peptides,
transit peptides, and signal peptides. In some embodiments, the
polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotide
sequence (e.g., an ORF) that encodes a signal peptide operably
linked to a nucleotide sequence that encodes a PBGD polypeptide
described herein.
[0441] In some embodiments, the "signal sequence" or "signal
peptide" is a polynucleotide or polypeptide, respectively, which is
from about 9 to 200 nucleotides (3-70 amino acids) in length that,
optionally, is incorporated at the 5' (or N-terminus) of the coding
region or the polypeptide, respectively. Addition of these
sequences results in trafficking the encoded polypeptide to a
desired site, such as the endoplasmic reticulum or the mitochondria
through one or more targeting pathways. Some signal peptides are
cleaved from the protein, for example by a signal peptidase after
the proteins are transported to the desired site.
[0442] In some embodiments, the polynucleotide of the invention
comprises a nucleotide sequence encoding a PBGD polypeptide,
wherein the nucleotide sequence further comprises a 5' nucleic acid
sequence encoding a heterologous signal peptide.
4. FUSION PROTEINS
[0443] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) can comprise more than one nucleic
acid sequence (e.g., an ORF) encoding a polypeptide of interest. In
some embodiments, polynucleotides of the invention comprise a
single ORF encoding a PBGD polypeptide, a functional fragment, or a
variant thereof. However, in some embodiments, the polynucleotide
of the invention can comprise more than one ORF, for example, a
first ORF encoding a PBGD polypeptide (a first polypeptide of
interest), a functional fragment, or a variant thereof, and a
second ORF expressing a second polypeptide of interest. In some
embodiments, two or more polypeptides of interest can be
genetically fused, i.e., two or more polypeptides can be encoded by
the same ORF. In some embodiments, the polynucleotide can comprise
a nucleic acid sequence encoding a linker (e.g., a G4S peptide
linker or another linker known in the art) between two or more
polypeptides of interest.
[0444] In some embodiments, a polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) can comprise two, three, four, or more
ORFs, each expressing a polypeptide of interest.
[0445] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) can comprise a first nucleic acid
sequence (e.g., a first ORF) encoding a PBGD polypeptide and a
second nucleic acid sequence (e.g., a second ORF) encoding a second
polypeptide of interest.
[0446] In some embodiments, the polynucleotide of the invention
(e.g., an mRNA) comprises a nucleic acid encoding a PBGD fusion
protein, wherein said fusion protein comprises an apolipoprotein A1
fused to PBGD. In a particular embodiment, the apolipoprotein A1
(ApoA1) fusion component is a mature form of human apolipoprotein
A1 without the native signal peptide and propeptide sequence. See
FIGS. 12A-D. In a particular embodiment, the ApoA1-PBGD fusion
protein comprises, consists, or consists essentially of the
sequence of SEQ ID NO: 154 (see FIG. 12A). In a particular
embodiment, the polypeptide of the invention is encoded by a
nucleic acid sequence encoding an ApoA1-PBGD fusion protein,
wherein said nucleic acid comprises, consists, or consists
essentially of the sequence of SEQ ID NO: 155 (see FIG. 12D).
[0447] In some embodiments, a polynucleotide of the invention can
comprise a portion encoding PBGD (e.g., a wild type PBGD or a
variant such as the SM gain of function variant), and, in some
embodiments, an ApoA1 component. For example, the polynucleotides
of the invention (e.g., mRNA) can comprise, for example,
polynucleotides encoding (i) human PBGD isoform 1 (human
housekeeping PBGD), (ii) human PBGD isoform 2 (human
erythroid-specific PBGD), (iii) SM variant (I291M and N340S double
mutant) of PBGD1, (iv) SM variant of PBGD2, (v) human
apolipoprotein A.sub.1 fused to human PBGD1, (vi) human
apolipoprotein A1 fused to PBGD2, (vii) human apolipoprotein A1
fused to SM variant of PBGD1, (viii) human apolipoprotein A1 fused
to SM variant of PBGD2, or (ix) combinations thereof. In some
embodiments, the polynucleotides have been sequence optimized
(e.g., see TABLE 2 and the sequences disclosed in International
Publication WO2010/036118, which is herein incorporated by
reference in its entirety).
5. SEQUENCE OPTIMIZATION OF NUCLEOTIDE SEQUENCE ENCODING A PBGD
POLYPEPTIDE
[0448] In some embodiments, the polynucleotide (e.g., a RNA, e.g.,
an mRNA) of the invention is sequence optimized. In some
embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the
invention comprises a nucleotide sequence (e.g., an ORF) encoding a
PBGD polypeptide, a nucleotide sequence (e.g., an ORF) encoding
another polypeptide of interest, a 5'-UTR, a 3'-UTR, a miRNA, a
nucleotide sequence encoding a linker, or any combination thereof,
that is sequence optimized.
[0449] A sequence optimized nucleotide sequence, e.g., a codon
optimized mRNA sequence encoding a PBGD polypeptide, is a sequence
comprising at least one synonymous nucleobase substitution with
respect to a reference sequence (e.g., a wild type nucleotide
sequence encoding a PBGD polypeptide).
[0450] A sequence optimized nucleotide sequence can be partially or
completely different in sequence from the reference sequence. For
example, a reference sequence encoding polyserine uniformly encoded
by TCT codons can be sequence optimized by having 100% of its
nucleobases substituted (for each codon, T in position 1 replaced
by A, C in position 2 replaced by G, and T in position 3 replaced
by C) to yield a sequence encoding polyserine which would be
uniformly encoded by AGC codons. The percentage of sequence
identity obtained from a global pairwise alignment between the
reference polyserine nucleic acid sequence and the sequence
optimized polyserine nucleic acid sequence would be 0%. However,
the protein products from both sequences would be 100%
identical.
[0451] Some sequence optimization (also sometimes referred to codon
optimization) methods are known in the art (and discussed in more
detail below) and can be useful to achieve one or more desired
results. These results can include, e.g., matching codon
frequencies in certain tissue targets and/or host organisms to
ensure proper folding; biasing G/C content to increase mRNA
stability or reduce secondary structures; minimizing tandem repeat
codons or base runs that can impair gene construction or
expression; customizing transcriptional and translational control
regions; inserting or removing protein trafficking sequences;
removing/adding post translation modification sites in an encoded
protein (e.g., glycosylation sites); adding, removing or shuffling
protein domains; inserting or deleting restriction sites; modifying
ribosome binding sites and mRNA degradation sites; adjusting
translational rates to allow the various domains of the protein to
fold properly; and/or reducing or eliminating problem secondary
structures within the polynucleotide. Sequence optimization tools,
algorithms and services are known in the art, non-limiting examples
include services from GeneArt (Life Technologies), DNA2.0 (Menlo
Park Calif.) and/or proprietary methods.
[0452] Codon options for each amino acid are given in TABLE 1.
TABLE-US-00001 TABLE 1 Codon Options Single Letter Amino Acid Code
Codon Options Isoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA,
CTG, TTA, TTG Valine V GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC
Methionine M ATG Cysteine C TGT, TGC Alanine A GCT, GCC, GCA, GCG
Glycine G GGT, GGC, GGA, GGG Proline P CCT, CCC, CCA, CCG Threonine
T ACT, ACC, ACA, ACG Serine S TCT, TCC, TCA, TCG, AGT, AGC Tyrosine
Y TAT, TAC Tryptophan W TGG Glutamine Q CAA, CAG Asparagine N AAT,
AAC Histidine H CAT, CAC Glutamic acid E GAA, GAG Aspartic acid D
GAT, GAC Ly sine K AAA, AAG Arginine R CGT, CGC, CGA, CGG, AGA, AGG
Selenocysteine Sec UGA in mRNA in presence of Selenocysteine
insertion element (SECIS) Stop codons Stop TAA, TAG, TGA
[0453] In some embodiments, a polynucleotide (e.g., a RNA, e.g., an
mRNA) of the invention comprises a sequence-optimized nucleotide
sequence (e.g., an ORF) encoding a PBGD polypeptide, a functional
fragment, or a variant thereof, wherein the PBGD polypeptide,
functional fragment, or a variant thereof encoded by the
sequence-optimized nucleotide sequence has improved properties
(e.g., compared to a PBGD polypeptide, functional fragment, or a
variant thereof encoded by a reference nucleotide sequence that is
not sequence optimized), e.g., improved properties related to
expression efficacy after administration in vivo. Such properties
include, but are not limited to, improving nucleic acid stability
(e.g., mRNA stability), increasing translation efficacy in the
target tissue, reducing the number of truncated proteins expressed,
improving the folding or prevent misfolding of the expressed
proteins, reducing toxicity of the expressed products, reducing
cell death caused by the expressed products, increasing and/or
decreasing protein aggregation.
[0454] In some embodiments, the sequence optimized nucleotide
sequence is codon optimized for expression in human subjects,
having structural and/or chemical features that avoid one or more
of the problems in the art, for example, features which are useful
for optimizing formulation and delivery of nucleic acid-based
therapeutics while retaining structural and functional integrity;
overcoming a threshold of expression; improving expression rates;
half-life and/or protein concentrations; optimizing protein
localization; and avoiding deleterious bio-responses such as the
immune response and/or degradation pathways.
[0455] In some embodiments, the polynucleotides of the invention
comprise a nucleotide sequence (e.g., a nucleotide sequence (e.g.,
an ORF) encoding a PBGD polypeptide, a nucleotide sequence (e.g.,
an ORF) encoding another polypeptide of interest, a 5'-UTR, a
3'-UTR, a microRNA binding site, a nucleic acid sequence encoding a
linker, or any combination thereof) that is sequence-optimized
according to a method comprising:
[0456] (i) substituting at least one codon in a reference
nucleotide sequence (e.g., an ORF encoding a PBGD polypeptide) with
an alternative codon to increase or decrease uridine content to
generate a uridine-modified sequence;
[0457] (ii) substituting at least one codon in a reference
nucleotide sequence (e.g., an ORF encoding a PBGD polypeptide) with
an alternative codon having a higher codon frequency in the
synonymous codon set;
[0458] (iii) substituting at least one codon in a reference
nucleotide sequence (e.g., an ORF encoding a PBGD polypeptide) with
an alternative codon to increase G/C content; or
[0459] (iv) a combination thereof.
[0460] In some embodiments, the sequence optimized nucleotide
sequence (e.g., an ORF encoding a PBGD polypeptide) has at least
one improved property with respect to the reference nucleotide
sequence.
[0461] In some embodiments, the sequence optimization method is
multiparametric and comprises one, two, three, four, or more
methods disclosed herein and/or other optimization methods known in
the art.
[0462] Features, which can be considered beneficial in some
embodiments of the invention, can be encoded by or within regions
of the polynucleotide and such regions can be upstream (5') to,
downstream (3') to, or within the region that encodes the PBGD
polypeptide. These regions can be incorporated into the
polynucleotide before and/or after sequence-optimization of the
protein encoding region or open reading frame (ORF). Examples of
such features include, but are not limited to, untranslated regions
(UTRs), microRNA sequences, Kozak sequences, oligo(dT) sequences,
poly-A tail, and detectable tags and can include multiple cloning
sites that can have XbaI recognition.
[0463] In some embodiments, the polynucleotide of the invention
comprises a 5' UTR, a 3' UTR and/or a miRNA binding site. In some
embodiments, the polynucleotide comprises two or more 5' UTRs
and/or 3' UTRs, which can be the same or different sequences. In
some embodiments, the polynucleotide comprises two or more miRNA,
which can be the same or different sequences. Any portion of the 5'
UTR, 3' UTR, and/or miRNA binding site, including none, can be
sequence-optimized and can independently contain one or more
different structural or chemical modifications, before and/or after
sequence optimization.
[0464] In some embodiments, after optimization, the polynucleotide
is reconstituted and transformed into a vector such as, but not
limited to, plasmids, viruses, cosmids, and artificial chromosomes.
For example, the optimized polynucleotide can be reconstituted and
transformed into chemically competent E. coli, yeast, neurospora,
maize, drosophila, etc. where high copy plasmid-like or chromosome
structures occur by methods described herein.
6. SEQUENCE-OPTIMIZED NUCLEOTIDE SEQUENCES ENCODING PBGD
POLYPEPTIDES
[0465] In some embodiments, the polynucleotide of the invention
comprises a sequence optimized nucleotide sequence encoding a PBGD
polypeptide disclosed herein. In some embodiments, the
polynucleotide of the invention comprises an open reading frame
(ORF) encoding a PBGD polypeptide, wherein the ORF has been
sequence optimized.
[0466] Exemplary sequence optimized nucleotide sequences encoding
human PBGD isoform 1 are set forth as SEQ ID Nos: 9-33 (PBGD-CO01,
PBGD-CO02, PBGD-CO03, PBGD-CO04, PBGD-CO05, PBGD-CO06, PBGD-CO07,
PBGD-CO08, PBGD-CO09, PBGD-CO10, PBGD-CO11, PBGD-CO12, PBGD-CO13,
PBGD-CO14, PBGD-CO15, PBGD-CO16, PBGD-CO17, PBGD-CO18, PBGD-CO19,
PBGD-CO20, PBGD-CO21, PBGD-CO22, PBGD-CO23, PBGD-CO24, and
PBGD-CO25, respectively. Further exemplary sequence optimized
nucleotide sequences encoding human PBGD isoform 1 are shown in
TABLE 2. In some embodiments, the sequence optimized PBGD sequences
set forth as SEQ ID Nos: 9-33 or shown in TABLE 2, fragments, and
variants thereof are used to practice the methods disclosed herein.
In some embodiments, the sequence optimized PBGD sequences set
forth as SEQ ID Nos: 9-33 or shown in TABLE 2, fragments and
variants thereof are combined with or alternatives to the wild-type
sequences disclosed in FIGS. 1-4
[0467] Exemplary sequence optimized nucleotide sequences encoding
human PBGD isoform 1 are set forth as SEQ ID Nos: 89 to 117
(PBGD-CO30, PBGD-CO31, PBGD-CO32, PBGD-CO33, PBGD-CO34, PBGD-CO35,
PBGD-CO36, PBGD-CO37, PBGD-CO38, PBGD-CO39, PBGD-CO40A, PBGD-CO41A,
PBGD-CO42A, PBGD-CO43A, PBGD-CO44A, PBGD-CO45A, PBGD-CO46A,
PBGD-CO47A, PBGD-CO40B, PBGD-CO41B, PBGD-CO42B, PBGD-CO43B,
PBGD-CO44B, PBGD-CO45B, PBGD-CO46B, PBGD-CO47B, PBGD-CO48,
PBGD-CO49, PBGD-CO50, respectively). Further exemplary sequence
optimized nucleotide sequences encoding human PBGD isoform 1 are
shown in TABLE 2. In some embodiments, the sequence optimized PBGD
sequences set forth as SEQ ID Nos: 89 to 117, or shown in TABLE 2,
fragments, and variants thereof are used to practice the methods
disclosed herein. In some embodiments, the sequence optimized PBGD
sequences set forth as SEQ ID Nos: 89 to 117, or shown in TABLE 2,
fragments and variants thereof are combined with or alternatives to
the wild-type sequences disclosed in FIGS. 1-4.
[0468] In some embodiments, a polynucleotide of the present
disclosure, for example a polynucleotide comprising an mRNA
nucleotide sequence encoding a PBGD polypeptide, comprises from 5'
to 3' end:
[0469] (i) a 5' cap provided herein, for example, CAP1;
[0470] (ii) a 5' UTR, such as the sequences provided herein, for
example, SEQ ID NO: 39;
[0471] (iii) an open reading frame encoding a PBGD polypeptide,
e.g., a sequence optimized nucleic acid sequence encoding PBGD set
forth as SEQ ID Nos: 9 to 33 and 89 to 117, or shown in TABLE
2;
[0472] (iv) at least one stop codon;
[0473] (v) a 3' UTR, such as the sequences provided herein, for
example, SEQ ID NOs 149 to 151; and
[0474] (vi) a poly-A tail provided above.
TABLE-US-00002 TABLE 2 Sequence optimized sequences for human PBGD,
isoform 1 SEQ ID No Name Sequence 9 PBGD-CO01 See Sequence Listing
10 PBGD-CO02 See Sequence Listing 11 PBGD-CO03 See Sequence Listing
12 PBGD-CO04 See Sequence Listing 13 PBGD-CO05 See Sequence Listing
14 PBGD-CO06 See Sequence Listing 15 PBGD-CO07 See Sequence Listing
16 PBGD-CO08 See Sequence Listing 17 PBGD-CO09 See Sequence Listing
18 PBGD-CO10 See Sequence Listing 19 PBGD-CO11 See Sequence Listing
20 PBGD-CO12 See Sequence Listing 21 PBGD-CO13 See Sequence Listing
22 PBGD-CO14 See Sequence Listing 23 PBGD-CO15 See Sequence Listing
24 PBGD-CO16 See Sequence Listing 25 PBGD-CO17 See Sequence Listing
26 PBGD-CO18 See Sequence Listing 27 PBGD-CO19 See Sequence Listing
28 PBGD-CO20 See Sequence Listing 29 PBGD-CO21 See Sequence Listing
30 PBGD-CO22 See Sequence Listing 31 PBGD-CO23 See Sequence Listing
32 PBGD-CO24 See Sequence Listing 33 PBGD-CO25 See Sequence Listing
89 PBGD-CO30
AUGAGCGGCAACGGCAACGCCGCAGCCACCGCCGAGGAAAACAGCCCCAAGAUGCGGGUGAUCAGAGUGG
GCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCUCCUACCC
CGGCCUGCAGUUCGAGAUCAUUGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGAGCCUGUUCACAAAAGAGCUGGAACACGCCCUGGAAAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCCCCUGGCUUCACCAUCGGCGCCAUCUGCAAGAGAGA
GAACCCCCACGACGCCGUGGUGUUCCACCCUAAGUUCGUGGGCAAGACACUGGAAACCCUGCCCGAGAAG
UCCGUGGUGGGCACCAGCAGCCUGCGGAGAGCCGCCCAGCUGCAGCGGAAGUUCCCCCACCUGGAAUUUC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAAUUUUCCGCUAUCAU
CCUGGCCACAGCCGGACUGCAGCGGAUGGGCUGGCACAACAGAGUGGGCCAGAUCCUGCACCCCGAGGAA
UGCAUGUACGCCGUGGGCCAGGGAGCCCUGGGCGUGGAAGUGCGGGCCAAGGACCAGGACAUCCUGGAUC
UGGUGGGCGUGCUGCAUGACCCCGAGACACUGCUGCGGUGUAUCGCCGAGCGGGCCUUCCUGCGGCACCU
GGAAGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGACAGCUGUACCUGACAGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCC
AGCACGAGGACGGCCCCGAGGACGACCCUCAGCUGGUCGGCAUCACCGCCCGGAACAUCCCCAGAGGCCC
CCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGCUGCUGUCCAAGGGCGCCAAGAACAUC
CUGGACGUGGCCCGGCAGCUGAACGACGCCCAC 90 PBGD-CO31
AUGAGCGGCAACGGCAACGCCGCCGCUACCGCCGAAGAGAACAGCCCAAAGAUGCGCGUGAUCAGGGUCG
GCACGCGCAAGUCCCAGCUCGCCCGGAUCCAAACCGAUAGCGUGGUGGCCACGCUCAAGGCGAGCUAUCC
GGGCUUACAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGAUAAGAUACUGGACACCGCCCUGUCCAAG
AUCGGCGAAAAGAGCCUGUUCACCAAGGAACUGGAGCACGCGCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCGACCGUGCUGCCGCCGGGAUUCACCAUCGGCGCCAUCUGCAAGAGGGA
GAAUCCGCACGAUGCCGUGGUGUUCCACCCAAAGUUCGUGGGCAAGACCUUGGAAACCCUGCCAGAGAAG
UCUGUGGUCGGCACCUCCAGCCUGCGGCGAGCCGCCCAGCUGCAGCGAAAGUUCCCGCACCUGGAGUUCA
GGUCCAUCCGCGGAAAUCUGAACACCAGGCUGCGCAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAU
CCUGGCCACCGCAGGCCUCCAAAGAAUGGGCUGGCAUAACCGAGUCGGCCAGAUCCUCCACCCGGAGGAG
UGCAUGUACGCAGUGGGCCAAGGCGCCCUGGGCGUCGAGGUGCGUGCCAAGGACCAGGACAUCCUGGACC
UGGUGGGCGUGCUCCACGAUCCAGAGACACUGCUGAGAUGCAUCGCGGAGCGCGCCUUCCUGCGCCAUCU
GGAGGGAGGCUGCUCCGUCCCGGUGGCCGUACAUACCGCCAUGAAGGACGGUCAGCUGUACCUCACCGGC
GGCGUAUGGUCCCUCGACGGUAGCGACAGCAUACAGGAGACGAUGCAGGCCACCAUCCACGUGCCGGCGC
AGCACGAGGAUGGACCAGAGGACGACCCGCAGCUGGUGGGUAUCACCGCCAGGAAUAUCCCGCGGGGACC
UCAGCUGGCCGCCCAGAACCUGGGCAUCUCCCUCGCCAACCUCCUGCUGAGCAAGGGCGCCAAGAACAUC
CUGGACGUGGCCAGGCAGCUCAACGAUGCCCAU 91 PBGD-CO32
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAAAACAGCCCGAAGAUGCGGGUGAUCAGGGUGG
GCACCAGGAAGUCCCAGCUCGCCCGGAUCCAGACCGACAGCGUGGUCGCCACCUUGAAGGCCUCCUACCC
GGGCCUCCAGUUCGAGAUCAUCGCCAUGUCCACAACCGGCGACAAGAUCCUGGAUACCGCCCUCAGCAAG
AUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAAUGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCUACCGUGCUGCCACCAGGCUUCACAAUCGGCGCCAUCUGCAAGAGAGA
GAACCCGCACGACGCCGUGGUGUUCCAUCCGAAGUUCGUGGGCAAGACCCUGGAAACCCUGCCGGAGAAG
UCCGUAGUGGGAACCUCAAGCCUGAGGCGCGCCGCCCAGCUCCAGAGGAAGUUCCCUCACCUGGAAUUCC
GGUCCAUCAGGGGCAACCUGAACACGCGCCUGCGGAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAU
CCUGGCCACAGCCGGCCUUCAGCGCAUGGGCUGGCACAACAGGGUGGGCCAGAUCCUGCACCCGGAAGAA
UGCAUGUACGCCGUGGGCCAAGGCGCCCUCGGCGUGGAAGUGCGUGCCAAGGACCAGGACAUCCUGGACC
UGGUGGGCGUGCUGCACGACCCUGAGACGCUGCUCAGGUGCAUCGCCGAACGCGCGUUCCUGCGGCACCU
GGAGGGAGGCUGCAGCGUCCCGGUGGCCGUCCACACCGCCAUGAAGGACGGCCAGCUCUACCUGACUGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUUCAGGAAACCAUGCAGGCCACCAUCCACGUGCCUGCCC
AGCACGAGGACGGCCCGGAGGACGACCCUCAACUGGUGGGCAUUACUGCGCGAAACAUCCCGCGCGGACC
UCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGCUCCUGUCCAAGGGCGCCAAGAACAUC
CUCGACGUGGCCAGGCAGCUGAACGACGCGCAC 92 PBGD-CO33
AUGAGCGGCAACGGAAACGCCGCCGCGACCGCGGAGGAGAACUCGCCUAAGAUGAGAGUGAUAAGGGUAG
GCACCCGGAAGUCUCAACUCGCCAGGAUCCAGACCGACAGCGUGGUGGCCACCCUCAAGGCCAGCUAUCC
AGGACUCCAGUUCGAAAUCAUCGCCAUGUCCACCACAGGCGAUAAGAUCCUGGACACCGCCCUGUCCAAG
AUCGGCGAGAAGUCCCUCUUCACCAAGGAACUGGAGCACGCCCUGGAGAAGAACGAGGUCGAUCUGGUCG
UGCACAGCCUGAAGGAUCUGCCUACCGUGCUCCCGCCGGGCUUCACCAUCGGCGCCAUCUGCAAGAGGGA
GAAUCCUCACGACGCCGUGGUGUUCCACCCGAAGUUCGUGGGCAAGACCCUGGAGACACUGCCAGAAAAG
UCGGUGGUGGGCACCAGCAGCCUGCGGCGGGCGGCCCAGCUGCAGCGGAAGUUCCCACACCUGGAGUUCA
GGUCCAUCCGUGGCAAUCUGAACACCCGGCUGCGUAAGCUGGACGAGCAGCAGGAAUUCAGCGCGAUCAU
CCUGGCAACCGCCGGUCUGCAAAGGAUGGGCUGGCACAACAGGGUGGGCCAGAUCCUGCACCCUGAGGAG
UGCAUGUACGCCGUGGGCCAGGGAGCCCUGGGCGUGGAAGUGCGGGCCAAGGACCAGGACAUCCUGGACC
UGGUGGGUGUGCUCCACGACCCUGAAACCCUGCUGCGGUGCAUCGCCGAAAGGGCCUUCCUGAGGCACCU
CGAGGGCGGCUGCAGCGUGCCGGUCGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGA
GGAGUGUGGAGCCUGGACGGCUCCGACUCCAUCCAGGAGACUAUGCAGGCCACCAUUCAUGUGCCGGCCC
AGCAUGAGGACGGUCCGGAGGACGAUCCACAGCUGGUCGGCAUCACCGCGCGGAACAUCCCAAGAGGCCC
GCAACUGGCCGCUCAGAACCUGGGCAUAUCCCUGGCCAACCUGCUCCUGAGCAAGGGCGCCAAGAACAUC
CUGGACGUGGCCAGGCAGCUGAAUGACGCCCAC 93 PBGD-CO34
AUGUCCGGCAACGGCAACGCCGCCGCUACCGCCGAGGAGAACUCCCCUAAGAUGCGGGUCAUCAGGGUGG
GCACCCGAAAGUCCCAACUUGCCCGGAUCCAGACCGACUCCGUCGUGGCCACCCUCAAGGCUAGCUAUCC
AGGCCUCCAGUUCGAAAUCAUCGCCAUGAGCACCACCGGCGACAAGAUUCUGGACACCGCCCUGUCCAAG
AUCGGCGAGAAGAGUCUGUUCACGAAGGAGCUCGAGCACGCCCUGGAAAAGAACGAGGUGGACCUGGUGG
UGCAUUCCCUGAAGGACCUGCCAACCGUGCUGCCGCCGGGCUUCACUAUAGGAGCCAUCUGCAAGCGGGA
GAACCCGCACGACGCGGUGGUGUUCCAUCCGAAGUUCGUGGGCAAGACUCUGGAAACCCUGCCGGAGAAG
UCCGUGGUGGGAACUAGCUCCCUGCGGCGGGCCGCCCAGCUGCAGAGGAAGUUCCCGCACCUGGAGUUCA
GGAGCAUACGCGGCAACCUGAACACCCGCCUGCGUAAGCUCGACGAGCAGCAGGAAUUCAGUGCCAUCAU
CCUGGCCACGGCGGGCCUGCAGCGGAUGGGCUGGCACAACAGGGUGGGCCAGAUCCUCCACCCGGAGGAA
UGUAUGUACGCCGUGGGCCAGGGCGCACUGGGCGUGGAGGUCCGCGCCAAGGACCAAGACAUCCUGGACC
UGGUCGGCGUGCUGCACGACCCUGAAACCCUGCUGAGGUGCAUUGCCGAGAGAGCCUUCCUGAGGCAUCU
GGAGGGCGGCUGCAGCGUGCCUGUGGCCGUGCACACAGCCAUGAAGGACGGUCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACUCCAUCCAGGAGACAAUGCAGGCCACCAUCCACGUCCCGGCCC
AACACGAGGACGGACCUGAGGACGAUCCUCAGCUGGUGGGCAUCACCGCCAGGAACAUCCCUCGGGGCCC
GCAGCUGGCCGCCCAGAACCUGGGCAUCUCCCUCGCCAACCUGCUGCUGUCCAAGGGCGCCAAGAACAUC
CUCGACGUGGCCAGACAGCUGAACGACGCCCAC 94 PBGD-CO35
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCGAAGAUGAGGGUGAUAAGGGUGG
GCACACGGAAGUCCCAGCUCGCCCGCAUCCAAACCGACUCCGUGGUGGCCACCCUCAAGGCCAGCUACCC
GGGCCUCCAAUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGUCUAAG
AUAGGCGAAAAGAGCCUGUUCACCAAGGAGCUGGAGCAUGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UCCACAGUCUCAAGGACCUGCCAACCGUGCUGCCGCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGUGA
GAACCCGCACGAUGCUGUGGUGUUCCACCCUAAGUUCGUGGGAAAGACCCUGGAGACGCUGCCGGAAAAG
AGCGUGGUCGGCACCUCCAGCCUGCGGAGGGCCGCCCAACUCCAGAGGAAGUUCCCGCACCUGGAGUUCA
GGAGCAUCCGCGGCAACCUGAACACCAGGCUGCGAAAGCUGGACGAGCAGCAGGAAUUCUCGGCCAUCAU
CCUCGCCACCGCCGGCUUGCAAAGAAUGGGCUGGCAUAAUCGCGUGGGCCAGAUCCUGCACCCUGAGGAG
UGCAUGUACGCCGUGGGCCAGGGUGCUCUGGGAGUGGAGGUGCGGGCCAAGGACCAGGAUAUCCUGGACC
UGGUCGGCGUGCUUCAUGACCCGGAGACGCUCCUGAGGUGCAUCGCCGAGCGGGCCUUCCUGAGACACCU
GGAGGGCGGCUGCUCCGUGCCAGUGGCCGUGCACACCGCCAUGAAGGACGGACAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGAAGCGACAGCAUCCAAGAAACCAUGCAGGCGACCAUUCACGUCCCUGCCC
AGCACGAGGAUGGACCAGAGGACGACCCGCAGCUGGUGGGCAUCACCGCCCGCAACAUCCCUAGAGGCCC
ACAGCUGGCCGCCCAGAAUCUGGGCAUCAGCCUGGCCAACCUGCUGCUGUCUAAGGGAGCCAAGAACAUC
CUGGACGUGGCCAGGCAGCUGAACGACGCCCAU 95 PBGD-CO36
AUGUCCGGCAACGGCAACGCCGCAGCCACCGCCGAGGAGAAUUCCCCGAAGAUGCGGGUGAUCCGGGUGG
GCACCAGAAAGAGCCAGCUCGCCCGCAUCCAAACCGACUCCGUGGUGGCCACCCUCAAGGCCUCCUACCC
AGGCUUGCAGUUCGAAAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUUGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCAUGCUCUGGAGAAGAACGAGGUGGACCUCGUGG
UGCACUCCCUGAAGGACCUGCCGACUGUGCUGCCGCCUGGCUUCACGAUCGGCGCCAUAUGCAAGCGGGA
AAACCCACACGACGCCGUGGUCUUCCACCCAAAGUUCGUGGGCAAGACCCUGGAAACCCUGCCGGAAAAG
AGCGUGGUCGGCACAAGCUCCCUGAGGAGAGCCGCCCAACUGCAAAGGAAGUUCCCUCACCUCGAGUUCA
GGUCCAUCCGGGGCAACCUGAACACCAGGCUGAGAAAGCUCGACGAACAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACGGCCGGCCUGCAGAGGAUGGGAUGGCAUAACAGGGUGGGCCAGAUCCUGCACCCGGAGGAG
UGCAUGUACGCCGUGGGCCAGGGAGCCCUCGGCGUGGAGGUCAGGGCCAAGGAUCAGGAUAUCCUGGACC
UGGUGGGCGUGCUGCACGAUCCUGAGACGCUGCUGAGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
AGAGGGCGGAUGCAGCGUGCCGGUCGCGGUCCACACCGCGAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGUCCCUGGACGGCAGCGAUUCAAUCCAGGAGACGAUGCAGGCCACCAUCCACGUGCCAGCCC
AGCACGAGGAUGGCCCGGAGGACGACCCGCAGCUGGUGGGCAUUACAGCCAGGAACAUCCCUCGGGGCCC
GCAGCUGGCCGCCCAGAAUCUGGGCAUCAGCCUGGCGAACCUGCUGCUCAGCAAGGGAGCGAAGAACAUC
CUGGACGUGGCCCGCCAGCUGAACGAUGCCCAC 96 PBGD-CO37
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCAAAGAUGCGGGUGAUCAGGGUGG
GCACCCGCAAGAGCCAACUCGCCAGAAUCCAGACCGACAGCGUGGUGGCCACCUUGAAGGCCAGCUACCC
GGGCCUCCAGUUCGAGAUCAUCGCUAUGUCCACCACCGGCGACAAGAUCCUGGACACCGCGCUGUCCAAG
AUCGGCGAAAAGAGCCUGUUCACCAAGGAACUGGAGCACGCCCUCGAGAAGAACGAGGUGGACCUGGUGG
UGCACUCCCUGAAGGACCUGCCGACGGUCCUGCCGCCGGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
AAACCCGCACGACGCUGUGGUGUUCCACCCAAAGUUCGUGGGCAAGACCCUGGAAACCCUGCCAGAAAAG
AGCGUGGUGGGCACCAGCAGCCUCAGGAGAGCCGCCCAGCUGCAGAGGAAGUUCCCGCACCUGGAGUUCA
GGAGCAUCAGGGGCAACCUGAACACCAGGCUGCGUAAGCUGGACGAGCAGCAGGAGUUCUCCGCCAUCAU
CCUCGCCACAGCCGGCCUCCAGAGGAUGGGUUGGCACAACAGGGUGGGCCAGAUCCUGCACCCGGAAGAG
UGCAUGUACGCAGUGGGCCAGGGCGCCCUUGGCGUGGAAGUGCGAGCCAAGGAUCAGGAUAUCCUGGACC
UGGUGGGCGUGCUGCACGACCCGGAAACUCUGCUGCGGUGCAUCGCCGAAAGGGCCUUCCUGCGCCACCU
CGAAGGCGGCUGUAGCGUGCCGGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUCGACGGCAGCGACAGCAUCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCGGCCC
AGCAUGAGGAUGGCCCGGAGGACGACCCUCAGCUGGUGGGCAUCACCGCCCGCAACAUCCCAAGAGGACC
GCAACUGGCCGCCCAGAACCUGGGCAUCUCCCUGGCCAACCUGCUCCUGAGCAAGGGCGCGAAGAACAUC
CUCGACGUCGCACGGCAGCUGAACGACGCCCAC 97 PBGD-CO38
AUGAGCGGCAACGGCAACGCCGCCGCGACGGCCGAGGAAAAUAGCCCGAAGAUGCGGGUGAUCAGGGUGG
GCACCAGGAAGUCCCAGCUCGCCAGGAUCCAGACCGACAGCGUGGUGGCCACCCUCAAGGCCUCCUACCC
GGGCCUCCAAUUCGAGAUCAUCGCCAUGUCCACCACCGGCGACAAGAUCCUCGACACCGCCCUGAGCAAG
AUCGGCGAAAAGUCGCUGUUCACCAAGGAGCUGGAGCACGCCCUCGAGAAGAACGAGGUGGACCUGGUAG
UGCACUCCCUAAAGGACCUGCCGACCGUGCUGCCGCCGGGCUUCACGAUCGGCGCCAUCUGCAAGCGCGA
GAACCCGCAUGAUGCCGUCGUUUUCCACCCUAAGUUCGUGGGCAAGACCCUGGAGACGCUGCCGGAGAAG
UCGGUGGUGGGAACCAGCAGCCUGAGGAGGGCCGCACAACUGCAGAGGAAGUUCCCGCAUCUGGAGUUCC
GCAGCAUUCGAGGCAACCUGAACACGCGCCUGAGAAAGCUCGAUGAACAGCAGGAGUUCAGCGCCAUCAU
UCUGGCCACUGCCGGACUGCAGCGGAUGGGCUGGCACAACAGAGUGGGCCAGAUCCUGCAUCCGGAAGAG
UGUAUGUACGCCGUGGGCCAGGGUGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGAUAUACUGGAUC
UGGUCGGCGUGCUCCACGACCCAGAAACACUCCUGAGGUGCAUCGCUGAGAGAGCCUUCCUCCGGCACCU
CGAGGGCGGCUGUUCCGUGCCGGUGGCCGUCCAUACCGCCAUGAAGGACGGUCAGCUGUACCUGACCGGA
GGCGUUUGGUCCCUGGACGGCAGCGACAGCAUCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCGGCGC
AGCACGAGGACGGCCCGGAAGACGACCCGCAGCUGGUCGGCAUCACGGCCAGAAACAUCCCGCGGGGCCC
GCAGCUGGCGGCCCAGAACCUGGGAAUCUCCCUGGCCAACCUGCUGCUGAGCAAGGGCGCGAAGAACAUC
CUGGACGUGGCCAGGCAGCUGAACGAUGCCCAC 98 PBGD-CO39
AUGAGCGGUAACGGCAACGCCGCCGCCACCGCCGAGGAGAACUCCCCGAAGAUGCGCGUGAUUCGGGUCG
GCACAAGAAAGUCUCAACUCGCCCGAAUCCAAACGGACAGCGUGGUGGCCACCCUCAAGGCGAGCUACCC
GGGCCUCCAGUUCGAAAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGUCGAAG
AUUGGCGAAAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAAGUCGACCUGGUCG
UGCACAGCCUGAAGGACCUGCCGACCGUUCUGCCGCCGGGCUUCACCAUCGGAGCCAUCUGCAAGCGGGA
GAAUCCGCACGACGCCGUGGUCUUCCACCCAAAGUUCGUGGGAAAGACCCUCGAGACACUGCCGGAGAAG
UCCGUGGUGGGAACCUCCUCCCUGCGGAGGGCCGCCCAACUGCAGCGGAAGUUCCCACACCUGGAAUUCC
GGUCCAUCAGAGGCAACCUCAACACCAGGCUGAGGAAGCUCGAUGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACAGCCGGACUGCAGCGCAUGGGCUGGCAUAACAGAGUGGGCCAGAUCCUCCACCCGGAGGAG
UGCAUGUACGCCGUGGGACAAGGCGCGCUGGGCGUGGAAGUUCGGGCCAAGGACCAGGAUAUCCUGGACC
UGGUGGGCGUGCUCCACGACCCAGAGACGCUGCUGCGGUGCAUCGCCGAGCGCGCCUUCCUGCGGCACCU
CGAGGGCGGCUGCAGCGUGCCGGUCGCUGUGCACACAGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGUCUGGACGGCAGCGACUCCAUCCAGGAGACUAUGCAAGCCACCAUCCAUGUGCCGGCCC
AACAUGAGGACGGCCCGGAGGACGACCCGCAACUGGUGGGCAUCACCGCCCGGAACAUCCCGAGGGGCCC
GCAGCUGGCCGCCCAGAACCUGGGCAUUAGCCUGGCCAACCUGCUCCUGAGCAAGGGCGCUAAGAACAUC
CUGGACGUCGCCAGACAGCUGAACGACGCCCAC 99 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCCG-
GGUGG CO40A
GCACCCGUAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCUCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCCCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAAACCCUGCCUGAGAAG
UCCGUCGUAGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCCCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCUACCGCCGGUCUGCAACGAAUGGGCUGGCACAAUAGGGUGGGCCAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCGGUGGGACAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCCUUGAUC
UGGUGGGCGUGCUGCACGACCCCGAGACGCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUUU
GGAGGGCGGAUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCUGCUC
AGCACGAAGACGGCCCAGAGGACGACCCCCAGCUGGUAGGCAUCACCGCCCGGAACAUCCCCCGGGGCCC
UCAGCUCGCCGCACAGAACCUUGGAAUCAGCCUGGCCAACCUGCUGUUGUCAAAGGGCGCCAAGAAUAUC
CUCGACGUGGCCCGGCAGCUGAACGACGCCCAC 100 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUG CO41A
GGCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCU-
AC
CCCGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGC
AAGAUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUG
GUGGUGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCGCCUGGCUUCACCAUCGGCGCCAUCUGCAAG
CGGGAGAACCCCCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACUCUGGAAACCCUGCCU
GAGAAGUCCGUGGUCGGAACAAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCCCACCUG
GAGUUCCGGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGC
GCCAUCAUCCUGGCCACAGCCGGCCUUCAGAGGAUGGGCUGGCACAAUCGGGUAGGCCAGAUCCUGCAC
CCCGAGGAGUGCAUGUACGCGGUAGGUCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGAC
AUCUUAGAUCUGGUUGGCGUGCUGCACGACCCCGAAACACUGCUGCGGUGCAUCGCCGAGCGGGCCUUC
CUGCGGCACCUCGAGGGCGGCUGCAGUGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUG
UACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACAAUGCAGGCCACCAUC
CACGUGCCAGCUCAGCACGAAGACGGACCAGAGGACGACCCCCAGUUAGUGGGAAUCACCGCCCGGAAC
AUCCCCCGGGGCCCUCAGCUCGCGGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGCUGCUGUCUAAG
GGCGCCAAGAACAUCCUAGACGUGGCCCGGCAGCUGAACGACGCCCAC 101 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO42A
GCACCAGGAAGUCACAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCGCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCCCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAAACCCUGCCAGAGAAG
UCUGUGGUCGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCCCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACCGCUGGCCUACAGCGGAUGGGCUGGCACAAUAGAGUUGGUCAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCCGUCGGCCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUACUAGACC
UCGUGGGCGUGCUGCACGACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
GGAAGGCGGUUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCGGCCC
AACACGAGGACGGACCUGAGGACGACCCCCAGCUUGUGGGAAUCACCGCCCGGAACAUCCCCCGGGGCCC
UCAACUGGCAGCCCAGAACUUAGGCAUAAGCCUGGCCAACCUGCUGCUGUCCAAGGGCGCCAAGAACAUC
CUCGAUGUGGCCCGGCAGCUGAACGACGCCCAC 102 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO43A
GCACCCGCAAGAGUCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCACCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCCCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACACUGGAAACCCUGCCGGAGAAG
UCCGUGGUAGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCCCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCAACAGCCGGCUUACAGCGUAUGGGCUGGCACAACAGGGUGGGACAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCUGUGGGCCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCUUAGAUC
UCGUCGGCGUGCUGCACGACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUCU
UGAGGGCGGAUGCUCCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACUAUGCAGGCCACCAUCCACGUGCCAGCCC
AGCACGAAGACGGCCCAGAGGACGACCCCCAGCUGGUGGGAAUCACCGCCCGGAACAUCCCCCGGGGCCC
UCAACUGGCCGCACAGAACCUAGGCAUCAGCCUGGCCAACCUGCUGCUCAGCAAGGGCGCCAAGAAUAUC
UUGGACGUGGCCCGGCAGCUGAACGACGCCCAC 103 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO44A
GCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGUCUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCUCCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCCCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAGACUCUGCCAGAGAAG
UCUGUAGUGGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCCCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACGGCCGGAUUACAGAGAAUGGGCUGGCACAACCGAGUGGGACAGAUCCUGCACCCCGAGGAG
UGCAUGUAUGCCGUUGGCCAAGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCCUCGAUC
UCGUGGGCGUGCUGCACGACCCCGAGACUUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
AGAGGGCGGCUGCUCGGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCAGCCC
AGCACGAGGAUGGCCCUGAAGACGACCCCCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCCCGGGGCCC
ACAAUUGGCCGCUCAGAACUUAGGCAUUAGCCUGGCCAACCUGCUGCUGUCUAAGGGCGCCAAGAACAUA
CUGGACGUGGCCCGGCAGCUGAACGACGCCCAC 104 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO45A
GCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGAGCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCCCCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCCCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAGACCCUGCCCGAGAAG
AGCGUGGUGGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCCCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCCGUGGGCCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCCUGGACC
UGGUGGGCGUGCUGCACGACCCCGAGACCCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
GGAGGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCC
AGCACGAGGACGGCCCCGAGGACGACCCCCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCCCGGGGCCC
CCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGCUGCUGAGCAAGGGCGCCAAGAACAUC
CUGGACGUGGCCCGGCAGCUGAACGACGCCCAC 105 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO46A
GCACCCGGAAGAGCCAGCUGGCGAGGAUCCAGACGGACAGCGUGGUGGCGACGCUGAAGGCGAGCUA-
CCC
GGGGCUGCAGUUCGAGAUCAUCGCGAUGAGCACGACGGGGGACAAGAUCCUGGACACGGCGCUGAGCAAG
AUCGGGGAGAAGAGCCUGUUCACGAAGGAGCUGGAGCACGCGCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCGACGGUGCUGCCGCCGGGGUUCACGAUCGGGGCGAUCUGCAAGAGGGA
GAACCCGCACGACGCGGUGGUGUUCCACCCGAAGUUCGUGGGGAAGACGCUGGAGACGCUGCCGGAGAAG
AGCGUGGUGGGGACGAGCAGCCUGAGGAGGGCGGCGCAGCUGCAGAGGAAGUUCCCGCACCUGGAGUUCA
GGAGCAUCAGGGGGAACCUGAACACGAGGCUGAGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCGAUCAU
CCUGGCGACGGCGGGGCUGCAGAGGAUGGGGUGGCACAACAGGGUGGGGCAGAUCCUGCACCCGGAGGAG
UGCAUGUACGCGGUGGGGCAGGGGGCGCUGGGGGUGGAGGUGAGGGCGAAGGACCAGGACAUCCUGGACC
UGGUGGGGGUGCUGCACGACCCGGAGACGCUGCUGAGGUGCAUCGCGGAGAGGGCGUUCCUGAGGCACCU
GGAGGGGGGGUGCAGCGUGCCGGUGGCGGUGCACACGGCGAUGAAGGACGGGCAGCUGUACCUGACGGGG
GGGGUGUGGAGCCUGGACGGGAGCGACAGCAUCCAGGAGACGAUGCAGGCGACGAUCCACGUGCCGGCGC
AGCACGAGGACGGGCCGGAGGACGACCCGCAGCUGGUGGGGAUCACGGCGAGGAACAUCCCGAGGGGGCC
GCAGCUGGCGGCGCAGAACCUGGGGAUCAGCCUGGCGAACCUGCUGCUGAGCAAGGGGGCGAAGAACAUC
CUGGACGUGGCGAGGCAGCUGAACGACGCGCAC 106 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO47A
GCACCCGGAAGAGCCAGCUGGCCCGCAUCCAGACCGACUCCGUCGUCGCCACCCUCAAGGCCUCCUA-
CCC
CGGCCUCCAGUUCGAGAUCAUCGCCAUGUCCACCACCGGCGACAAGAUCCUCGACACCGCCCUCUCCAAG
AUCGGCGAGAAGUCCCUCUUCACCAAGGAGCUCGAGCACGCCCUCGAGAAGAACGAGGUCGACCUCGUCG
UCCACUCCCUCAAGGACCUCCCCACCGUCCUCCCCCCCGGCUUCACCAUCGGGGCCAUCUGCAAGCGCGA
GAACCCCCACGACGCCGUCGUCUUCCACCCCAAGUUCGUCGGCAAGACCCUCGAGACCCUCCCCGAGAAG
UCCGUCGUCGGCACCUCCUCCCUCCGCCGCGCCGCCCAGCUCCAGCGCAAGUUCCCCCACCUCGAGUUCC
GCUCCAUCCGCGGCAACCUCAACACCCGCCUCCGCAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAU
GCUCGCCACCGCCGGCCUCCAGCGCAUGGGCUGGCACAACCGCGUCGGCCAGAUCCUCCACCCCGAGGAG
UGCAUGUACGCCGUCGGCCAGGGCGCCCUCGGCGUCGAGGUCCGCGCCAAGGACCAGGACAUCCUCGACC
UCGUCGGCCUCCUCCACGACCCCGAGACCCUCCUCCGCUGCAUCGCCGACCGCCCCUUCCUCCGCCACCU
GGAGGGCGGCUGCUCCGUCCCCGUCGCCGUCCACACCGCCAUGAAGGACGGCCAGCUCUACCUCACCGGC
GGCGUCUGGUCCCUCGACGGCUCCGACUCCAUCCAGGAGACCAUGCAGGCCACCAUCCACGUCCCCGCCC
AGCACGAGGACGGCCCCGAGGACGACCCCCAGCUCGUCGGCAUCACCGCCCGCAACAUCCCCCGCGGCCC
GCAGCUCGCCGCCCAGAACCUCGGCAUCUCCCUCGCCAACCUCCUCCUCUCCAAGGGCGCCAAGAACAUC
CUCGACGUCGCCCGCCAGCUCAACGACGCCCAC 107 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO40A
GCACCCGUAAGAGCCAGCUGGCCCCGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCUCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCUCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAAACCCUGCCUGAGAAG
UCCGUCGUAGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCACACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCUACCGCCGGUCUGCAACGAAUGGGCUGGCACAAUAGGGUGGGCCAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCGGUGGGACAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCCUUGAUC
UGGUGGGCGUGCUGCACGACCCCGAGACGCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUUU
GGAGGGCGGAUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCUGCUC
AGCACGAAGACGGCCCAGAGGACGACCCUCAGCUGGUAGGCAUCACCGCCCGGAACAUCCCUCGGGGCCC
UCAGCUCGCCGCACAGAACCUUGGAAUCAGCCUGGCCAACCUGCUGUUGUCAAAGGGCGCCAAGAAUAUC
GUCGACGUGGCCCGGCAGCUGAACGACGCCCAC 108 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO41B
GCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCGCCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCGCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACUCUGGAAACCCUGCCUGAGAAG
UCCGUGGUCGGAACAAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCACACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACAGCCGGCCUUCAGAGGAUGGGCUGGCACAAUCGGGUAGGCCAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCGGUAGGUCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCUUAGAUC
UGGUUGGCGUGCUGCACGACCCCGAAACACUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
CGAGGGCGGCUGCAGUGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCAGCUC
AGCACGAAGACGGACCAGAGGACGACCCACAGUUAGUGGGAAUCACCGCCCGGAACAUCCCGCGGGGCCC
UCAGCUCGCGGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGCUGCUGUCUAAGGGCGCCAAGAACAUC
CUAGACGUGGCCCGGCAGCUGAACGACGCCCAC 109 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO42B
GCACCAGGAAGUCACAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCGCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCGCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAAACCCUGCCAGAGAAG
UCUGUGGUCGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCGCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACCGCUGGCCUACAGCGGAUGGGCUGGCACAAUAGAGUUGGUCAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCCGUCGGCCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUACUAGACC
UCGUGGGCGUGCUGCACGACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
GGAAGGCGGUUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCGGCCC
AACACGAGGACGGACCUGAGGACGACCCACAGCUUGUGGGAAUCACCGCCCGGAACAUCCCACGGGGCCC
UCAACUGGCAGCCCAGAACUUAGGCAUAAGCCUGGCCAACCUGCUGCUGUCCAAGGGCGCCAAGAACAUC
CUCGAUGUGGCCCGGCAGCUGAACGACGCCCAC 110 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO43B
GCACCCGCAAGAGUCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCACCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCACACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACACUGGAAACCCUGCCGGAGAAG
UCCGUGGUAGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCGCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCAACAGCCGGCUUACAGCGUAUGGGCUGGCACAACAGGGUGGGACAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCUGUGGGCCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCUUAGAUC
UCGUCGGCGUGCUGCACGACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUCU
UGAGGGCGGAUGCUCCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACUAUGCAGGCCACCAUCCACGUGCCAGCCC
AGCACGAAGACGGCCCAGAGGACGACCCACAGCUGGUGGGAAUCACCGCCCGGAACAUCCCGCGGGGCCC
UCAACUGGCCGCACAGAACCUAGGCAUCAGCCUGGCCAACCUGCUGCUCAGCAAGGGCGCCAAGAAUAUC
UUGGACGUGGCCCGGCAGCUGAACGACGCCCAC 111 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO44B
GCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGUCUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCUCCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCACACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAGACUCUGCCAGAGAAG
UCUGUAGUGGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCUCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACGGCCGGAUUACAGAGAAUGGGCUGGCACAACCGAGUGGGACAGAUCCUGCACCCCGAGGAG
UGCAUGUAUGCCGUUGGCCAAGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCCUCGAUC
UCGUGGGCGUGCUGCACGACCCCGAGACUUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
AGAGGGCGGCUGCUCGGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCAGCCC
AGCACGAGGAUGGCCCUGAAGACGACCCACAGCUGGUGGGCAUCACCGCCCGGAACAUCCCGCGGGGCCC
ACAAUUGGCCGCUCAGAACUUAGGCAUUAGCCUGGCCAACCUGCUGCUGUCUAAGGGCGCCAAGAACAUA
CUGGACGUGGCCCGGCAGCUGAACGACGCCCAC 112 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO45B
GCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCGACAGCGUGGUGGCCACCCUGAAGGCCAGCUA-
CCC
CGGCCUGCAGUUCGAGAUCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCAAG
AUCGGCGAGAAGAGCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCCACCGUGCUGCCGCCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGA
GAACCCGCACGACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAGACCCUGCCCGAGAAG
AGCGUGGUGGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCUCACCUGGAGUUCC
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCCUGCACCCCGAGGAG
UGCAUGUACGCCGUGGGCCAGGGCGCCCUGGGCGUGGAGGUGCGGGCCAAGGACCAGGACAUCCUGGACC
UGGUGGGCGUGCUGCACGACCCCGAGACCCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACCU
GGAGGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACAGCAUCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCC
AGCACGAGGACGGCCCCGAGGACGACCCUCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCACGGGGCCC
UCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGCUGCUGAGCAAGGGCGCCAAGAACAUC
CUGGACGUGGCCCGGCAGCUGAACGACGCCCAC 113 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO46B
GCACCCGGAAGAGCCAGCUGGCGAGGAUCCAGACGGACAGCGUGGUGGCGACGCUGAAGGCGAGCUA-
CCC
GGGGCUGCAGUUCGAGAUCAUCGCGAUGAGCACGACGGGCGACAAGAUCCUGGACACGGCGCUGAGCAAG
AUCGGGGAGAAGAGCCUGUUCACGAAGGAGCUGGAGCACGCGCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACAGCCUGAAGGACCUGCCGACGGUGCUGCCGCCGGGGUUCACGAUCGGGGCGAUCUGCAAGAGGGA
GAACCCGCACGACGCGGUGGUGUUCCACCCGAAGUUCGUGGGGAAGACGCUGGAGACGCUGCCGGAGAAG
AGCGUGGUGGGGACGAGCAGCCUGAGGAGGGCGGCGCAGCUGCAGAGGAAGUUCCCGCACCUGGAGUUCA
GGAGCAUCAGGGGUAACCUGAACACGAGGCUGAGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCGAUCAU
CCUGGCGACGGCGGGGCUGCAGAGGAUGGGGUGGCACAACAGGGUGGGGCAGAUCCUGCACCCGGAGGAG
UGCAUGUACGCGGUGGGGCAGGGCGCGCUGGGCGUGGAGGUGAGGGCGAAGGACCAGGACAUCCUGGACC
UGGUGGGCGUGCUGCACGACCCGGAGACGCUGCUGAGGUGCAUCGCGGAGAGGGCGUUCCUGAGGCACCU
GGAGGGCGGGUGCAGCGUGCCGGUGGCGGUGCACACGGCGAUGAAGGACGGGCAGCUGUACCUGACGGGA
GGGGUGUGGAGCCUGGACGGGAGCGACAGCAUCCAGGAGACGAUGCAGGCGACGAUCCACGUGCCGGCGC
AGCACGAGGACGGGCCGGAGGACGACCCGCAGCUGGUGGGGAUCACGGCGAGGAACAUCCCGAGGGGUCC
GCAGCUGGCGGCGCAGAACCUGGGGAUCAGCCUGGCGAACCUGCUGCUGAGCAAGGGAGCGAAGAACAUC
CUGGACGUGGCGAGGCAGCUGAACGACGCGCAC 114 PBGD-
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGCGGGUGAUCC-
GGGUGG CO47B
GCACCCGGAAGAGCCAGCUGGCCCGCAUCCAGACCGACUCCGUCGUCGCCACCCUCAAGGCCUCCUA-
CCC
CGGCCUCCAGUUCGAGAUCAUCGCCAUGUCCACCACCGGCGACAAGAUCCUCGACACCGCCCUCUCCAAG
AUCGGCGAGAAGUCCCUCUUCACCAAGGAGCUCGAGCACGCCCUCGAGAAGAACGAGGUCGACCUCGUCG
UCCACUCCCUCAAGGACCUCCCCACCGUCCUCCCACCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGCGA
GAACCCUCACGACGCCGUCGUCUUCCACCCCAAGUUCGUCGGCAAGACCCUCGAGACCCUCCCCGAGAAG
UCCGUCGUCGGCACCUCCUCCCUCCGCCGCGCCGCCCAGCUCCAGCGCAAGUUCCCACACCUCGAGUUCC
GCUCCAUCCGCGGCAACCUCAACACCCGCCUCCGCAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAU
CCUCGCCACCGCCGGCCUCCAGCGCAUGGGCUGGCACAACCGCGUCGGCCAGAUCCUCCACCCCGAGGAG
UGCAUGUACGCCGUCGGCCAGGGCGCCCUCGGCGUCGAGGUCCGCGCCAAGGACCAGGACAUCCUCGACC
UCGUCGGCGUCCUCCACGACCCCGAGACCCUCCUCCGCUGCAUCGCCGAGCGCGCCUUCCUCCGCCACCU
CGAGGGCGGCUGCUCCGUCCCCGUCGCCGUCCACACCGCCAUGAAGGACGGCCAGCUCUACCUCACCGGC
GGCGUCUGGUCCCUCGACGGCUCCGACUCCAUCCAGGAGACCAUGCAGGCCACCAUCCACGUCCCCGCCC
AGCACGAGGACGGCCCCGAGGACGACCCUCAGCUCGUCGGCAUCACCGCCCGCAACAUCCCGCGCGGCCC
UCAGCUCGCCGCCCAGAACCUCGGCAUCUCCCUCGCCAACCUCCUCCUCUCCAAGGGCGCCAAGAACAUC
CUCGACGUCGCCCGCCAGCUCAACGACGCCCAC 115 PBGD-CO48
AUGUCCGGAAACGGAAACGCCGCCGCUACCGCCGAGGAAAAUAGCCCGAAGAUGAGAGUGAUCCGGGUGG
GUACCAGGAAGUCCCAGCUCGCCAGGAUCCAAACGGACUCGGUGGUGGCCACCCUCAAGGCUAGCUACCC
GGGCCUGCAAUUCGAGAUCAUUGCUAUGUCCACCACCGGCGACAAGAUCCUGGAUACGGCCCUGUCCAAG
AUCGGCGAAAAGAGCCUCUUCACCAAGGAGCUGGAACACGCGCUCGAGAAGAACGAGGUGGACCUGGUCG
UCCACAGCCUCAAGGACCUGCCUACGGUGCUGCCGCCGGGAUUCACCAUCGGCGCCAUCUGUAAGCGGGA
GAAUCCGCACGACGCCGUGGUGUUCCACCCUAAGUUCGUGGGCAAGACCCUCGAGACACUGCCGGAAAAG
UCCGUCGUGGGCACCUCCUCCCUGAGAAGGGCCGCUCAGCUCCAGAGAAAGUUCCCGCACCUGGAAUUCA
GGAGCAUCCGGGGCAACCUGAAUACCCGGCUUCGCAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCCUGCACCCGGAGGAG
UGCAUGUAUGCCGUGGGUCAGGGAGCCCUGGGCGUGGAGGUCCGGGCCAAGGACCAGGACAUCCUGGACC
UCGUGGGCGUGCUCCACGACCCUGAAACCCUCCUGAGGUGCAUCGCCGAGAGGGCCUUCCUCCGGCACCU
GGAGGGCGGCUGUUCCGUCCCUGUGGCCGUGCAUACCGCCAUGAAGGACGGACAGCUGUACCUGACCGGC
GGCGUGUGGUCCCUGGACGGCUCCGACAGCAUCCAGGAAACCAUGCAGGCCACUAUCCACGUGCCGGCCC
AGCACGAAGACGGCCCAGAGGAUGACCCGCAACUGGUCGGCAUUACCGCCAGGAACAUACCAAGGGGCCC
GCAGCUGGCCGCCCAGAACCUGGGCAUCUCCCUGGCCAACCUGCUGCUGUCCAAGGGAGCCAAGAACAUU
CUGGACGUGGCCAGGCAGCUCAAUGAUGCCCAC 116 PBGD-CO49
AUGAGCGGCAACGGCAACGCCGCCGCCACCGCAGAGGAGAAUAGCCCGAAGAUGAGGGUGAUCCGAGUGG
GCACCAGGAAGUCCCAGCUUGCGCGAAUUCAGACCGACAGCGUGGUGGCCACCCUCAAGGCCUCCUACCC
GGGACUCCAGUUCGAGAUCAUCGCCAUGAGCACCACGGGAGACAAGAUCCUGGACACCGCCCUGUCCAAG
AUCGGCGAAAAGAGCCUCUUCACCAAGGAGCUGGAGCACGCCCUGGAGAAGAACGAGGUCGAUCUGGUGG
UGCACAGCCUGAAGGACCUGCCGACCGUCCUGCCGCCGGGAUUCACCAUCGGUGCCAUCUGUAAGCGGGA
GAACCCGCACGACGCCGUGGUGUUCCACCCGAAGUUCGUCGGCAAGACCCUGGAAACCCUGCCGGAGAAG
UCCGUGGUGGGCACCAGCAGCCUGAGGCGGGCCGCCCAGCUGCAGCGGAAGUUCCCGCACCUGGAAUUCA
GGAGCAUCCGGGGCAACCUGAACACCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACCGCAGGCCUCCAGCGCAUGGGAUGGCACAACAGGGUAGGCCAAAUCCUGCACCCGGAGGAG
UGUAUGUACGCCGUGGGCCAGGGAGCCCUGGGCGUGGAGGUGAGAGCCAAGGACCAGGACAUCCUAGACC
UGGUCGGCGUGCUGCACGACCCGGAGACACUGCUGAGAUGCAUCGCGGAGAGAGCCUUCCUGCGACACCU
GGAGGGCGGCUGCUCCGUGCCGGUGGCCGUGCACACCGCCAUGAAGGACGGCCAGCUGUAUCUGACCGGC
GGCGUGUGGAGCCUGGACGGCAGCGACUCCAUCCAAGAAACCAUGCAGGCUACCAUCCACGUGCCGGCCC
AGCACGAGGAUGGACCAGAGGACGAUCCUCAACUGGUGGGCAUCACUGCCAGGAACAUCCCAAGAGGCCC
GCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGCUGCUGUCCAAGGGCGCCAAGAACAUU
CUCGAUGUGGCCAGGCAGCUGAACGAUGCCCAC 117 PBGD-CO50
AUGUCGGGAAACGGCAACGCCGCCGCCACGGCCGAGGAGAACAGCCCGAAGAUGAGAGUGAUUAGGGUGG
GCACCCGGAAGUCCCAACUCGCGCGGAUCCAGACCGACUCCGUGGUGGCCACCCUCAAGGCCAGCUACCC
GGGCCUCCAGUUCGAGAUUAUCGCCAUGUCCACCACAGGCGACAAGAUCCUCGACACCGCACUCUCGAAG
AUCGGCGAGAAGUCCCUGUUCACCAAGGAACUGGAGCACGCCCUGGAGAAGAACGAGGUGGACCUGGUGG
UGCACUCCCUGAAGGACCUGCCGACCGUGCUCCCACCAGGCUUCACCAUCGGCGCAAUCUGUAAGCGCGA
GAAUCCGCACGACGCCGUGGUGUUCCACCCAAAGUUCGUGGGCAAGACCCUCGAAACCCUCCCGGAAAAG
AGCGUGGUGGGUACCAGCUCCCUGCGGAGAGCUGCCCAGCUGCAGAGAAAGUUCCCGCAUCUGGAAUUCA
GGAGCAUCAGGGGAAAUCUGAAUACCAGACUGCGCAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAU
CCUGGCCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACAGGGUGGGCCAGAUACUGCAUCCGGAGGAG
UGUAUGUACGCCGUGGGCCAGGGCGCCCUCGGCGUGGAGGUGAGAGCCAAGGACCAAGACAUCCUGGACC
UAGUGGGCGUGCUGCAUGACCCUGAAACCCUGCUCAGGUGCAUCGCCGAGAGGGCCUUCCUGCGGCACCU
GGAGGGCGGCUGCAGCGUGCCGGUGGCCGUCCACACCGCCAUGAAGGACGGCCAGCUGUACCUGACCGGC
GGCGUCUGGAGCCUGGACGGAUCCGACAGCAUCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCGGCCC
AGCACGAGGACGGCCCUGAGGACGACCCUCAGCUGGUGGGCAUCACCGCUAGGAACAUCCCAAGGGGCCC
GCAGCUGGCCGCCCAGAACCUCGGCAUCAGCCUGGCCAACCUGCUGCUGUCCAAGGGCGCCAAGAAUAUC
CUGGACGUGGCCAGGCAGCUGAACGACGCCCAC
[0475] The sequence optimized nucleotide sequences disclosed herein
are distinct from the corresponding wild type nucleotide acid
sequences and from other known sequence optimized nucleotide
sequences, e.g., these sequence optimized nucleic acids have unique
compositional characteristics.
[0476] In some embodiments, the percentage of uracil or thymine
nucleobases in a sequence optimized nucleotide sequence (e.g.,
encoding a PBGD polypeptide, a functional fragment, or a variant
thereof) is modified (e.g., reduced) with respect to the percentage
of uracil or thymine nucleobases in the reference wild-type
nucleotide sequence. Such a sequence is referred to as a
uracil-modified or thymine-modified sequence. The percentage of
uracil or thymine content in a nucleotide sequence can be
determined by dividing the number of uracils or thymines in a
sequence by the total number of nucleotides and multiplying by 100.
In some embodiments, the sequence optimized nucleotide sequence has
a lower uracil or thymine content than the uracil or thymine
content in the reference wild-type sequence. In some embodiments,
the uracil or thymine content in a sequence-optimized nucleotide
sequence of the invention is greater than the uracil or thymine
content in the reference wild-type sequence and still maintain
beneficial effects, e.g., increased expression and/or reduced
Toll-Like Receptor (TLR) response when compared to the reference
wild-type sequence.
[0477] The uracil or thymine content of wild-type PBGD isoform 1 is
about 20%. In some embodiments, the uracil or thymine content of a
uracil- or thymine-modified sequence encoding a PBGD polypeptide is
less than 20%. In some embodiments, the uracil or thymine content
of a uracil- or thymine-modified sequence encoding a PBGD
polypeptide of the invention is less than 19%, less that 18%, less
than 17%, less than 16%, less than 15%, less than 14%, less than
13%, less than 12%, less than 11%, or less than 10%. In some
embodiments, the uracil or thymine content is not less than 18%,
17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10%. The uracil or thymine
content of a sequence disclosed herein, i.e., its total uracil or
thymine content is abbreviated herein as % U.sub.TL or %
T.sub.TL.
[0478] A uracil- or thymine-modified sequence encoding a PBGD
polypeptide of the invention can also be described according to its
uracil or thymine content relative to the uracil or thymine content
in the corresponding wild-type nucleic acid sequence (% U.sub.WT or
% T.sub.WT), or according to its uracil or thymine content relative
to the theoretical minimum uracil or thymine content of a nucleic
acid encoding the wild-type protein sequence (% U.sub.TM or (%
T.sub.TM).
[0479] The phrases "uracil or thymine content relative to the
uracil or thymine content in the wild type nucleic acid sequence,"
refers to a parameter determined by dividing the number of uracils
or thymines in a sequence-optimized nucleic acid by the total
number of uracils or thymines in the corresponding wild-type
nucleic acid sequence and multiplying by 100. This parameter is
abbreviated herein as % U.sub.WT or % T.sub.WT.
[0480] In some embodiments, the % U.sub.WT or % T.sub.WT of a
uracil- or thymine-modified sequence encoding a PBGD polypeptide of
the invention is above 50%, above 55%, above 60%, above 65%, above
70%, above 75%, above 80%, above 85%, above 90%, or above 95%.
[0481] Uracil- or thymine-content relative to the uracil or thymine
theoretical minimum, refers to a parameter determined by dividing
the number of uracils or thymines in a sequence-optimized
nucleotide sequence by the total number of uracils or thymines in a
hypothetical nucleotide sequence in which all the codons in the
hypothetical sequence are replaced with synonymous codons having
the lowest possible uracil or thymine content and multiplying by
100. This parameter is abbreviated herein as % U.sub.TM or %
T.sub.TM.
[0482] For DNA it is recognized that thymine is present instead of
uracil, and one would substitute T where U appears. Thus, all the
disclosures related to, e.g., % U.sub.TM, % U.sub.WT, or
.sub.%U.sub.TL, with respect to RNA are equally applicable to %
T.sub.TM, % T.sub.WT, or % T.sub.TL with respect to DNA.
[0483] In some embodiments, the % U.sub.TM of a uracil-modified
sequence encoding a PBGD polypeptide of the invention is between
about 118% and about 132%.
[0484] In some embodiments, a uracil-modified sequence encoding a
PBGD polypeptide of the invention has a reduced number of
consecutive uracils with respect to the corresponding wild-type
nucleic acid sequence. For example, two consecutive leucines can be
encoded by the sequence CUUUUG, which includes a four uracil
cluster. Such a subsequence can be substituted, e.g., with CUGCUC,
which removes the uracil cluster.
[0485] Phenylalanine can be encoded by UUC or UUU. Thus, even if
phenylalanines encoded by UUU are replaced by UUC, the synonymous
codon still contains a uracil pair (UU). Accordingly, the number of
phenylalanines in a sequence establishes a minimum number of uracil
pairs (UU) that cannot be eliminated without altering the number of
phenylalanines in the encoded polypeptide. For example, if the
polypeptide (e.g., wild type PBGD isoform 1) has, e.g., 7, 8, or 9
phenylalanines, the absolute minimum number of uracil pairs (UU) in
that a uracil-modified sequence encoding the polypeptide (e.g.,
wild type PBGD isoform 1) can contain is 7, 8, or 9,
respectively.
[0486] Wild type PBGD isoform 1 contains 32 uracil pairs (UU), and
four uracil triplets (UUU). In some embodiments, a uracil-modified
sequence encoding a PBGD polypeptide of the invention has a reduced
number of uracil triplets (UUU) with respect to the wild-type
nucleic acid sequence. In some embodiments, a uracil-modified
sequence encoding a PBGD polypeptide of the invention contains 4,
3, 2, 1 or no uracil triplets (UUU).
[0487] In some embodiments, a uracil-modified sequence encoding a
PBGD polypeptide has a reduced number of uracil pairs (UU) with
respect to the number of uracil pairs (UU) in the wild-type nucleic
acid sequence. In some embodiments, a uracil-modified sequence
encoding a PBGD polypeptide of the invention has a number of uracil
pairs (UU) corresponding to the minimum possible number of uracil
pairs (UU) in the wild-type nucleic acid sequence, e.g., 9 uracil
pairs in the case of wild type PBGD isoform 1.
[0488] In some embodiments, a uracil-modified sequence encoding a
PBGD polypeptide of the invention has at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 24
uracil pairs (UU) less than the number of uracil pairs (UU) in the
wild-type nucleic acid sequence. In some embodiments, a
uracil-modified sequence encoding a PBGD polypeptide of the
invention has between 8 and 16 uracil pairs (UU).
[0489] The phrase "uracil pairs (UU) relative to the uracil pairs
(UU) in the wild type nucleic acid sequence," refers to a parameter
determined by dividing the number of uracil pairs (UU) in a
sequence-optimized nucleotide sequence by the total number of
uracil pairs (UU) in the corresponding wild-type nucleotide
sequence and multiplying by 100. This parameter is abbreviated
herein as % UU.sub.wt.
[0490] In some embodiments, a uracil-modified sequence encoding a
PBGD polypeptide of the invention has a % UU.sub.wt less than 90%,
less than 85%, less than 80%, less than 75%, less than 70%, less
than 65%, less than 60%, less than 65%, less than 60%, less than
55%, less than 50%, less than 40%, less than 30%, or less than
20%.
[0491] In some embodiments, a uracil-modified sequence encoding a
PBGD polypeptide has a % UU.sub.wt between 20% and 55%. In a
particular embodiment, a uracil-modified sequence encoding a PBGD
polypeptide of the invention has a % UU.sub.wt between 25% and
55%.
[0492] In some embodiments, the polynucleotide of the invention
comprises a uracil-modified sequence encoding a PBGD polypeptide
disclosed herein. In some embodiments, the uracil-modified sequence
encoding a PBGD polypeptide comprises at least one chemically
modified nucleobase, e.g., 5-methoxyuracil. In some embodiments, at
least 95% of a nucleobase (e.g., uracil) in a uracil-modified
sequence encoding a PBGD polypeptide of the invention are modified
nucleobases. In some embodiments, at least 95% of uracil in a
uracil-modified sequence encoding a PBGD polypeptide is
5-methoxyuracil. In some embodiments, the polynucleotide comprising
a uracil-modified sequence further comprises a miRNA binding site,
e.g., a miRNA binding site that binds to miR-142 and/or a miRNA
binding site that binds to miR-126. In some embodiments, the
polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a
uracil-modified sequence disclosed herein is formulated with a
delivery agent comprising, e.g., a compound having the Formula (I),
e.g., any of Compounds 1-232, e.g., Compound 18; a compound having
the Formula (III), (IV), (V), or (VI), e.g., any of Compounds
233-342, e.g., Compound 236; or a compound having the Formula
(VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or any
combination thereof. In some embodiments, the delivery agent
comprises Compound 18, DSPC, Cholesterol, and Compound 428, e.g.,
with a mole ratio of about 50:10:38.5:1.5.
[0493] In some embodiments, the "guanine content of the sequence
optimized ORF encoding PBGD with respect to the theoretical maximum
guanine content of a nucleotide sequence encoding the PBGD
polypeptide," abbreviated as % G.sub.TMX is at least 69%, at least
70%, at least 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or about 100%. In some embodiments,
the % G.sub.TMX is between about 70% and about 80%, between about
71% and about 79%, between about 71% and about 78%, or between
about 71% and about 77%.
[0494] In some embodiments, the "cytosine content of the ORF
relative to the theoretical maximum cytosine content of a
nucleotide sequence encoding the PBGD polypeptide," abbreviated as
% C.sub.TMX, is at least 59%, at least 60%, at least about 65%, at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, or about 100%.
In some embodiments, the % C.sub.TMX is between about 60% and about
80%, between about 62% and about 80%, between about 63% and about
79%, or between about 68% and about 76%.
[0495] In some embodiments, the "guanine and cytosine content (G/C)
of the ORF relative to the theoretical maximum G/C content in a
nucleotide sequence encoding the PBGD polypeptide," abbreviated as
% G/C.sub.TMX is at least about 81%, at least about 85%, at least
about 90%, at least about 95%, or about 100%. The % G/C.sub.TMX is
between about 80% and about 100%, between about 85% and about 99%,
between about 90% and about 97%, or between about 91% and about
96%.
[0496] In some embodiments, the "G/C content in the ORF relative to
the G/C content in the corresponding wild-type ORF," abbreviated as
% G/C.sub.WT is at least 102%, at least 103%, at least 104%, at
least 105%, at least 106%, at least 107%, at least 110%, at least
115%, or at least 120%.
[0497] In some embodiments, the average G/C content in the 3rd
codon position in the ORF is at least 20%, at least 21%, at least
22%, at least 23%, at least 24%, at least 25%, at least 26%, at
least 27%, at least 28%, at least 29%, or at least 30% higher than
the average G/C content in the 3rd codon position in the
corresponding wild-type ORF.
[0498] In some embodiments, the polynucleotide of the invention
comprises an open reading frame (ORF) encoding a PBGD polypeptide,
wherein the ORF has been sequence optimized, and wherein each of %
U.sub.TL, % U.sub.WT, % U.sub.TM, % G.sub.TL, % G.sub.WT, %
G.sub.TMX, % C.sub.TL, % C.sub.WT, % C.sub.TMX, % G/C.sub.TL, %
G/C.sub.WT, or % G/C.sub.TMX, alone or in a combination thereof is
in a range between (i) a maximum corresponding to the parameter's
maximum value (MAX) plus about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,
5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 standard deviations
(STD DEV), and (ii) a minimum corresponding to the parameter's
minimum value (MIN) less 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,
5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 standard deviations (STD
DEV).
7. METHODS FOR SEQUENCE OPTIMIZATION
[0499] In some embodiments, a polynucleotide, e.g., mRNA, of the
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide, e.g., the wild-type sequence,
functional fragment, or variant thereof) is sequence optimized. A
sequence optimized nucleotide sequence (nucleotide sequence is also
referred to as "nucleic acid" herein) comprises at least one codon
modification with respect to a reference sequence (e.g., a
wild-type sequence encoding a PBGD polypeptide). Thus, in a
sequence optimized nucleic acid, at least one codon is different
from a corresponding codon in a reference sequence (e.g., a
wild-type sequence).
[0500] In general, sequence optimized nucleic acids are generated
by at least a step comprising substituting codons in a reference
sequence with synonymous codons (i.e., codons that encode the same
amino acid). Such substitutions can be effected, for example, by
applying a codon substitution map (i.e., a table providing the
codons that will encode each amino acid in the codon optimized
sequence), or by applying a set of rules (e.g., if glycine is next
to neutral amino acid, glycine would be encoded by a certain codon,
but if it is next to a polar amino acid, it would be encoded by
another codon). In addition to codon substitutions (i.e., "codon
optimization") the sequence optimization methods disclosed herein
comprise additional optimization steps which are not strictly
directed to codon optimization such as the removal of deleterious
motifs (destabilizing motif substitution). Compositions and
formulations comprising these sequence optimized nucleic acids
(e.g., a RNA, e.g., an mRNA) can be administered to a subject in
need thereof to facilitate in vivo expression of functionally
active PBGD.
[0501] The recombinant expression of large molecules in cell
cultures can be a challenging task with numerous limitations (e.g.,
poor protein expression levels, stalled translation resulting in
truncated expression products, protein misfolding, etc.). These
limitations can be reduced or avoided by administering the
polynucleotides (e.g., a RNA, e.g., an mRNA), which encode a
functionally active PBGD or compositions or formulations comprising
the same to a patient suffering from AIP, so the synthesis and
delivery of the PBGD polypeptide to treat AIP takes place
endogenously.
[0502] Changing from an in vitro expression system (e.g., cell
culture) to in vivo expression requires the redesign of the nucleic
acid sequence encoding the therapeutic agent. Redesigning a
naturally occurring gene sequence by choosing different codons
without necessarily altering the encoded amino acid sequence can
often lead to dramatic increases in protein expression levels
(Gustafsson et al., 2004, Trends Biotechnol 22:346-53). Variables
such as codon adaptation index (CAI), mRNA secondary structures,
cis-regulatory sequences, GC content and many other similar
variables have been shown to somewhat correlate with protein
expression levels (Villalobos et al., 2006, BMC Bioinformatics
7:285). However, due to the degeneracy of the genetic code, there
are numerous different nucleic acid sequences that can all encode
the same therapeutic agent. Each amino acid is encoded by up to six
synonymous codons; and the choice between these codons influences
gene expression. In addition, codon usage (i.e., the frequency with
which different organisms use codons for expressing a polypeptide
sequence) differs among organisms (for example, recombinant
production of human or humanized therapeutic antibodies frequently
takes place in hamster cell cultures).
[0503] In some embodiments, a reference nucleic acid sequence can
be sequence optimized by applying a codon map. The skilled artisan
will appreciate that T bases are present in DNA, whereas the T
bases would be replaced by U bases in corresponding RNAs. For
example, a sequence optimized nucleic acid disclosed herein in DNA
form, e.g., a vector or an in-vitro translation (IVT) template,
would have its T bases transcribed as U based in its corresponding
transcribed mRNA. In this respect, both sequence optimized DNA
sequences (comprising T) and their corresponding RNA sequences
(comprising U) are considered sequence optimized nucleic acid of
the present invention. A skilled artisan would also understand that
equivalent codon-maps can be generated by replaced one or more
bases with non-natural bases. Thus, e.g., a TTC codon (DNA map)
would correspond to a UUC codon (RNA map), which in turn can
correspond to a .psi..psi.C codon (RNA map in which U has been
replaced with pseudouridine).
[0504] In one embodiment, a reference sequence encoding PBGD can be
optimized by replacing all the codons encoding a certain amino acid
with only one of the alternative codons provided in a codon map.
For example, all the valines in the optimized sequence would be
encoded by GTG or GTC or GTT.
[0505] Sequence optimized polynucleotides of the invention can be
generated using one or more optimization methods, or a combination
thereof. Sequence optimization methods which can be used to
sequence optimize nucleic acid sequences are described in detail
herein. This list of methods is not comprehensive or limiting.
[0506] It will be appreciated that the design principles and rules
described for each one of the sequence optimization methods
discussed below can be combined in many different ways, for example
high G/C content sequence optimization for some regions or uridine
content sequence optimization for other regions of the reference
nucleic acid sequence, as well as targeted nucleotide mutations to
minimize secondary structure throughout the sequence or to
eliminate deleterious motifs.
[0507] The choice of potential combinations of sequence
optimization methods can be, for example, dependent on the specific
chemistry used to produce a synthetic polynucleotide. Such a choice
can also depend on characteristics of the protein encoded by the
sequence optimized nucleic acid, e.g., a full sequence, a
functional fragment, or a fusion protein comprising PBGD, etc. In
some embodiments, such a choice can depend on the specific tissue
or cell targeted by the sequence optimized nucleic acid (e.g., a
therapeutic synthetic mRNA).
[0508] The mechanisms of combining the sequence optimization
methods or design rules derived from the application and analysis
of the optimization methods can be either simple or complex. For
example, the combination can be:
[0509] (i) Sequential: Each sequence optimization method or set of
design rules applies to a different subsequence of the overall
sequence, for example reducing uridine at codon positions 1 to 30
and then selecting high frequency codons for the remainder of the
sequence.
[0510] (ii) Hierarchical: Several sequence optimization methods or
sets of design rules are combined in a hierarchical, deterministic
fashion. For example, use the most GC-rich codons, breaking ties
(which are common) by choosing the most frequent of those
codons.
[0511] (iii) Multifactorial/Multiparametric: Machine learning or
other modeling techniques are used to design a single sequence that
best satisfies multiple overlapping and possibly contradictory
requirements. This approach would require the use of a computer
applying a number of mathematical techniques, for example, genetic
algorithms.
[0512] Ultimately, each one of these approaches can result in a
specific set of rules which in many cases can be summarized in a
single codon table, i.e., a sorted list of codons for each amino
acid in the target protein (i.e., PBGD), with a specific rule or
set of rules indicating how to select a specific codon for each
amino acid position.
a. Uridine Content Optimization
[0513] The presence of local high concentrations of uridine in a
nucleic acid sequence can have detrimental effects on translation,
e.g., slow or prematurely terminated translation, especially when
modified uridine analogs are used in the production of synthetic
mRNAs. Furthermore, high uridine content can also reduce the in
vivo half-life of synthetic mRNAs due to TLR activation.
[0514] Accordingly, a nucleic acid sequence can be sequence
optimized using a method comprising at least one uridine content
optimization step. Such a step comprises, e.g., substituting at
least one codon in the reference nucleic acid with an alternative
codon to generate a uridine-modified sequence, wherein the
uridine-modified sequence has at least one of the following
properties:
[0515] (i) increase or decrease in global uridine content;
[0516] (ii) increase or decrease in local uridine content (i.e.,
changes in uridine content are limited to specific
subsequences);
[0517] (iii) changes in uridine distribution without altering the
global uridine content;
[0518] (iv) changes in uridine clustering (e.g., number of
clusters, location of clusters, or distance between clusters);
or
[0519] (v) combinations thereof.
[0520] In some embodiments, the sequence optimization process
comprises optimizing the global uridine content, i.e., optimizing
the percentage of uridine nucleobases in the sequence optimized
nucleic acid with respect to the percentage of uridine nucleobases
in the reference nucleic acid sequence. For example, 30% of
nucleobases can be uridines in the reference sequence and 10% of
nucleobases can be uridines in the sequence optimized nucleic
acid.
[0521] In other embodiments, the sequence optimization process
comprises reducing the local uridine content in specific regions of
a reference nucleic acid sequence, i.e., reducing the percentage of
uridine nucleobases in a subsequence of the sequence optimized
nucleic acid with respect to the percentage of uridine nucleobases
in the corresponding subsequence of the reference nucleic acid
sequence. For example, the reference nucleic acid sequence can have
a 5'-end region (e.g., 30 codons) with a local uridine content of
30%, and the uridine content in that same region could be reduced
to 10% in the sequence optimized nucleic acid.
[0522] In specific embodiments, codons can be replaced in the
reference nucleic acid sequence to reduce or modify, for example,
the number, size, location, or distribution of uridine clusters
that could have deleterious effects on protein translation.
Although as a general rule it is desirable to reduce the uridine
content of the reference nucleic acid sequence, in certain
embodiments the uridine content, and in particular the local
uridine content, of some subsequences of the reference nucleic acid
sequence can be increased.
[0523] The reduction of uridine content to avoid adverse effects on
translation can be done in combination with other optimization
methods disclosed here to achieve other design goals. For example,
uridine content optimization can be combined with ramp design,
since using the rarest codons for most amino acids will, with a few
exceptions, reduce the U content.
[0524] In some embodiments, the uridine-modified sequence is
designed to induce a lower Toll-Like Receptor (TLR) response when
compared to the reference nucleic acid sequence. Several TLRs
recognize and respond to nucleic acids. Double-stranded (ds)RNA, a
frequent viral constituent, has been shown to activate TLR3. See
Alexopoulou et al. (2001) Nature, 413:732-738 and Wang et al.
(2004) Nat. Med., 10:1366-1373. Single-stranded (ss)RNA activates
TLR7. See Diebold et al. (2004) Science 303:1529-1531. RNA
oligonucleotides, for example RNA with phosphorothioate
internucleotide linkages, are ligands of human TLR8. See Heil et
al. (2004) Science 303:1526-1529. DNA containing unmethylated CpG
motifs, characteristic of bacterial and viral DNA, activate TLR9.
See Hemmi et al. (2000) Nature, 408: 740-745.
[0525] As used herein, the term "TLR response" is defined as the
recognition of single-stranded RNA by a TLR7 receptor, and in some
embodiments encompasses the degradation of the RNA and/or
physiological responses caused by the recognition of the
single-stranded RNA by the receptor. Methods to determine and
quantitate the binding of an RNA to a TLR7 are known in the art.
Similarly, methods to determine whether an RNA has triggered a
TLR7-mediated physiological response (e.g., cytokine secretion) are
well known in the art. In some embodiments, a TLR response can be
mediated by TLR3, TLR8, or TLR9 instead of TLR7.
[0526] Suppression of TLR7-mediated response can be accomplished
via nucleoside modification. RNA undergoes over hundred different
nucleoside modifications in nature (see the RNA Modification
Database, available at mods.ma.albany.edu). Human rRNA, for
example, has ten times more pseudouridine (P) and 25 times more
2'-O-methylated nucleosides than bacterial rRNA. Bacterial mRNA
contains no nucleoside modifications, whereas mammalian mRNAs have
modified nucleosides such as 5-methylcytidine (m5C),
N6-methyladenosine (m6A), inosine and many 2'-O-methylated
nucleosides in addition to N7-methylguanosine (m7G).
[0527] Uracil and ribose, the two defining features of RNA, are
both necessary and sufficient for TLR7 stimulation, and short
single-stranded RNA (ssRNA) act as TLR7 agonists in a
sequence-independent manner as long as they contain several
uridines in close proximity. See Diebold et al. (2006) Eur. J.
Immunol. 36:3256-3267, which is herein incorporated by reference in
its entirety. Accordingly, one or more of the optimization methods
disclosed herein comprises reducing the uridine content (locally
and/or globally) and/or reducing or modifying uridine clustering to
reduce or to suppress a TLR7-mediated response.
[0528] In some embodiments, the TLR response (e.g., a response
mediated by TLR7) caused by the uridine-modified sequence is at
least about 10%, at least about 15%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, or at least about 100% lower than the TLR
response caused by the reference nucleic acid sequence.
[0529] In some embodiments, the TLR response caused by the
reference nucleic acid sequence is at least about 1-fold, at least
about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold,
at least about 1.4-fold, at least about 1.5-fold, at least about
1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at
least about 1.9-fold, at least about 2-fold, at least about 3-fold,
at least about 4-fold, at least about 5-fold, at least about
6-fold, at least about 7-fold, at least about 8-fold, at least
about 9-fold, or at least about 10-fold higher than the TLR
response caused by the uridine-modified sequence.
[0530] In some embodiments, the uridine content (average global
uridine content) (absolute or relative) of the uridine-modified
sequence is higher than the uridine content (absolute or relative)
of the reference nucleic acid sequence. Accordingly, in some
embodiments, the uridine-modified sequence contains at least about
5%, at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or at least about 100% more uridine
that the reference nucleic acid sequence.
[0531] In other embodiments, the uridine content (average global
uridine content) (absolute or relative) of the uridine-modified
sequence is lower than the uridine content (absolute or relative)
of the reference nucleic acid sequence. Accordingly, in some
embodiments, the uridine-modified sequence contains at least about
5%, at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or at least about 100% less uridine
that the reference nucleic acid sequence.
[0532] In some embodiments, the uridine content (average global
uridine content) (absolute or relative) of the uridine-modified
sequence is less than 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%,
41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%,
28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%,
15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%
of the total nucleobases in the uridine-modified sequence. In some
embodiments, the uridine content of the uridine-modified sequence
is between about 10% and about 20%. In some particular embodiments,
the uridine content of the uridine-modified sequence is between
about 12% and about 16%.
[0533] In some embodiments, the uridine content of the reference
nucleic acid sequence can be measured using a sliding window. In
some embodiments, the length of the sliding window is 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40
nucleobases. In some embodiments, the sliding window is over 40
nucleobases in length. In some embodiments, the sliding window is
20 nucleobases in length. Based on the uridine content measured
with a sliding window, it is possible to generate a histogram
representing the uridine content throughout the length of the
reference nucleic acid sequence and sequence optimized nucleic
acids.
[0534] In some embodiments, a reference nucleic acid sequence can
be modified to reduce or eliminate peaks in the histogram that are
above or below a certain percentage value. In some embodiments, the
reference nucleic acid sequence can be modified to eliminate peaks
in the sliding-window representation which are above 65%, 60%, 55%,
50%, 45%, 40%, 35%, or 30% uridine. In another embodiment, the
reference nucleic acid sequence can be modified so no peaks are
over 30% uridine in the sequence optimized nucleic acid, as
measured using a 20 nucleobase sliding window. In some embodiments,
the reference nucleic acid sequence can be modified so no more or
no less than a predetermined number of peaks in the sequence
optimized nucleic sequence, as measured using a 20 nucleobase
sliding window, are above or below a certain threshold value. For
example, in some embodiments, the reference nucleic acid sequence
can be modified so no peaks or no more than 1, 2, 3, 4, 5, 6, 7, 8,
9 or 10 peaks in the sequence optimized nucleic acid are above 10%,
15%, 20%, 25% or 30% uridine. In another embodiment, the sequence
optimized nucleic acid contains between 0 peaks and 2 peaks with
uridine contents 30% of higher.
[0535] In some embodiments, a reference nucleic acid sequence can
be sequence optimized to reduce the incidence of consecutive
uridines. For example, two consecutive leucines could be encoded by
the sequence CUUUUG, which would include a four uridine cluster.
Such subsequence could be substituted with CUGCUC, which would
effectively remove the uridine cluster. Accordingly, a reference
nucleic sequence can be sequence optimized by reducing or
eliminating uridine pairs (UU), uridine triplets (UUU) or uridine
quadruplets (UUUU). Higher order combinations of U are not
considered combinations of lower order combinations. Thus, for
example, UUUU is strictly considered a quadruplet, not two
consecutive U pairs; or UUUUUU is considered a sextuplet, not three
consecutive U pairs, or two consecutive U triplets, etc.
[0536] In some embodiments, all uridine pairs (UU) and/or uridine
triplets (UUU) and/or uridine quadruplets (UUUU) can be removed
from the reference nucleic acid sequence. In other embodiments,
uridine pairs (UU) and/or uridine triplets (UUU) and/or uridine
quadruplets (UUUU) can be reduced below a certain threshold, e.g.,
no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 occurrences in the sequence optimized nucleic
acid. In a particular embodiment, the sequence optimized nucleic
acid contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 uridine pairs. In another particular
embodiment, the sequence optimized nucleic acid contains no uridine
pairs and/or triplets.
[0537] Phenylalanine codons, i.e., UUC or UUU, comprise a uridine
pair or triplet and therefore sequence optimization to reduce
uridine content can at most reduce the phenylalanine U triplet to a
phenylalanine U pair. In some embodiments, the occurrence of
uridine pairs (UU) and/or uridine triplets (UUU) refers only to
non-phenylalanine U pairs or triplets. Accordingly, in some
embodiments, non-phenylalanine uridine pairs (UU) and/or uridine
triplets (UUU) can be reduced below a certain threshold, e.g., no
more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 occurrences in the sequence optimized nucleic
acid. In a particular embodiment, the sequence optimized nucleic
acid contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 non-phenylalanine uridine pairs and/or
triplets. In another particular embodiment, the sequence optimized
nucleic acid contains no non-phenylalanine uridine pairs and/or
triplets.
[0538] In some embodiments, the reduction in uridine combinations
(e.g., pairs, triplets, quadruplets) in the sequence optimized
nucleic acid can be expressed as a percentage reduction with
respect to the uridine combinations present in the reference
nucleic acid sequence.
[0539] In some embodiments, a sequence optimized nucleic acid can
contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,
13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, or 65% of the total number of uridine pairs present
in the reference nucleic acid sequence. In some embodiments, a
sequence optimized nucleic acid can contain about 1%, 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,
19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the
total number of uridine triplets present in the reference nucleic
acid sequence. In some embodiments, a sequence optimized nucleic
acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, or 65% of the total number of uridine
quadruplets present in the reference nucleic acid sequence.
[0540] In some embodiments, a sequence optimized nucleic acid can
contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,
13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, or 65% of the total number of non-phenylalanine
uridine pairs present in the reference nucleic acid sequence. In
some embodiments, a sequence optimized nucleic acid can contain
about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, or 65% of the total number of non-phenylalanine uridine
triplets present in the reference nucleic acid sequence.
[0541] In some embodiments, the uridine content in the sequence
optimized sequence can be expressed with respect to the theoretical
minimum uridine content in the sequence. The term "theoretical
minimum uridine content" is defined as the uridine content of a
nucleic acid sequence as a percentage of the sequence's length
after all the codons in the sequence have been replaced with
synonymous codon with the lowest uridine content. In some
embodiments, the uridine content of the sequence optimized nucleic
acid is identical to the theoretical minimum uridine content of the
reference sequence (e.g., a wild type sequence). In some aspects,
the uridine content of the sequence optimized nucleic acid is about
100%, about 105%, about 110%, about 115%, about 120%, about 125%,
about 130%, about 135%, about 140%, about 145%, about 150%, about
155%, about 160%, about 165%, about 170%, about 175%, about 180%,
about 185%, about 190%, about 195%, about 200%, about 210%, about
220%, about 230%, about 240% or about 250% of the theoretical
minimum uridine content of the reference sequence (e.g., a wild
type sequence).
[0542] In some embodiments, the uridine content of the sequence
optimized nucleic acid is identical to the theoretical minimum
uridine content of the reference sequence (e.g., a wild type
sequence).
[0543] The reference nucleic acid sequence (e.g., a wild type
sequence) can comprise uridine clusters which due to their number,
size, location, distribution or combinations thereof have negative
effects on translation. As used herein, the term "uridine cluster"
refers to a subsequence in a reference nucleic acid sequence or
sequence optimized nucleic sequence with contains a uridine content
(usually described as a percentage) which is above a certain
threshold. Thus, in certain embodiments, if a subsequence comprises
more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60% or 65% uridine content, such subsequence would be considered a
uridine cluster.
[0544] The negative effects of uridine clusters can be, for
example, eliciting a TLR7 response. Thus, in some implementations
of the nucleic acid sequence optimization methods disclosed herein
it is desirable to reduce the number of clusters, size of clusters,
location of clusters (e.g., close to the 5' and/or 3' end of a
nucleic acid sequence), distance between clusters, or distribution
of uridine clusters (e.g., a certain pattern of cluster along a
nucleic acid sequence, distribution of clusters with respect to
secondary structure elements in the expressed product, or
distribution of clusters with respect to the secondary structure of
an mRNA).
[0545] In some embodiments, the reference nucleic acid sequence
comprises at least one uridine cluster, wherein said uridine
cluster is a subsequence of the reference nucleic acid sequence
wherein the percentage of total uridine nucleobases in said
subsequence is above a predetermined threshold. In some
embodiments, the length of the subsequence is at least about 10, at
least about 15, at least about 20, at least about 25, at least
about 30, at least about 35, at least about 40, at least about 45,
at least about 50, at least about 55, at least about 60, at least
about 65, at least about 70, at least about 75, at least about 80,
at least about 85, at least about 90, at least about 95, or at
least about 100 nucleobases. In some embodiments, the subsequence
is longer than 100 nucleobases. In some embodiments, the threshold
is 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% uridine
content. In some embodiments, the threshold is above 25%.
[0546] For example, an amino acid sequence comprising A, D, G, S
and R could be encoded by the nucleic acid sequence GCU, GAU, GGU,
AGU, CGU. Although such sequence does not contain any uridine
pairs, triplets, or quadruplets, one third of the nucleobases would
be uridines. Such a uridine cluster could be removed by using
alternative codons, for example, by using GCC, GAC, GGC, AGC, and
CGC, which would contain no uridines.
[0547] In other embodiments, the reference nucleic acid sequence
comprises at least one uridine cluster, wherein said uridine
cluster is a subsequence of the reference nucleic acid sequence
wherein the percentage of uridine nucleobases of said subsequence
as measured using a sliding window that is above a predetermined
threshold. In some embodiments, the length of the sliding window is
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
or 40 nucleobases. In some embodiments, the sliding window is over
40 nucleobases in length. In some embodiments, the threshold is 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% uridine content. In
some embodiments, the threshold is above 25%.
[0548] In some embodiments, the reference nucleic acid sequence
comprises at least two uridine clusters. In some embodiments, the
uridine-modified sequence contains fewer uridine-rich clusters than
the reference nucleic acid sequence. In some embodiments, the
uridine-modified sequence contains more uridine-rich clusters than
the reference nucleic acid sequence. In some embodiments, the
uridine-modified sequence contains uridine-rich clusters with are
shorter in length than corresponding uridine-rich clusters in the
reference nucleic acid sequence. In other embodiments, the
uridine-modified sequence contains uridine-rich clusters which are
longer in length than the corresponding uridine-rich cluster in the
reference nucleic acid sequence.
[0549] See, Kariko et al. (2005) Immunity 23:165-175; Kormann et
al. (2010) Nature Biotechnology 29:154-157; or Sahin et al. (2014)
Nature Reviews Drug Discovery|AOP, published online 19 Sep. 2014m
doi:10.1038/nrd4278; all of which are herein incorporated by
reference their entireties.
b. Guanine/Cytosine (G/C) Content
[0550] A reference nucleic acid sequence can be sequence optimized
using methods comprising altering the Guanine/Cytosine (G/C)
content (absolute or relative) of the reference nucleic acid
sequence. Such optimization can comprise altering (e.g., increasing
or decreasing) the global G/C content (absolute or relative) of the
reference nucleic acid sequence; introducing local changes in G/C
content in the reference nucleic acid sequence (e.g., increase or
decrease G/C in selected regions or subsequences in the reference
nucleic acid sequence); altering the frequency, size, and
distribution of G/C clusters in the reference nucleic acid
sequence, or combinations thereof.
[0551] In some embodiments, the sequence optimized nucleic acid
encoding PBGD comprises an overall increase in G/C content
(absolute or relative) relative to the G/C content (absolute or
relative) of the reference nucleic acid sequence. In some
embodiments, the overall increase in G/C content (absolute or
relative) is at least about 5%, at least about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, or at
least about 100% relative to the G/C content (absolute or relative)
of the reference nucleic acid sequence.
[0552] In some embodiments, the sequence optimized nucleic acid
encoding PBGD comprises an overall decrease in G/C content
(absolute or relative) relative to the G/C content of the reference
nucleic acid sequence. In some embodiments, the overall decrease in
G/C content (absolute or relative) is at least about 5%, at least
about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or at least about 100% relative to the G/C content
(absolute or relative) of the reference nucleic acid sequence.
[0553] In some embodiments, the sequence optimized nucleic acid
encoding PBGD comprises a local increase in Guanine/Cytosine (G/C)
content (absolute or relative) in a subsequence (i.e., a G/C
modified subsequence) relative to the G/C content (absolute or
relative) of the corresponding subsequence in the reference nucleic
acid sequence. In some embodiments, the local increase in G/C
content (absolute or relative) is by at least about 5%, at least
about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or at least about 100% relative to the G/C content
(absolute or relative) of the corresponding subsequence in the
reference nucleic acid sequence.
[0554] In some embodiments, the sequence optimized nucleic acid
encoding PBGD comprises a local decrease in Guanine/Cytosine (G/C)
content (absolute or relative) in a subsequence (i.e., a G/C
modified subsequence) relative to the G/C content (absolute or
relative) of the corresponding subsequence in the reference nucleic
acid sequence. In some embodiments, the local decrease in G/C
content (absolute or relative) is by at least about 5%, at least
about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or at least about 100% relative to the G/C content
(absolute or relative) of the corresponding subsequence in the
reference nucleic acid sequence.
[0555] In some embodiments, the G/C content (absolute or relative)
is increased or decreased in a subsequence which is at least about
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100 nucleobases in length.
[0556] In some embodiments, the G/C content (absolute or relative)
is increased or decreased in a subsequence which is at least about
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,
360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,
490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,
620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,
750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,
880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000
nucleobases in length.
[0557] In some embodiments, the G/C content (absolute or relative)
is increased or decreased in a subsequence which is at least about
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,
2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200,
3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300,
4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400,
5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500,
6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600,
7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700,
8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800,
9900, or 10000 nucleobases in length.
[0558] The increases or decreases in G and C content (absolute or
relative) described herein can be conducted by replacing synonymous
codons with low G/C content with synonymous codons having higher
G/C content, or vice versa. For example, L has 6 synonymous codons:
two of them have 2 G/C (CUC, CUG), 3 have a single G/C (UUG, CUU,
CUA), and one has no G/C (UUA). So if the reference nucleic acid
had a CUC codon in a certain position, G/C content at that position
could be reduced by replacing CUC with any of the codons having a
single G/C or the codon with no G/C.
[0559] See, U.S. Publ. Nos. US20140228558, US20050032730 A1;
Gustafsson et al. (2012) Protein Expression and Purification 83:
37-46; all of which are incorporated herein by reference in their
entireties.
c. Codon Frequency--Codon Usage Bias
[0560] Numerous codon optimization methods known in the art are
based on the substitution of codons in a reference nucleic acid
sequence with codons having higher frequencies. Thus, in some
embodiments, a nucleic acid sequence encoding PBGD disclosed herein
can be sequence optimized using methods comprising the use of
modifications in the frequency of use of one or more codons
relative to other synonymous codons in the sequence optimized
nucleic acid with respect to the frequency of use in the non-codon
optimized sequence.
[0561] As used herein, the term "codon frequency" refers to codon
usage bias, i.e., the differences in the frequency of occurrence of
synonymous codons in coding DNA/RNA. It is generally acknowledged
that codon preferences reflect a balance between mutational biases
and natural selection for translational optimization. Optimal
codons help to achieve faster translation rates and high accuracy.
As a result of these factors, translational selection is expected
to be stronger in highly expressed genes. In the field of
bioinformatics and computational biology, many statistical methods
have been proposed and used to analyze codon usage bias. See, e.g.,
Comeron & Aguade (1998) J. Mol. Evol. 47: 268-74. Methods such
as the "frequency of optimal codons" (Fop) (Ikemura (1981) J. Mol.
Biol. 151 (3): 389-409), the "Relative Codon Adaptation" (RCA) (Fox
& Eril (2010) DNA Res. 17 (3): 185-96) or the "Codon Adaptation
Index" (CAI) (Sharp & Li (1987) Nucleic Acids Res. 15 (3):
1281-95) are used to predict gene expression levels, while methods
such as the "effective number of codons" (Nc) and Shannon entropy
from information theory are used to measure codon usage evenness.
Multivariate statistical methods, such as correspondence analysis
and principal component analysis, are widely used to analyze
variations in codon usage among genes (Suzuki et al. (2008) DNA
Res. 15 (6): 357-65; Sandhu et al., In Silico Biol. 2008;
8(2):187-92).
[0562] The nucleic acid sequence encoding a PBGD polypeptide
disclosed herein (e.g., a wild type nucleic acid sequence, a mutant
nucleic acid sequence, a chimeric nucleic sequence, etc. which can
be, for example, an mRNA), can be codon optimized using methods
comprising substituting at least one codon in the reference nucleic
acid sequence with an alternative codon having a higher or lower
codon frequency in the synonymous codon set; wherein the resulting
sequence optimized nucleic acid has at least one optimized property
with respect to the reference nucleic acid sequence.
[0563] In some embodiments, at least about 5%, at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, at least about 99%, or 100% of the codons in the
reference nucleic acid sequence encoding PBGD are substituted with
alternative codons, each alternative codon having a codon frequency
higher than the codon frequency of the substituted codon in the
synonymous codon set.
[0564] In some embodiments, at least one codon in the reference
nucleic acid sequence encoding PBGD is substituted with an
alternative codon having a codon frequency higher than the codon
frequency of the substituted codon in the synonymous codon set, and
at least one codon in the reference nucleic acid sequence is
substituted with an alternative codon having a codon frequency
lower than the codon frequency of the substituted codon in the
synonymous codon set.
[0565] In some embodiments, at least about 5%, at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, or at least about 75%
of the codons in the reference nucleic acid sequence encoding PBGD
are substituted with alternative codons, each alternative codon
having a codon frequency higher than the codon frequency of the
substituted codon in the synonymous codon set.
[0566] In some embodiments, at least one alternative codon having a
higher codon frequency has the highest codon frequency in the
synonymous codon set. In other embodiments, all alternative codons
having a higher codon frequency have the highest codon frequency in
the synonymous codon set.
[0567] In some embodiments, at least one alternative codon having a
lower codon frequency has the lowest codon frequency in the
synonymous codon set. In some embodiments, all alternative codons
having a higher codon frequency have the highest codon frequency in
the synonymous codon set.
[0568] In some specific embodiments, at least one alternative codon
has the second highest, the third highest, the fourth highest, the
fifth highest or the sixth highest frequency in the synonymous
codon set. In some specific embodiments, at least one alternative
codon has the second lowest, the third lowest, the fourth lowest,
the fifth lowest, or the sixth lowest frequency in the synonymous
codon set.
[0569] Optimization based on codon frequency can be applied
globally, as described above, or locally to the reference nucleic
acid sequence encoding a PBGD polypeptide. In some embodiments,
when applied locally, regions of the reference nucleic acid
sequence can modified based on codon frequency, substituting all or
a certain percentage of codons in a certain subsequence with codons
that have higher or lower frequencies in their respective
synonymous codon sets. Thus, in some embodiments, at least about
5%, at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 99%, or 100% of the
codons in a subsequence of the reference nucleic acid sequence are
substituted with alternative codons, each alternative codon having
a codon frequency higher than the codon frequency of the
substituted codon in the synonymous codon set.
[0570] In some embodiments, at least one codon in a subsequence of
the reference nucleic acid sequence encoding a PBGD polypeptide is
substituted with an alternative codon having a codon frequency
higher than the codon frequency of the substituted codon in the
synonymous codon set, and at least one codon in a subsequence of
the reference nucleic acid sequence is substituted with an
alternative codon having a codon frequency lower than the codon
frequency of the substituted codon in the synonymous codon set.
[0571] In some embodiments, at least about 5%, at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, or at least about 75%
of the codons in a subsequence of the reference nucleic acid
sequence encoding a PBGD polypeptide are substituted with
alternative codons, each alternative codon having a codon frequency
higher than the codon frequency of the substituted codon in the
synonymous codon set.
[0572] In some embodiments, at least one alternative codon
substituted in a subsequence of the reference nucleic acid sequence
encoding a PBGD polypeptide and having a higher codon frequency has
the highest codon frequency in the synonymous codon set. In other
embodiments, all alternative codons substituted in a subsequence of
the reference nucleic acid sequence and having a lower codon
frequency have the lowest codon frequency in the synonymous codon
set.
[0573] In some embodiments, at least one alternative codon
substituted in a subsequence of the reference nucleic acid sequence
encoding a PBGD polypeptide and having a lower codon frequency has
the lowest codon frequency in the synonymous codon set. In some
embodiments, all alternative codons substituted in a subsequence of
the reference nucleic acid sequence and having a higher codon
frequency have the highest codon frequency in the synonymous codon
set.
[0574] In specific embodiments, a sequence optimized nucleic acid
encoding a PBGD polypeptide can comprise a subsequence having an
overall codon frequency higher or lower than the overall codon
frequency in the corresponding subsequence of the reference nucleic
acid sequence at a specific location, for example, at the 5' end or
3' end of the sequence optimized nucleic acid, or within a
predetermined distance from those region (e.g., at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95 or 100 codons from the 5' end or 3' end of
the sequence optimized nucleic acid).
[0575] In some embodiments, a sequence optimized nucleic acid
encoding a PBGD polypeptide can comprise more than one subsequence
having an overall codon frequency higher or lower than the overall
codon frequency in the corresponding subsequence of the reference
nucleic acid sequence. A skilled artisan would understand that
subsequences with overall higher or lower overall codon frequencies
can be organized in innumerable patterns, depending on whether the
overall codon frequency is higher or lower, the length of the
subsequence, the distance between subsequences, the location of the
subsequences, etc.
[0576] See, U.S. Pat. Nos. 5,082,767, 8,126,653, 7,561,973,
8,401,798; U.S. Publ. No. US 20080046192, US 20080076161; Int'l.
Publ. No. WO2000018778; Welch et al. (2009) PLoS ONE 4(9): e7002;
Gustafsson et al. (2012) Protein Expression and Purification 83:
37-46; Chung et al. (2012) BMC Systems Biology 6:134; all of which
are incorporated herein by reference in their entireties.
d. Destabilizing Motif Substitution
[0577] There is a variety of motifs that can affect sequence
optimization, which fall into various non-exclusive categories, for
example:
[0578] (i) Primary sequence based motifs: Motifs defined by a
simple arrangement of nucleotides.
[0579] (ii) Structural motifs: Motifs encoded by an arrangement of
nucleotides that tends to form a certain secondary structure.
[0580] (iii) Local motifs: Motifs encoded in one contiguous
subsequence.
[0581] (iv) Distributed motifs: Motifs encoded in two or more
disjoint subsequences.
[0582] (v) Advantageous motifs: Motifs which improve nucleotide
structure or function.
[0583] (vi) Disadvantageous motifs: Motifs with detrimental effects
on nucleotide structure or function.
[0584] There are many motifs that fit into the category of
disadvantageous motifs. Some examples include, for example,
restriction enzyme motifs, which tend to be relatively short, exact
sequences such as the restriction site motifs forXbaI (TCTAGA),
EcoRI (GAATTC), EcoRII (CCWGG, wherein W means A or T, per the
IUPAC ambiguity codes), or HindIII (AAGCTT); enzyme sites, which
tend to be longer and based on consensus not exact sequence, such
in the T7 RNA polymerase (GnnnnWnCRnCTCnCnnWnD, wherein n means any
nucleotide, R means A or G, W means A or T, D means A or G or T but
not C); structural motifs, such as GGGG repeats (Kim et al. (1991)
Nature 351(6324):331-2); or other motifs such as CUG-triplet
repeats (Querido et al. (2014) J. Cell Sci. 124:1703-1714).
[0585] Accordingly, the nucleic acid sequence encoding a PBGD
polypeptide disclosed herein can be sequence optimized using
methods comprising substituting at least one destabilizing motif in
a reference nucleic acid sequence, and removing such
disadvantageous motif or replacing it with an advantageous
motif.
[0586] In some embodiments, the optimization process comprises
identifying advantageous and/or disadvantageous motifs in the
reference nucleic sequence, wherein such motifs are, e.g., specific
subsequences that can cause a loss of stability in the reference
nucleic acid sequence prior or during the optimization process. For
example, substitution of specific bases during optimization can
generate a subsequence (motif) recognized by a restriction enzyme.
Accordingly, during the optimization process the appearance of
disadvantageous motifs can be monitored by comparing the sequence
optimized sequence with a library of motifs known to be
disadvantageous. Then, the identification of disadvantageous motifs
could be used as a post-hoc filter, i.e., to determine whether a
certain modification which potentially could be introduced in the
reference nucleic acid sequence should be actually implemented or
not.
[0587] In some embodiments, the identification of disadvantageous
motifs can be used prior to the application of the sequence
optimization methods disclosed herein, i.e., the identification of
motifs in the reference nucleic acid sequence encoding a PBGD
polypeptide and their replacement with alternative nucleic acid
sequences can be used as a preprocessing step, for example, before
uridine reduction.
[0588] In other embodiments, the identification of disadvantageous
motifs and their removal is used as an additional sequence
optimization technique integrated in a multiparametric nucleic acid
optimization method comprising two or more of the sequence
optimization methods disclosed herein. When used in this fashion, a
disadvantageous motif identified during the optimization process
would be removed, for example, by substituting the lowest possible
number of nucleobases in order to preserve as closely as possible
the original design principle(s) (e.g., low U, high frequency,
etc.).
[0589] See, e.g., U.S. Publ. Nos. US20140228558, US20050032730, or
US20140228558, which are herein incorporated by reference in their
entireties.
e. Limited Codon Set Optimization
[0590] In some particular embodiments, sequence optimization of a
reference nucleic acid sequence encoding a PBGD polypeptide can be
conducted using a limited codon set, e.g., a codon set wherein less
than the native number of codons is used to encode the 20 natural
amino acids, a subset of the 20 natural amino acids, or an expanded
set of amino acids including, for example, non-natural amino
acids.
[0591] The genetic code is highly similar among all organisms and
can be expressed in a simple table with 64 entries which would
encode the 20 standard amino acids involved in protein translation
plus start and stop codons. The genetic code is degenerate, i.e.,
in general, more than one codon specifies each amino acid. For
example, the amino acid leucine is specified by the UUA, UUG, CUU,
CUC, CUA, or CUG codons, while the amino acid serine is specified
by UCA, UCG, UCC, UCU, AGU, or AGC codons (difference in the first,
second, or third position). Native genetic codes comprise 62 codons
encoding naturally occurring amino acids. Thus, in some embodiments
of the methods disclosed herein optimized codon sets (genetic
codes) comprising less than 62 codons to encode 20 amino acids can
comprise 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48,
47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31,
30, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 codons.
[0592] In some embodiments, the limited codon set comprises less
than 20 codons. For example, if a protein contains less than 20
types of amino acids, such protein could be encoded by a codon set
with less than 20 codons. Accordingly, in some embodiments, an
optimized codon set comprises as many codons as different types of
amino acids are present in the protein encoded by the reference
nucleic acid sequence. In some embodiments, the optimized codon set
comprises 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2 or even 1 codon.
[0593] In some embodiments, at least one amino acid selected from
the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr, and Val, i.e., amino
acids which are naturally encoded by more than one codon, is
encoded with less codons than the naturally occurring number of
synonymous codons. For example, in some embodiments, Ala can be
encoded in the sequence optimized nucleic acid by 3, 2 or 1 codons;
Cys can be encoded in the sequence optimized nucleic acid by 1
codon; Asp can be encoded in the sequence optimized nucleic acid by
1 codon; Glu can be encoded in the sequence optimized nucleic acid
by 1 codon; Phe can be encoded in the sequence optimized nucleic
acid by 1 codon; Gly can be encoded in the sequence optimized
nucleic acid by 3 codons, 2 codons or 1 codon; His can be encoded
in the sequence optimized nucleic acid by 1 codon; Ile can be
encoded in the sequence optimized nucleic acid by 2 codons or 1
codon; Lys can be encoded in the sequence optimized nucleic acid by
1 codon; Leu can be encoded in the sequence optimized nucleic acid
by 5 codons, 4 codons, 3 codons, 2 codons or 1 codon; Asn can be
encoded in the sequence optimized nucleic acid by 1 codon; Pro can
be encoded in the sequence optimized nucleic acid by 3 codons, 2
codons, or 1 codon; Gln can be encoded in the sequence optimized
nucleic acid by 1 codon; Arg can be encoded in the sequence
optimized nucleic acid by 5 codons, 4 codons, 3 codons, 2 codons,
or 1 codon; Ser can be encoded in the sequence optimized nucleic
acid by 5 codons, 4 codons, 3 codons, 2 codons, or 1 codon; Thr can
be encoded in the sequence optimized nucleic acid by 3 codons, 2
codons, or 1 codon; Val can be encoded in the sequence optimized
nucleic acid by 3 codons, 2 codons, or 1 codon; and, Tyr can be
encoded in the sequence optimized nucleic acid by 1 codon.
[0594] In some embodiments, at least one amino acid selected from
the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr, and Val, i.e., amino
acids which are naturally encoded by more than one codon, is
encoded by a single codon in the limited codon set.
[0595] In some specific embodiments, the sequence optimized nucleic
acid is a DNA and the limited codon set consists of 20 codons,
wherein each codon encodes one of 20 amino acids. In some
embodiments, the sequence optimized nucleic acid is a DNA and the
limited codon set comprises at least one codon selected from the
group consisting of GCT, GCC, GCA, and GCG; at least a codon
selected from the group consisting of CGT, CGC, CGA, CGG, AGA, and
AGG; at least a codon selected from AAT or ACC; at least a codon
selected from GAT or GAC; at least a codon selected from TGT or
TGC; at least a codon selected from CAA or CAG; at least a codon
selected from GAA or GAG; at least a codon selected from the group
consisting of GGT, GGC, GGA, and GGG; at least a codon selected
from CAT or CAC; at least a codon selected from the group
consisting of ATT, ATC, and ATA; at least a codon selected from the
group consisting of TTA, TTG, CTT, CTC, CTA, and CTG; at least a
codon selected from AAA or AAG; an ATG codon; at least a codon
selected from TTT or TTC; at least a codon selected from the group
consisting of CCT, CCC, CCA, and CCG; at least a codon selected
from the group consisting of TCT, TCC, TCA, TCG, AGT, and AGC; at
least a codon selected from the group consisting of ACT, ACC, ACA,
and ACG; a TGG codon; at least a codon selected from TAT or TAC;
and, at least a codon selected from the group consisting of GTT,
GTC, GTA, and GTG.
[0596] In other embodiments, the sequence optimized nucleic acid is
an RNA (e.g., an mRNA) and the limited codon set consists of 20
codons, wherein each codon encodes one of 20 amino acids. In some
embodiments, the sequence optimized nucleic acid is an RNA and the
limited codon set comprises at least one codon selected from the
group consisting of GCU, GCC, GCA, and GCG; at least a codon
selected from the group consisting of CGU, CGC, CGA, CGG, AGA, and
AGG; at least a codon selected from AAU or ACC; at least a codon
selected from GAU or GAC; at least a codon selected from UGU or
UGC; at least a codon selected from CAA or CAG; at least a codon
selected from GAA or GAG; at least a codon selected from the group
consisting of GGU, GGC, GGA, and GGG; at least a codon selected
from CAU or CAC; at least a codon selected from the group
consisting of AUU, AUC, and AUA; at least a codon selected from the
group consisting of UUA, UUG, CUU, CUC, CUA, and CUG; at least a
codon selected from AAA or AAG; an AUG codon; at least a codon
selected from UUU or UUC; at least a codon selected from the group
consisting of CCU, CCC, CCA, and CCG; at least a codon selected
from the group consisting of UCU, UCC, UCA, UCG, AGU, and AGC; at
least a codon selected from the group consisting of ACU, ACC, ACA,
and ACG; a UGG codon; at least a codon selected from UAU or UAC;
and, at least a codon selected from the group consisting of GUU,
GUC, GUA, and GUG.
[0597] In some specific embodiments, the limited codon set has been
optimized for in vivo expression of a sequence optimized nucleic
acid (e.g., a synthetic mRNA) following administration to a certain
tissue or cell.
[0598] In some embodiments, the optimized codon set (e.g., a 20
codon set encoding 20 amino acids) complies at least with one of
the following properties:
[0599] (i) the optimized codon set has a higher average G/C content
than the original or native codon set; or,
[0600] (ii) the optimized codon set has a lower average U content
than the original or native codon set; or,
[0601] (iii) the optimized codon set is composed of codons with the
highest frequency; or,
[0602] (iv) the optimized codon set is composed of codons with the
lowest frequency; or,
[0603] (v) a combination thereof.
[0604] In some specific embodiments, at least one codon in the
optimized codon set has the second highest, the third highest, the
fourth highest, the fifth highest or the sixth highest frequency in
the synonymous codon set. In some specific embodiments, at least
one codon in the optimized codon has the second lowest, the third
lowest, the fourth lowest, the fifth lowest, or the sixth lowest
frequency in the synonymous codon set.
[0605] As used herein, the term "native codon set" refers to the
codon set used natively by the source organism to encode the
reference nucleic acid sequence. As used herein, the term "original
codon set" refers to the codon set used to encode the reference
nucleic acid sequence before the beginning of sequence
optimization, or to a codon set used to encode an optimized variant
of the reference nucleic acid sequence at the beginning of a new
optimization iteration when sequence optimization is applied
iteratively or recursively.
[0606] In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of
codons in the codon set are those with the highest frequency. In
other embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in
the codon set are those with the lowest frequency.
[0607] In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of
codons in the codon set are those with the highest uridine content.
In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons
in the codon set are those with the lowest uridine content.
[0608] In some embodiments, the average G/C content (absolute or
relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%
higher than the average G/C content (absolute or relative) of the
original codon set. In some embodiments, the average G/C content
(absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 100% lower than the average G/C content (absolute or
relative) of the original codon set.
[0609] In some embodiments, the uracil content (absolute or
relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%
higher than the average uracil content (absolute or relative) of
the original codon set. In some embodiments, the uracil content
(absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or 100% lower than the average uracil content (absolute or
relative) of the original codon set.
[0610] See also U.S. Appl. Publ. No. 2011/0082055, and Int'l. Publ.
No. WO2000018778, both of which are incorporated herein by
reference in their entireties.
8. CHARACTERIZATION OF SEQUENCE OPTIMIZED NUCLEIC ACIDS
[0611] In some embodiments of the invention, the polynucleotide
(e.g., a RNA, e.g., an mRNA) comprising a sequence optimized
nucleic acid disclosed herein encoding a PBGD polypeptide can be
tested to determine whether at least one nucleic acid sequence
property (e.g., stability when exposed to nucleases) or expression
property has been improved with respect to the non-sequence
optimized nucleic acid.
[0612] As used herein, "expression property" refers to a property
of a nucleic acid sequence either in vivo (e.g., translation
efficacy of a synthetic mRNA after administration to a subject in
need thereof) or in vitro (e.g., translation efficacy of a
synthetic mRNA tested in an in vitro model system). Expression
properties include but are not limited to the amount of protein
produced by an mRNA encoding a PBGD polypeptide after
administration, and the amount of soluble or otherwise functional
protein produced. In some embodiments, sequence optimized nucleic
acids disclosed herein can be evaluated according to the viability
of the cells expressing a protein encoded by a sequence optimized
nucleic acid sequence (e.g., a RNA, e.g., an mRNA) encoding a PBGD
polypeptide disclosed herein.
[0613] In a particular embodiment, a plurality of sequence
optimized nucleic acids disclosed herein (e.g., a RNA, e.g., an
mRNA) containing codon substitutions with respect to the
non-optimized reference nucleic acid sequence can be characterized
functionally to measure a property of interest, for example an
expression property in an in vitro model system, or in vivo in a
target tissue or cell.
a. Optimization of Nucleic Acid Sequence Intrinsic Properties
[0614] In some embodiments of the invention, the desired property
of the polynucleotide is an intrinsic property of the nucleic acid
sequence. For example, the nucleotide sequence (e.g., a RNA, e.g.,
an mRNA) can be sequence optimized for in vivo or in vitro
stability. In some embodiments, the nucleotide sequence can be
sequence optimized for expression in a particular target tissue or
cell. In some embodiments, the nucleic acid sequence is sequence
optimized to increase its plasma half-life by preventing its
degradation by endo and exonucleases.
[0615] In other embodiments, the nucleic acid sequence is sequence
optimized to increase its resistance to hydrolysis in solution, for
example, to lengthen the time that the sequence optimized nucleic
acid or a pharmaceutical composition comprising the sequence
optimized nucleic acid can be stored under aqueous conditions with
minimal degradation.
[0616] In other embodiments, the sequence optimized nucleic acid
can be optimized to increase its resistance to hydrolysis in dry
storage conditions, for example, to lengthen the time that the
sequence optimized nucleic acid can be stored after lyophilization
with minimal degradation.
b. Nucleic Acids Sequence Optimized for Protein Expression
[0617] In some embodiments of the invention, the desired property
of the polynucleotide is the level of expression of a PBGD
polypeptide encoded by a sequence optimized sequence disclosed
herein. Protein expression levels can be measured using one or more
expression systems. In some embodiments, expression can be measured
in cell culture systems, e.g., CHO cells or HEK293 cells. In some
embodiments, expression can be measured using in vitro expression
systems prepared from extracts of living cells, e.g., rabbit
reticulocyte lysates, or in vitro expression systems prepared by
assembly of purified individual components. In other embodiments,
the protein expression is measured in an in vivo system, e.g.,
mouse, rabbit, monkey, etc.
[0618] In some embodiments, protein expression in solution form can
be desirable. Accordingly, in some embodiments, a reference
sequence can be sequence optimized to yield a sequence optimized
nucleic acid sequence having optimized levels of expressed proteins
in soluble form. Levels of protein expression and other properties
such as solubility, levels of aggregation, and the presence of
truncation products (i.e., fragments due to proteolysis,
hydrolysis, or defective translation) can be measured according to
methods known in the art, for example, using electrophoresis (e.g.,
native or SDS-PAGE) or chromatographic methods (e.g., HPLC, size
exclusion chromatography, etc.).
c. Optimization of Target Tissue or Target Cell Viability
[0619] In some embodiments, the expression of heterologous
therapeutic proteins encoded by a nucleic acid sequence can have
deleterious effects in the target tissue or cell, reducing protein
yield, or reducing the quality of the expressed product (e.g., due
to the presence of protein fragments or precipitation of the
expressed protein in inclusion bodies), or causing toxicity.
[0620] Accordingly, in some embodiments of the invention, the
sequence optimization of a nucleic acid sequence disclosed herein,
e.g., a nucleic acid sequence encoding a PBGD polypeptide, can be
used to increase the viability of target cells expressing the
protein encoded by the sequence optimized nucleic acid.
[0621] Heterologous protein expression can also be deleterious to
cells transfected with a nucleic acid sequence for autologous or
heterologous transplantation. Accordingly, in some embodiments of
the present disclosure the sequence optimization of a nucleic acid
sequence disclosed herein can be used to increase the viability of
target cells expressing the protein encoded by the sequence
optimized nucleic acid sequence. Changes in cell or tissue
viability, toxicity, and other physiological reaction can be
measured according to methods known in the art.
d. Reduction of Immune and/or Inflammatory Response
[0622] In some cases, the administration of a sequence optimized
nucleic acid encoding PBGD polypeptide or a functional fragment
thereof can trigger an immune response, which could be caused by
(i) the therapeutic agent (e.g., an mRNA encoding a PBGD
polypeptide), or (ii) the expression product of such therapeutic
agent (e.g., the PBGD polypeptide encoded by the mRNA), or (iv) a
combination thereof. Accordingly, in some embodiments of the
present disclosure the sequence optimization of nucleic acid
sequence (e.g., an mRNA) disclosed herein can be used to decrease
an immune or inflammatory response triggered by the administration
of a nucleic acid encoding a PBGD polypeptide or by the expression
product of PBGD encoded by such nucleic acid.
[0623] In some aspects, an inflammatory response can be measured by
detecting increased levels of one or more inflammatory cytokines
using methods known in the art, e.g., ELISA. The term "inflammatory
cytokine" refers to cytokines that are elevated in an inflammatory
response. Examples of inflammatory cytokines include interleukin-6
(IL-6), CXCL1 (chemokine (C--X--C motif) ligand 1; also known as
GRO.alpha., interferon-.gamma. (IFN.gamma.), tumor necrosis factor
.alpha. (TNF.alpha.), interferon .gamma.-induced protein 10
(IP-10), or granulocyte-colony stimulating factor (G-CSF). The term
inflammatory cytokines includes also other cytokines associated
with inflammatory responses known in the art, e.g., interleukin-1
(IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12),
interleukin-13 (Il-13), interferon .alpha. (IFN-.alpha.), etc.
9. MODIFIED NUCLEOTIDE SEQUENCES ENCODING PBGD POLYPEPTIDES
[0624] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) comprises a chemically modified
nucleobase, e.g., 5-methoxyuracil. In some embodiments, the mRNA is
a uracil-modified sequence comprising an ORF encoding a PBGD
polypeptide, wherein the mRNA comprises a chemically modified
nucleobase, e.g., 5-methoxyuracil.
[0625] In certain aspects of the invention, when the
5-methoxyuracil base is connected to a ribose sugar, as it is in
polynucleotides, the resulting modified nucleoside or nucleotide is
referred to as 5-methoxyuridine. In some embodiments, uracil in the
polynucleotide is at least about 25%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least 90%, at least 95%, at least 99%,
or about 100% 5-methoxyuracil. In one embodiment, uracil in the
polynucleotide is at least 95% 5-methoxyuracil. In another
embodiment, uracil in the polynucleotide is 100%
5-methoxyuracil.
[0626] In embodiments where uracil in the polynucleotide is at
least 95% 5-methoxyuracil, overall uracil content can be adjusted
such that the polynucleotide of the invention (e.g., a RNA, e.g.,
an mRNA) provides suitable protein expression levels while inducing
little to no immune response. In some embodiments, the uracil
content of the ORF is between about 105% and about 145%, about 105%
and about 140%, about 110% and about 140%, about 110% and about
145%, about 115% and about 135%, about 105% and about 135%, about
110% and about 135%, about 115% and about 145%, or about 115% and
about 140% of the theoretical minimum uracil content in the
corresponding wild-type ORF (% U.sub.TM). In other embodiments, the
uracil content of the ORF is between about 117% and about 134% or
between 118% and 132% of the % U.sub.TM. In some embodiments, the
uracil content of the ORF encoding a PBGD polypeptide is about
115%, about 120%, about 125%, about 130%, about 135%, about 140%,
about 145%, or about 150% of the % U.sub.TM. In this context, the
term "uracil" can refer to 5-methoxyuracil and/or naturally
occurring uracil.
[0627] In some embodiments, the uracil content in the ORF of the
mRNA encoding a PBGD polypeptide of the invention is less than
about 50%, about 40%, about 30%, or about 20% of the total
nucleobase content in the ORF. In some embodiments, the uracil
content in the ORF is between about 15% and about 25% of the total
nucleobase content in the ORF. In other embodiments, the uracil
content in the ORF is between about 20% and about 30% of the total
nucleobase content in the ORF. In one embodiment, the uracil
content in the ORF of the mRNA encoding a PBGD polypeptide is less
than about 20% of the total nucleobase content in the open reading
frame. In this context, the term "uracil" can refer to
5-methoxyuracil and/or naturally occurring uracil.
[0628] In further embodiments, the ORF of the mRNA encoding a PBGD
polypeptide having 5-methoxyuracil and adjusted uracil content has
increased Cytosine (C), Guanine (G), or Guanine/Cytosine (G/C)
content (absolute or relative). In some embodiments, the overall
increase in C, G, or G/C content (absolute or relative) of the ORF
is at least about 2%, at least about 3%, at least about 4%, at
least about 5%, at least about 6%, at least about 7%, at least
about 10%, at least about 15%, at least about 20%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 95%, or at
least about 100% relative to the G/C content (absolute or relative)
of the wild-type ORF. In some embodiments, the G, the C, or the G/C
content in the ORF is less than about 100%, less than about 90%,
less than about 85%, or less than about 80% of the theoretical
maximum G, C, or G/C content of the corresponding wild type
nucleotide sequence encoding the PBGD polypeptide (% G.sub.TMX; %
C.sub.TMX, or % G/C.sub.TMX). In other embodiments, the G, the C,
or the G/C content in the ORF is between about 70% and about 80%,
between about 71% and about 79%, between about 71% and about 78%,
or between about 71% and about 77% of the % G.sub.TMX, % C.sub.TMX,
or % G/C.sub.TMX. In some embodiments, the increases in G and/or C
content (absolute or relative) described herein can be conducted by
replacing synonymous codons with low G, C, or G/C content with
synonymous codons having higher G, C, or G/C content. In other
embodiments, the increase in G and/or C content (absolute or
relative) is conducted by replacing a codon ending with U with a
synonymous codon ending with G or C.
[0629] In further embodiments, the ORF of the mRNA encoding a PBGD
polypeptide of the invention comprises 5-methoxyuracil and has an
adjusted uracil content containing less uracil pairs (UU) and/or
uracil triplets (UUU) and/or uracil quadruplets (UUUU) than the
corresponding wild-type nucleotide sequence encoding the PBGD
polypeptide. In some embodiments, the ORF of the mRNA encoding a
PBGD polypeptide of the invention contains no uracil pairs and/or
uracil triplets and/or uracil quadruplets. In some embodiments,
uracil pairs and/or uracil triplets and/or uracil quadruplets are
reduced below a certain threshold, e.g., no more than 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
occurrences in the ORF of the mRNA encoding the PBGD polypeptide.
In a particular embodiment, the ORF of the mRNA encoding the PBGD
polypeptide of the invention contains less than 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
non-phenylalanine uracil pairs and/or triplets. In another
embodiment, the ORF of the mRNA encoding the PBGD polypeptide
contains no non-phenylalanine uracil pairs and/or triplets.
[0630] In further embodiments, the ORF of the mRNA encoding a PBGD
polypeptide of the invention comprises 5-methoxyuracil and has an
adjusted uracil content containing less uracil-rich clusters than
the corresponding wild-type nucleotide sequence encoding the PBGD
polypeptide. In some embodiments, the ORF of the mRNA encoding the
PBGD polypeptide of the invention contains uracil-rich clusters
that are shorter in length than corresponding uracil-rich clusters
in the corresponding wild-type nucleotide sequence encoding the
PBGD polypeptide.
[0631] In further embodiments, alternative lower frequency codons
are employed. At least about 5%, at least about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 99%, or 100% of the codons in the PBGD polypeptide-encoding
ORF of the 5-methoxyuracil-comprising mRNA are substituted with
alternative codons, each alternative codon having a codon frequency
lower than the codon frequency of the substituted codon in the
synonymous codon set. The ORF also has adjusted uracil content, as
described above. In some embodiments, at least one codon in the ORF
of the mRNA encoding the PBGD polypeptide is substituted with an
alternative codon having a codon frequency lower than the codon
frequency of the substituted codon in the synonymous codon set.
[0632] In some embodiments, the adjusted uracil content, PBGD
polypeptide-encoding ORF of the 5-methoxyuracil-comprising mRNA
exhibits expression levels of PBGD when administered to a mammalian
cell that are higher than expression levels of PBGD from the
corresponding wild-type mRNA. In other embodiments, the expression
levels of PBGD when administered to a mammalian cell are increased
relative to a corresponding mRNA containing at least 95%
5-methoxyuracil and having a uracil content of about 160%, about
170%, about 180%, about 190%, or about 200% of the theoretical
minimum. In yet other embodiments, the expression levels of PBGD
when administered to a mammalian cell are increased relative to a
corresponding mRNA, wherein at least about 50%, at least about 60%,
at least about 70%, at least about 80%, at least about 90%, or
about 100% of uracils are 1-methylpseudouracil or pseudouracils. In
some embodiments, the mammalian cell is a mouse cell, a rat cell,
or a rabbit cell. In other embodiments, the mammalian cell is a
monkey cell or a human cell. In some embodiments, the human cell is
a HeLa cell, a BJ fibroblast cell, or a peripheral blood
mononuclear cell (PBMC). In some embodiments, PBGD is expressed
when the mRNA is administered to a mammalian cell in vivo. In some
embodiments, the mRNA is administered to mice, rabbits, rats,
monkeys, or humans. In one embodiment, mice are null mice. In some
embodiments, the mRNA is administered to mice in an amount of about
0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, or about 0.15 mg/kg.
In some embodiments, the mRNA is administered intravenously or
intramuscularly. In other embodiments, the PBGD polypeptide is
expressed when the mRNA is administered to a mammalian cell in
vitro. In some embodiments, the expression is increased by at least
about 2-fold, at least about 5-fold, at least about 10-fold, at
least about 50-fold, at least about 500-fold, at least about
1500-fold, or at least about 3000-fold. In other embodiments, the
expression is increased by at least about 10%, about 20%, about
30%, about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or
about 100%.
[0633] In some embodiments, adjusted uracil content, PBGD
polypeptide-encoding ORF of the 5-methoxyuracil-comprising mRNA
exhibits increased stability. In some embodiments, the mRNA
exhibits increased stability in a cell relative to the stability of
a corresponding wild-type mRNA under the same conditions. In some
embodiments, the mRNA exhibits increased stability including
resistance to nucleases, thermal stability, and/or increased
stabilization of secondary structure. In some embodiments,
increased stability exhibited by the mRNA is measured by
determining the half-life of the mRNA (e.g., in a plasma, serum,
cell, or tissue sample) and/or determining the area under the curve
(AUC) of the protein expression by the mRNA over time (e.g., in
vitro or in vivo). An mRNA is identified as having increased
stability if the half-life and/or the AUC is greater than the
half-life and/or the AUC of a corresponding wild-type mRNA under
the same conditions.
[0634] In some embodiments, the mRNA of the present invention
induces a detectably lower immune response (e.g., innate or
acquired) relative to the immune response induced by a
corresponding wild-type mRNA under the same conditions. In other
embodiments, the mRNA of the present disclosure induces a
detectably lower immune response (e.g., innate or acquired)
relative to the immune response induced by an mRNA that encodes for
a PBGD polypeptide but does not comprise 5-methoxyuracil under the
same conditions, or relative to the immune response induced by an
mRNA that encodes for a PBGD polypeptide and that comprises
5-methoxyuracil but that does not have adjusted uracil content
under the same conditions. The innate immune response can be
manifested by increased expression of pro-inflammatory cytokines,
activation of intracellular PRRs (RIG-I, MDA5, etc.), cell death,
and/or termination or reduction in protein translation. In some
embodiments, a reduction in the innate immune response can be
measured by expression or activity level of Type 1 interferons
(e.g., IFN-.alpha., IFN-.beta., IFN-.kappa., IFN-.delta.,
IFN-.epsilon., IFN-.tau., IFN-.omega., and IFN-.zeta.) or the
expression of interferon-regulated genes such as the toll-like
receptors (e.g., TLR7 and TLR8), and/or by decreased cell death
following one or more administrations of the mRNA of the invention
into a cell.
[0635] In some embodiments, the expression of Type-1 interferons by
a mammalian cell in response to the mRNA of the present disclosure
is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 99%, 99.9%, or greater than 99.9% relative to a corresponding
wild-type mRNA, to an mRNA that encodes a PBGD polypeptide but does
not comprise 5-methoxyuracil, or to an mRNA that encodes a PBGD
polypeptide and that comprises 5-methoxyuracil but that does not
have adjusted uracil content. In some embodiments, the interferon
is IFN-.beta.. In some embodiments, cell death frequency caused by
administration of mRNA of the present disclosure to a mammalian
cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than
the cell death frequency observed with a corresponding wild-type
mRNA, an mRNA that encodes for a PBGD polypeptide but does not
comprise 5-methoxyuracil, or an mRNA that encodes for a PBGD
polypeptide and that comprises 5-methoxyuracil but that does not
have adjusted uracil content. In some embodiments, the mammalian
cell is a BJ fibroblast cell. In other embodiments, the mammalian
cell is a splenocyte. In some embodiments, the mammalian cell is
that of a mouse or a rat. In other embodiments, the mammalian cell
is that of a human. In one embodiment, the mRNA of the present
disclosure does not substantially induce an innate immune response
of a mammalian cell into which the mRNA is introduced.
[0636] In some embodiments, the polynucleotide is an mRNA that
comprises an ORF that encodes a PBGD polypeptide, wherein uracil in
the mRNA is at least about 95% 5-methoxyuracil, wherein the uracil
content of the ORF is between about 115% and about 135% of the
theoretical minimum uracil content in the corresponding wild-type
ORF, and wherein the uracil content in the ORF encoding the PBGD
polypeptide is less than about 30% of the total nucleobase content
in the ORF. In some embodiments, the ORF that encodes the PBGD
polypeptide is further modified to increase G/C content of the ORF
(absolute or relative) by at least about 40%, as compared to the
corresponding wild-type ORF. In yet other embodiment, the ORF
encoding the PBGD polypeptide contains less than 20
non-phenylalanine uracil pairs and/or triplets. In some
embodiments, at least one codon in the ORF of the mRNA encoding the
PBGD polypeptide is further substituted with an alternative codon
having a codon frequency lower than the codon frequency of the
substituted codon in the synonymous codon set. In some embodiments,
the expression of the PBGD polypeptide encoded by an mRNA
comprising an ORF wherein uracil in the mRNA is at least about 95%
5-methoxyuracil, and wherein the uracil content of the ORF is
between about 115% and about 135% of the theoretical minimum uracil
content in the corresponding wild-type ORF, is increased by at
least about 10-fold when compared to expression of the PBGD
polypeptide from the corresponding wild-type mRNA. In some
embodiments, the mRNA comprises an open ORF wherein uracil in the
mRNA is at least about 95% 5-methoxyuracil, and wherein the uracil
content of the ORF is between about 115% and about 135% of the
theoretical minimum uracil content in the corresponding wild-type
ORF, and wherein the mRNA does not substantially induce an innate
immune response of a mammalian cell into which the mRNA is
introduced.
10. METHODS FOR MODIFYING POLYNUCLEOTIDES
[0637] The invention includes modified polynucleotides comprising a
polynucleotide described herein (e.g., a polynucleotide, e.g., an
mRNA, comprising a nucleotide sequence encoding a PBGD
polypeptide). The modified polynucleotides can be chemically
modified and/or structurally modified. When the polynucleotides of
the present invention are chemically and/or structurally modified
the polynucleotides can be referred to as "modified
polynucleotides."
[0638] The present disclosure provides for modified nucleosides and
nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as
mRNA polynucleotides) encoding a PBGD polypeptide. A "nucleoside"
refers to a compound containing a sugar molecule (e.g., a pentose
or ribose) or a derivative thereof in combination with an organic
base (e.g., a purine or pyrimidine) or a derivative thereof (also
referred to herein as "nucleobase"). A "nucleotide" refers to a
nucleoside including a phosphate group. Modified nucleotides can be
synthesized by any useful method, such as, for example, chemically,
enzymatically, or recombinantly, to include one or more modified or
non-natural nucleosides. Polynucleotides can comprise a region or
regions of linked nucleosides. Such regions can have variable
backbone linkages. The linkages can be standard phosphodiester
linkages, in which case the polynucleotides would comprise regions
of nucleotides.
[0639] The modified polynucleotides disclosed herein can comprise
various distinct modifications. In some embodiments, the modified
polynucleotides contain one, two, or more (optionally different)
nucleoside or nucleotide modifications. In some embodiments, a
modified polynucleotide, introduced to a cell can exhibit one or
more desirable properties, e.g., improved protein expression,
reduced immunogenicity, or reduced degradation in the cell, as
compared to an unmodified polynucleotide.
a. Structural Modifications
[0640] In some embodiments, a polynucleotide of the present
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide) is structurally modified. As used
herein, a "structural" modification is one in which two or more
linked nucleosides are inserted, deleted, duplicated, inverted or
randomized in a polynucleotide without significant chemical
modification to the nucleotides themselves. Because chemical bonds
will necessarily be broken and reformed to effect a structural
modification, structural modifications are of a chemical nature and
hence are chemical modifications. However, structural modifications
will result in a different sequence of nucleotides. For example,
the polynucleotide "ATCG" can be chemically modified to
"AT-5meC-G". The same polynucleotide can be structurally modified
from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been
inserted, resulting in a structural modification to the
polynucleotide.
b. Chemical Modifications
[0641] In some embodiments, the polynucleotides of the present
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide) are chemically modified. As used
herein in reference to a polynucleotide, the terms "chemical
modification" or, as appropriate, "chemically modified" refer to
modification with respect to adenosine (A), guanosine (G), uridine
(U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in
one or more of their position, pattern, percent or population,
including, but not limited to, its nucleobase, sugar, backbone, or
any combination thereof. Generally, herein, these terms are not
intended to refer to the ribonucleotide modifications in naturally
occurring 5'-terminal mRNA cap moieties.
[0642] In some embodiments, the polynucleotides of the invention
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) can have a uniform chemical modification of all
or any of the same nucleoside type or a population of modifications
produced by downward titration of the same starting modification in
all or any of the same nucleoside type, or a measured percent of a
chemical modification of all any of the same nucleoside type but
with random incorporation, such as where all uridines are replaced
by a uridine analog, e.g., 5-methoxyuridine. In another embodiment,
the polynucleotides can have a uniform chemical modification of
two, three, or four of the same nucleoside type throughout the
entire polynucleotide (such as all uridines and/or all cytidines,
etc. are modified in the same way).
[0643] Modified nucleotide base pairing encompasses not only the
standard adenine-thymine, adenine-uracil, or guanine-cytosine base
pairs, but also base pairs formed between nucleotides and/or
modified nucleotides comprising non-standard or modified bases,
wherein the arrangement of hydrogen bond donors and hydrogen bond
acceptors permits hydrogen bonding between a non-standard base and
a standard base or between two complementary non-standard base
structures. One example of such non-standard base pairing is the
base pairing between the modified nucleobase inosine and adenine,
cytosine or uracil. Any combination of base/sugar or linker can be
incorporated into polynucleotides of the present disclosure.
[0644] The skilled artisan will appreciate that, except where
otherwise noted, polynucleotide sequences set forth in the instant
application will recite "T"s in a representative DNA sequence but
where the sequence represents RNA, the "T"s would be substituted
for "U"s.
[0645] Modifications of polynucleotides (e.g., RNA polynucleotides,
such as mRNA polynucleotides) that are useful in the compositions,
methods and synthetic processes of the present disclosure include,
but are not limited to the following nucleotides, nucleosides, and
nucleobases: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;
2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl
carbamoyladenosine; N6-glycinylcarbamoyladenosine;
N6-isopentenyladenosine; N6-methyladenosine;
N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine;
1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); 2-methyladenosine; 2-methylthio-N6
isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl
carbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); Isopentenyladenosine;
N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine;
N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine;
N6,N6-dimethyladenosine; N6-acetyladenosine;
N6-hydroxynorvalylcarbamoyladenosine;
N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;
2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;
N1-methyl-adenosine; N6, N6 (dimethyl)adenine;
N6-cis-hydroxy-isopentenyl-adenosine; .alpha.-thio-adenosine; 2
(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6
(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;
2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;
2-(propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP;
2'-Deoxy-2'-.alpha.-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine
TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine;
6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8
(alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine;
8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine;
8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine;
8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza
adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine;
7-deaza-8-aza-adenosine; 7-methyladenine; 1-Deazaadenosine TP;
2'Fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-Amino-ATP;
2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP;
2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP;
2'-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;
2'-b-Ethynyladenosine TP; 2-Bromoadenosine TP;
2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;
2'-Deoxy-2',2'-difluoroadenosine TP;
2'-Deoxy-2'-a-mercaptoadenosine TP;
2'-Deoxy-2'-a-thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine
TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine
TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine
TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine
TP; 2'-Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;
2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;
2-methylthio-adenine; 2-Trifluoromethyladenosine TP;
3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;
3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;
3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine
TP; 4'-Ethynyladenosine TP; 5'-Homo-adenosine TP; 8-Aza-ATP;
8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP;
9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine;
7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;
2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;
2-thiocytidine; 3-methylcytidine; 5-formylcytidine;
5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;
2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine;
5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine;
N4-acetyl-2'-O-methylcytidine; N4-methylcytidine;
N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine;
5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine;
.alpha.-thio-cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP;
2'-Azido-2'-deoxy-CTP; 2'-Deoxy-2'-a-aminocytidine TP;
2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3
(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine;
3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5 (halo)cytosine; 5
(methyl)cytosine; 5 (propynyl)cytosine; 5
(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;
5-(halo)cytosine; 5-(propynyl)cytosine;
5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine;
5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine;
deaza cytosine; N4 (acetyl)cytosine;
1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;
2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;
2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;
4-methoxy-pseudoisocytidine;
4-thio-1-methyl-1-deaza-pseudoisocytidine;
4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;
5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;
Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine
TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP;
2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP;
2'O-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP;
2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP;
2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine
TP; 2'-Deoxy-2'-a-mercaptocytidine TP;
2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine
TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP;
2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP;
2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP;
2'-Deoxy-2'-b-thiomethoxycytidine TP;
2'-O-Methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP;
4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine
TP; 5-(1-Propynyl)ara-cytidine TP;
5-(2-Chloro-phenyl)-2-thiocytidine TP;
5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP;
5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP;
5'-Homo-cytidine TP; 5-Methoxycytidine TP;
5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP;
N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine;
N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyosine;
1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine;
2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine;
2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine;
7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine;
N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine;
N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;
N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;
8-oxo-guanosine; N1-methyl-guanosine; .alpha.-thio-guanosine; 2
(propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy-GTP;
2'-Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP;
2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine;
6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7
(alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine;
7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8
(alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8
(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine;
8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;
8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza
guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;
1-methyl-6-thio-guanosine; 6-methoxy-guanosine;
6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;
6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;
7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;
N2-methyl-6-thio-guanosine; 1-Me-GTP;
2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine
TP; 2'-a-Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP;
2'-b-Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP;
2'-Deoxy-2',2'-difluoroguanosine TP;
2'-Deoxy-2'-a-mercaptoguanosine TP;
2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine
TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine
TP; 2'-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine
TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine
TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP;
4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP;
5'-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP;
N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;
1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine;
2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;
Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine;
deaza thymidine; deoxy-thymidine; 2'-O-methyluridine;
2-thiouridine; 3-methyluridine; 5-carboxymethyluridine;
5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine;
5-taurinomethyluridine; Dihydrouridine; Pseudouridine;
(3-(3-amino-3-carboxypropyl)uridine;
1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;
1-methylpseudouridine; 1-ethyl-pseudouridine; 2'-O-methyluridine;
2'-O-methylpseudouridine; 2'-O-methyluridine;
2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine;
3,2'-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine;
5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine
methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine;
5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine;
5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;
5-carboxyhydroxymethyluridine methyl ester;
5-carboxymethylaminomethyl-2'-O-methyluridine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyluridine;
5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP;
5-methoxycarbonylmethyl-2'-O-methyluridine;
5-methoxycarbonylmethyl-2-thiouridine;
5-methoxycarbonylmethyluridine; 5-methyluridine,),
5-methoxyuridine; 5-methyl-2-thiouridine;
5-methylaminomethyl-2-selenouridine;
5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine;
5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic
acid-methyl ester-Uridine TP; N1-methyl-pseudo-uracil;
N1-ethyl-pseudo-uracil; uridine 5-oxyacetic acid; uridine
5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine
TP; 5-(iso-Pentenylaminomethyl)-2-thiouridine TP;
5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP;
5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;
.alpha.-thio-uridine; 1
(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1
(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1
(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminocarbonylethylenyl)-pseudouracil; 1 substituted
2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1
substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;
1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;
1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;
1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP;
1-Methyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2'
deoxy uridine; 2' fluorouridine; 2-(thio)uracil;
2,4-(dithio)pseudouracil; 2' methyl, 2'amino, 2'azido,
2'fluro-guanosine; 2'-Amino-2'-deoxy-UTP; 2'-Azido-2'-deoxy-UTP;
2'-Azido-deoxyuridine TP; 2'-O-methylpseudouridine; 2' deoxy
uridine; 2' fluorouridine; 2'-Deoxy-2'-a-aminouridine TP;
2'-Deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3
carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio)pseudouracil;
4-(thio)uracil; 4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5
(2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5
(dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5
(methoxycarbonylmethyl)-2-(thio)uracil; 5
(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5
(methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5
(methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4
(dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5
(propynyl)uracil; 5 (trifluoromethyl)uracil;
5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil;
5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil;
5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil;
5-(allylamino)uracil; 5-(cyanoalkyl)uracil;
5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;
5-(guanidiniumalkyl)uracil; 5-(halo)uracil;
5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;
5-(methoxycarbonylmethyl)-2-(thio)uracil;
5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil;
5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;
5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4
(dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil;
5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil;
5-(methylaminomethyl)-2,4(dithio)uracil;
5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;
5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;
5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil;
6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3
(methyl)uracil; P seudo-UTP-1-2-ethanoic acid; Pseudouracil;
4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine;
1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine;
1-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine;
1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine;
2-thio-1-methyl-1-deaza-pseudouridine;
2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;
2-thio-dihydropseudouridine; 2-thio-dihydrouridine;
2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;
4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;
4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;
(+)1-(2-Hydroxypropyl)pseudouridine TP;
(2R)-1-(2-Hydroxypropyl)pseudouridine TP;
(2S)-1-(2-Hydroxypropyl)pseudouridine TP;
(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine
TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP;
(Z)-5-(2-Bromo-vinyl)uridine TP;
1-(2,2,2-Trifluoroethyl)-pseudo-UTP;
1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;
1-(2,2-Diethoxyethyl)pseudouridine TP;
1-(2,4,6-Trimethylbenzyl)pseudouridine TP;
1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;
1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;
1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;
1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine
TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;
1-(3,4-Dimethoxybenzyl)pseudouridine TP;
1-(3-Amino-3-carboxypropyl)pseudo-UTP;
1-(3-Amino-propyl)pseudo-UTP;
1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;
1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;
1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;
1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine
TP; 1-(4-Chlorobenzyl)pseudouridine TP;
1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine
TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP;
1-(4-Methoxybenzyl)pseudouridine TP;
1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP;
1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP;
1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP;
1(4-Nitro-phenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)pseudouridine
TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP;
1-(4-Trifluoromethylbenzyl)pseudouridine TP;
1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;
1,6-Dimethyl-pseudo-UTP;
1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouri-
dine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine
TP; 1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;
1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;
1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;
1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;
1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;
1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;
1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;
1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;
1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;
1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;
1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;
1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;
1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;
1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;
1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine
TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;
1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;
1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine
TP; 1-Methoxymethylpseudouridine TP;
1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;
1-Methyl-6-(4-morpholino)-pseudo-UTP;
1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted
phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;
1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;
1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;
1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;
1-Methyl-6-ethoxy-pseudo-UTP;
1-Methyl-6-ethylcarboxylate-pseudo-UTP;
1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;
1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;
1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;
1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;
1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;
1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;
1-Methyl-6-trifluoromethoxy-pseudo-UTP;
1-Methyl-6-trifluoromethyl-pseudo-UTP;
1-Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP;
1-Phenyl-pseudo-UTP; 1-Pivaloylpseudouridine TP;
1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;
1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP;
1-tert-Butyl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP;
1-Thiomorpholinomethylpseudouridine TP;
1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;
1-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP;
2'-bromo-deoxyuridine TP; 2'-F-5-Methyl-2'-deoxy-UTP;
2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP;
2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP;
2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP;
2'-Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine
TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP;
2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP;
2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP;
2'-Deoxy-2'-b-mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine
TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine;
2'-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP;
4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine
TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP;
5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homo-uridine TP;
5-iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;
5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine
TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;
6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;
6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;
6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;
6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP;
6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP;
6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;
6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP;
6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP;
6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP;
6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP;
6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP;
6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP;
Alpha-thio-pseudo-UTP; Pseudouridine 1-(4-methylbenzenesulfonic
acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine
TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP
1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;
Pseudouridine TP
1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic
acid; Pseudouridine TP
1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine
TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP
1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid
diethyl ester; Pseudo-UTP-N1-3-propionic acid;
Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;
Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;
Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;
Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;
undermodified hydroxywybutosine; 4-demethylwyosine;
2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;
2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido,
2'fluro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluro-adenine;
2'methyl, 2'amino, 2'azido, 2'fluro-uridine;
2'-amino-2'-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl;
2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose;
2'-fluoro-modified bases; 2'-O-methyl-ribose;
2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;
2-pyridinone; 3 nitropyrrole;
3-(methyl)-7-(propynyl)isocarbostyrilyl;
3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;
4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl;
5 nitroindole; 5 substituted pyrimidines;
5-(methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine;
6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine;
6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aza)indolyl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,
propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;
Aminoindolyl; Anthracenyl;
bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;
Isocarbostyrilyl; Isoguanisine; N2-substituted purines;
N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated
derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;
Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;
O-alkylated derivative;
ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin
TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;
Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;
pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,
2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;
Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted
1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine;
Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine;
7-deaza-2-amino-purine; pyridin-4-one ribonucleoside;
2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP;
2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine
TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and
N6-(19-Amino-pentaoxanonadecyl)adenosine TP.
[0646] In some embodiments, the polynucleotide (e.g., RNA
polynucleotide, such as mRNA polynucleotide) includes a combination
of at least two (e.g., 2, 3, 4 or more) of the aforementioned
modified nucleobases.
[0647] In some embodiments, the mRNA comprises at least one
chemically modified nucleoside. In some embodiments, the at least
one chemically modified nucleoside is selected from the group
consisting of pseudouridine (.psi.), 2-thiouridine (s2U),
4'-thiouridine, 5-methylcytosine,
2-thio-1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,
2-thio-dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,
4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,
4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,
5-methyluridine, 5-methoxyuridine, 2'-O-methyl uridine,
1-methyl-pseudouridine (m1.psi.), 1-ethyl-pseudouridine (e1.psi.),
5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C),
.alpha.-thio-guanosine, .alpha.-thio-adenosine, 5-cyano uridine,
4'-thio uridine 7-deaza-adenine, 1-methyl-adenosine (m1A),
2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and
2,6-Diaminopurine, (I), 1-methyl-inosine (m1I), wyosine (imG),
methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine
(preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1),
7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G),
8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine,
2-geranylthiouridine, 2-lysidine, 2-selenouridine,
3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine,
3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine,
5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester,
5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine,
5-aminomethyluridine, 5-carbamoylhydroxymethyluridine,
5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine,
5-carboxymethylaminomethyl-2-geranylthiouridine,
5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine,
5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine,
7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine,
7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine,
N4,N4-dimethylcytidine, N6-formyladenosine,
N6-hydroxymethyladenosine, agmatidine, cyclic
N6-threonylcarbamoyladenosine, glutamyl-queuosine, methylated
undermodified hydroxywybutosine, N4,N4,2'-O-trimethylcytidine,
geranylated 5-methylaminomethyl-2-thiouridine, geranylated
5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQ0base,
preQ1base, and two or more combinations thereof. In some
embodiments, the at least one chemically modified nucleoside is
selected from the group consisting of pseudouridine,
1-methylpseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine,
5-methoxyuridine, and a combination thereof. In some embodiments,
the polynucleotide (e.g., RNA polynucleotide, such as mRNA
polynucleotide) includes a combination of at least two (e.g., 2, 3,
4 or more) of the aforementioned modified nucleobases.
[0648] (i) Base Modifications
[0649] In certain embodiments, the chemical modification is at
nucleobases in the polynucleotides (e.g., RNA polynucleotide, such
as mRNA polynucleotide). In some embodiments, modified nucleobases
in the polynucleotide (e.g., RNA polynucleotide, such as mRNA
polynucleotide) are selected from the group consisting of
1-methyl-pseudouridine (m1.psi.), 1-ethyl-pseudouridine (e1.psi.),
5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine
(w), .alpha.-thio-guanosine and .alpha.-thio-adenosine. In some
embodiments, the polynucleotide includes a combination of at least
two (e.g., 2, 3, 4 or more) of the aforementioned modified
nucleobases.
[0650] In some embodiments, the polynucleotide (e.g., RNA
polynucleotide, such as mRNA polynucleotide) comprises
pseudouridine (v) and 5-methyl-cytidine (m5C). In some embodiments,
the polynucleotide (e.g., RNA polynucleotide, such as mRNA
polynucleotide) comprises 1-methyl-pseudouridine (m1.psi.). In some
embodiments, the polynucleotide (e.g., RNA polynucleotide, such as
mRNA polynucleotide) comprises 1-ethyl-pseudouridine (e1.psi.). In
some embodiments, the polynucleotide (e.g., RNA polynucleotide,
such as mRNA polynucleotide) comprises 1-methyl-pseudouridine
(m1.psi.) and 5-methyl-cytidine (m5C). In some embodiments, the
polynucleotide (e.g., RNA polynucleotide, such as mRNA
polynucleotide) comprises 1-ethyl-pseudouridine (e1.psi.) and
5-methyl-cytidine (m5C). In some embodiments, the polynucleotide
(e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises
2-thiouridine (s2U). In some embodiments, the polynucleotide (e.g.,
RNA polynucleotide, such as mRNA polynucleotide) comprises
2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the
polynucleotide (e.g., RNA polynucleotide, such as mRNA
polynucleotide) comprises methoxy-uridine (mo5U). In some
embodiments, the polynucleotide (e.g., RNA polynucleotide, such as
mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and
5-methyl-cytidine (m5C). In some embodiments, the polynucleotide
(e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises
2'-O-methyl uridine. In some embodiments, the polynucleotide (e.g.,
RNA polynucleotide, such as mRNA polynucleotide) comprises
2'-O-methyl uridine and 5-methyl-cytidine (m5C). In some
embodiments, the polynucleotide (e.g., RNA polynucleotide, such as
mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some
embodiments, the polynucleotide (e.g., RNA polynucleotide, such as
mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and
5-methyl-cytidine (m5C).
[0651] In some embodiments, the polynucleotide (e.g., mRNA
polynucleotide, such as mRNA polynucleotide) is uniformly modified
(e.g., fully modified, modified throughout the entire sequence) for
a particular modification. For example, a polynucleotide can be
uniformly modified with 5-methyl-cytidine (m5C), meaning that all
cytosine residues in the mRNA sequence are replaced with
5-methyl-cytidine (m5C). Similarly, a polynucleotide can be
uniformly modified for any type of nucleoside residue present in
the sequence by replacement with a modified residue such as any of
those set forth above.
[0652] In some embodiments, the chemically modified nucleosides in
the open reading frame are selected from the group consisting of
uridine, adenine, cytosine, guanine, and any combination
thereof.
[0653] In some embodiments, the modified nucleobase is a modified
cytosine. Examples of nucleobases and nucleosides having a modified
cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine
(m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine),
5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine,
2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine.
[0654] In some embodiments, a modified nucleobase is a modified
uridine. Example nucleobases and nucleosides having a modified
uridine include 5-cyano uridine or 4'-thio uridine.
[0655] In some embodiments, a modified nucleobase is a modified
adenine. Example nucleobases and nucleosides having a modified
adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A),
2-methyl-adenine (m2A), N6-methyl-adenine (m6A), and
2,6-Diaminopurine.
[0656] In some embodiments, a modified nucleobase is a modified
guanine. Example nucleobases and nucleosides having a modified
guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG),
methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine
(preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1),
7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G),
8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
[0657] In some embodiments, the nucleobase modified nucleotides in
the polynucleotide (e.g., RNA polynucleotide, such as mRNA
polynucleotide) are 5-methoxyuridine.
[0658] In some embodiments, the polynucleotide (e.g., RNA
polynucleotide, such as mRNA polynucleotide) includes a combination
of at least two (e.g., 2, 3, 4 or more) of modified
nucleobases.
[0659] In some embodiments, at least 95% of a type of nucleobases
(e.g., uracil) in a polynucleotide of the invention (e.g., an mRNA
polynucleotide encoding PBGD) are modified nucleobases. In some
embodiments, at least 95% of uracil in a polynucleotide of the
present invention (e.g., an mRNA polynucleotide encoding PBGD) is
5-methoxyuracil.
[0660] In some embodiments, the polynucleotide (e.g., RNA
polynucleotide, such as mRNA polynucleotide) comprises
5-methoxyuridine (5mo5U) and 5-methyl-cytidine (m5C).
[0661] In some embodiments, the polynucleotide (e.g., RNA
polynucleotide, such as mRNA polynucleotide) is uniformly modified
(e.g., fully modified, modified throughout the entire sequence) for
a particular modification. For example, a polynucleotide can be
uniformly modified with 5-methoxyuridine, meaning that
substantially all uridine residues in the mRNA sequence are
replaced with 5-methoxyuridine. Similarly, a polynucleotide can be
uniformly modified for any type of nucleoside residue present in
the sequence by replacement with a modified residue such as any of
those set forth above.
[0662] In some embodiments, the modified nucleobase is a modified
cytosine.
[0663] In some embodiments, a modified nucleobase is a modified
uracil. Example nucleobases and nucleosides having a modified
uracil include 5-methoxyuracil.
[0664] In some embodiments, a modified nucleobase is a modified
adenine.
[0665] In some embodiments, a modified nucleobase is a modified
guanine.
[0666] In some embodiments, the nucleobases, sugar, backbone, or
any combination thereof in the open reading frame encoding a PBGD
polypeptide are chemically modified by at least 10%, at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, at least 99%, or
100%.
[0667] In some embodiments, the uridine nucleosides in the open
reading frame encoding a PBGD polypeptide are chemically modified
by at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, at least 99%, or 100%.
[0668] In some embodiments, the adenosine nucleosides in the open
reading frame encoding a PBGD polypeptide are chemically modified
by at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, at least 99%, or 100%.
[0669] In some embodiments, the cytidine nucleosides in the open
reading frame encoding a PBGD polypeptide are chemically modified
by at least at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, at least 99%, or 100%.
[0670] In some embodiments, the guanosine nucleosides in the open
reading frame encoding a PBGD polypeptide are chemically modified
by at least at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, at least 99%, or 100%.
[0671] In some embodiments, the polynucleotides can include any
useful linker between the nucleosides. Such linkers, including
backbone modifications, that are useful in the composition of the
present disclosure include, but are not limited to the following:
3'-alkylene phosphonates, 3'-amino phosphoramidate, alkene
containing backbones, aminoalkylphosphoramidates,
aminoalkylphosphotriesters, boranophosphates,
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--NH--CH.sub.2--, chiral phosphonates, chiral
phosphorothioates, formacetyl and thioformacetyl backbones,
methylene (methylimino), methylene formacetyl and thioformacetyl
backbones, methyleneimino and methylenehydrazino backbones,
morpholino linkages, --N(CH.sub.3)--CH.sub.2--CH.sub.2--,
oligonucleosides with heteroatom internucleoside linkage,
phosphinates, phosphoramidates, phosphorodithioates,
phosphorothioate intemucleoside linkages, phosphorothioates,
phosphotriesters, PNA, siloxane backbones, sulfamate backbones,
sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide
backbones, thionoalkylphosphonates, thionoalkylphosphotriesters,
and thionophosphoramidates.
[0672] (ii) Sugar Modifications
[0673] The modified nucleosides and nucleotides (e.g., building
block molecules), which can be incorporated into a polynucleotide
(e.g., RNA or mRNA, as described herein), can be modified on the
sugar of the ribonucleic acid. For example, the 2' hydroxyl group
(OH) can be modified or replaced with a number of different
substituents. Exemplary substitutions at the 2'-position include,
but are not limited to, H, halo, optionally substituted C.sub.1-6
alkyl; optionally substituted C.sub.1-6 alkoxy; optionally
substituted C.sub.6-10 aryloxy; optionally substituted C.sub.3-8
cycloalkyl; optionally substituted C.sub.3-8 cycloalkoxy;
optionally substituted C.sub.6-10 aryloxy; optionally substituted
C.sub.6-10 aryl-C.sub.1-6 alkoxy, optionally substituted C.sub.1-12
(heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described
herein); a polyethyleneglycol (PEG),
--O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR, where R is H or
optionally substituted alkyl, and n is an integer from 0 to 20
(e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1
to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2
to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4
to 8, from 4 to 10, from 4 to 16, and from 4 to 20); "locked"
nucleic acids (LNA) in which the 2'-hydroxyl is connected by a
C.sub.1-6 alkylene or C.sub.1-6 heteroalkylene bridge to the
4'-carbon of the same ribose sugar, where exemplary bridges
included methylene, propylene, ether, or amino bridges; aminoalkyl,
as defined herein; aminoalkoxy, as defined herein; amino as defined
herein; and amino acid, as defined herein
[0674] Generally, RNA includes the sugar group ribose, which is a
5-membered ring having an oxygen. Exemplary, non-limiting modified
nucleotides include replacement of the oxygen in ribose (e.g., with
S, Se, or alkylene, such as methylene or ethylene); addition of a
double bond (e.g., to replace ribose with cyclopentenyl or
cyclohexenyl); ring contraction of ribose (e.g., to form a
4-membered ring of cyclobutane or oxetane); ring expansion of
ribose (e.g., to form a 6- or 7-membered ring having an additional
carbon or heteroatom, such as for anhydrohexitol, altritol,
mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has
a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and
"unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or
S-GNA, where ribose is replaced by glycol units attached to
phosphodiester bonds), threose nucleic acid (TNA, where ribose is
replace with .alpha.-L-threofuranosyl-(3'.fwdarw.2')), and peptide
nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the
ribose and phosphodiester backbone). The sugar group can also
contain one or more carbons that possess the opposite
stereochemical configuration than that of the corresponding carbon
in ribose. Thus, a polynucleotide molecule can include nucleotides
containing, e.g., arabinose, as the sugar. Such sugar modifications
are taught International Patent Publication Nos. WO2013052523 and
WO2014093924, the contents of each of which are incorporated herein
by reference in their entireties.
[0675] (iii) Combinations of Modifications
[0676] The polynucleotides of the invention (e.g., a polynucleotide
comprising a nucleotide sequence encoding a PBGD polypeptide or a
functional fragment or variant thereof) can include a combination
of modifications to the sugar, the nucleobase, and/or the
intemucleoside linkage. These combinations can include any one or
more modifications described herein.
[0677] Combinations of modified nucleotides can be used to form the
polynucleotides of the invention. Unless otherwise noted, the
modified nucleotides can be completely substituted for the natural
nucleotides of the polynucleotides of the invention. As a
non-limiting example, the natural nucleotide uridine can be
substituted with a modified nucleoside described herein. In another
non-limiting example, the natural nucleotide uridine can be
partially substituted or replaced (e.g., about 0.1%, 1%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or 99.9%) with at least one of the modified
nucleoside disclosed herein. Any combination of base/sugar or
linker can be incorporated into the polynucleotides of the
invention and such modifications are taught in International Patent
Publications WO2013052523 and WO2014093924, and U.S. Publ. Nos. US
20130115272 and US20150307542, the contents of each of which are
incorporated herein by reference in its entirety.
11. UNTRANSLATED REGIONS (UTRS)
[0678] Untranslated regions (UTRs) are nucleic acid sections of a
polynucleotide before a start codon (5'UTR) and after a stop codon
(3'UTR) that are not translated. In some embodiments, a
polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger
RNA (mRNA)) of the invention comprising an open reading frame (ORF)
encoding a PBGD polypeptide further comprises UTR (e.g., a 5'UTR or
functional fragment thereof, a 3'UTR or functional fragment
thereof, or a combination thereof).
[0679] A UTR can be homologous or heterologous to the coding region
in a polynucleotide. In some embodiments, the UTR is homologous to
the ORF encoding the PBGD polypeptide. In some embodiments, the UTR
is heterologous to the ORF encoding the PBGD polypeptide. In some
embodiments, the polynucleotide comprises two or more 5'UTRs or
functional fragments thereof, each of which has the same or
different nucleotide sequences. In some embodiments, the
polynucleotide comprises two or more 3'UTRs or functional fragments
thereof, each of which has the same or different nucleotide
sequences.
[0680] In some embodiments, the 5'UTR or functional fragment
thereof, 3' UTR or functional fragment thereof, or any combination
thereof is sequence optimized.
[0681] In some embodiments, the 5'UTR or functional fragment
thereof, 3' UTR or functional fragment thereof, or any combination
thereof comprises at least one chemically modified nucleobase,
e.g., 1-methylpseudouridine or 5-methoxyuracil.
[0682] UTRs can have features that provide a regulatory role, e.g.,
increased or decreased stability, localization and/or translation
efficiency. A polynucleotide comprising a UTR can be administered
to a cell, tissue, or organism, and one or more regulatory features
can be measured using routine methods. In some embodiments, a
functional fragment of a 5'UTR or 3'UTR comprises one or more
regulatory features of a full length 5' or 3' UTR,
respectively.
[0683] Natural 5'UTRs bear features that play roles in translation
initiation. They harbor signatures like Kozak sequences that are
commonly known to be involved in the process by which the ribosome
initiates translation of many genes. Kozak sequences have the
consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine)
three bases upstream of the start codon (AUG), which is followed by
another `G`. 5'UTRs also have been known to form secondary
structures that are involved in elongation factor binding.
[0684] By engineering the features typically found in abundantly
expressed genes of specific target organs, one can enhance the
stability and protein production of a polynucleotide. For example,
introduction of 5'UTR of liver-expressed mRNA, such as albumin,
serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha
fetoprotein, erythropoietin, or Factor VIII, can enhance expression
of polynucleotides in hepatic cell lines or liver. Likewise, use of
5'UTR from other tissue-specific mRNA to improve expression in that
tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin,
Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for
myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1,
i-NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue
(e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial
cells (e.g., SP-A/B/C/D).
[0685] In some embodiments, UTRs are selected from a family of
transcripts whose proteins share a common function, structure,
feature or property. For example, an encoded polypeptide can belong
to a family of proteins (i.e., that share at least one function,
structure, feature, localization, origin, or expression pattern),
which are expressed in a particular cell, tissue or at some time
during development. The UTRs from any of the genes or mRNA can be
swapped for any other UTR of the same or different family of
proteins to create a new polynucleotide.
[0686] In some embodiments, the 5'UTR and the 3'UTR can be
heterologous. In some embodiments, the 5'UTR can be derived from a
different species than the 3'UTR. In some embodiments, the 3'UTR
can be derived from a different species than the 5'UTR.
[0687] Co-owned International Patent Application No.
PCT/US2014/021522 (Publ. No. WO/2014/164253, incorporated herein by
reference in its entirety) provides a listing of exemplary UTRs
that can be utilized in the polynucleotide of the present invention
as flanking regions to an ORF.
[0688] Exemplary UTRs of the application include, but are not
limited to, one or more 5'UTR and/or 3'UTR derived from the nucleic
acid sequence of: a globin, such as an .alpha.- or .beta.-globin
(e.g., aXenopus, mouse, rabbit, or human globin); a strong Kozak
translational initiation signal; a CYBA (e.g., human cytochrome
b-245.alpha. polypeptide); an albumin (e.g., human albumin7); a
HSD17B4 (hydroxysteroid (17-3) dehydrogenase); a virus (e.g., a
tobacco etch virus (TEV), a Venezuelan equine encephalitis virus
(VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV
immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B
virus), a sindbis virus, or a PAV barley yellow dwarf virus); a
heat shock protein (e.g., hsp70); a translation initiation factor
(e.g., elF4G); a glucose transporter (e.g., hGLUT1 (human glucose
transporter 1)); an actin (e.g., human .alpha. or .beta. actin); a
GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a
topoisomerase (e.g., a 5'UTR of a TOP gene lacking the 5' TOP motif
(the oligopyrimidine tract)); a ribosomal protein Large 32 (L32); a
ribosomal protein (e.g., human or mouse ribosomal protein, such as,
for example, rps9); an ATP synthase (e.g., ATP5A.sub.1 or the
.beta. subunit of mitochondrial H.sup.+-ATP synthase); a growth
hormone e (e.g., bovine (bGH) or human (hGH)); an elongation factor
(e.g., elongation factor 1 .alpha.1 (EEF1A1)); a manganese
superoxide dismutase (MnSOD); a myocyte enhancer factor 2A (MEF2A);
a 0-F1-ATPase, a creatine kinase, a myoglobin, a granulocyte-colony
stimulating factor (G-CSF); a collagen (e.g., collagen type I,
alpha 2 (Col1A2), collagen type I, alpha 1 (Col1A1), collagen type
VI, alpha 2 (Col6A.sub.2), collagen type VI, alpha 1
(Col6A.sub.1)); a ribophorin (e.g., ribophorin I (RPNI)); a low
density lipoprotein receptor-related protein (e.g., LRP1); a
cardiotrophin-like cytokine factor (e.g., Nntl); calreticulin
(Calr); a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1
(Plodl); and a nucleobindin (e.g., Nucbl).
[0689] In some embodiments, the 5'UTR is selected from the group
consisting of a .beta.-globin 5'UTR; a 5'UTR containing a strong
Kozak translational initiation signal; a cytochrome b-245 .alpha.
polypeptide (CYBA) 5'UTR; a hydroxysteroid (17-.beta.)
dehydrogenase (HSD17B4) 5'UTR; a Tobacco etch virus (TEV) 5'UTR; a
Venezuelen equine encephalitis virus (TEEV) 5'UTR; a 5' proximal
open reading frame of rubella virus (RV) RNA encoding nonstructural
proteins; a Dengue virus (DEN) 5'UTR; a heat shock protein 70
(Hsp70) 5'UTR; a eIF4G 5'UTR; a GLUT1 5'UTR; functional fragments
thereof and any combination thereof.
[0690] In some embodiments, the 3'UTR is selected from the group
consisting of a .beta.-globin 3'UTR; a CYBA 3'UTR; an albumin
3'UTR; a growth hormone (GH) 3'UTR; a VEEV 3'UTR; a hepatitis B
virus (HBV) 3'UTR; .alpha.-globin 3'UTR; a DEN 3'UTR; a PAV barley
yellow dwarf virus (BYDV-PAV) 3'UTR; an elongation factor 1 ul
(EEF1A.sub.1) 3'UTR; a manganese superoxide dismutase (MnSOD)
3'UTR; a .beta. subunit of mitochondrial H(+)-ATP synthase
(.beta.-mRNA) 3'UTR; a GLUT1 3'UTR; a MEF2A 3'UTR; a
.beta.-F1-ATPase 3'UTR; functional fragments thereof and
combinations thereof.
[0691] Wild-type UTRs derived from any gene or mRNA can be
incorporated into the polynucleotides of the invention. In some
embodiments, a UTR can be altered relative to a wild type or native
UTR to produce a variant UTR, e.g., by changing the orientation or
location of the UTR relative to the ORF; or by inclusion of
additional nucleotides, deletion of nucleotides, swapping or
transposition of nucleotides. In some embodiments, variants of 5'
or 3' UTRs can be utilized, for example, mutants of wild type UTRs,
or variants wherein one or more nucleotides are added to or removed
from a terminus of the UTR.
[0692] Additionally, one or more synthetic UTRs can be used in
combination with one or more non-synthetic UTRs. See, e.g., Mandal
and Rossi, Nat. Protoc. 2013 8(3):568-82, and sequences available
at www.addgene.org/Derrick_Rossi/, the contents of each are
incorporated herein by reference in their entirety.
[0693] UTRs or portions thereof can be placed in the same
orientation as in the transcript from which they were selected or
can be altered in orientation or location. Hence, a 5' and/or 3'
UTR can be inverted, shortened, lengthened, or combined with one or
more other 5' UTRs or 3' UTRs.
[0694] In some embodiments, the polynucleotide comprises multiple
UTRs, e.g., a double, a triple or a quadruple 5'UTR or 3'UTR. For
example, a double UTR comprises two copies of the same UTR either
in series or substantially in series. For example, a double
beta-globin 3'UTR can be used (see US2010/0129877, the contents of
which are incorporated herein by reference in its entirety).
[0695] In certain embodiments, the polynucleotides of the invention
comprise a 5'UTR and/or a 3'UTR selected from any of the UTRs
disclosed herein. In some embodiments, the 5'UTR comprises:
TABLE-US-00003 5'UTR-001 (Upstream UTR) (SEQ ID NO. 39)
(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC); 5'UTR-002
(Upstream UTR) (SEQ ID NO. 40)
(GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC); 5'UTR-003
(Upstream UTR) (See SEQ ID NO. 41); 5'UTR-004 (Upstream UTR) (SEQ
ID NO. 42) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC); 5'UTR-005
(Upstream UTR) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC)
(SEQ ID NO. 43); 5'UTR-006 (Upstream UTR); (See SEQ ID NO. 44)
5'UTR-007 (Upstream UTR) (SEQ ID NO. 45)
(GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC); 5'UTR-008 (Upstream
UTR) (SEQ ID NO. 46)
(GGGAAUUAACAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC); 5'UTR-009
(Upstream UTR) (SEQ ID NO. 47)
(GGGAAAUUAGACAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC); UTR 5'UTR-010,
Upstream (SEQ ID NO. 48)
(GGGAAAUAAGAGAGUAAAGAACAGUAAGAAGAAAUAUAAGAGCCACC); 5'UTR-011
(Upstream UTR) (SEQ ID NO. 49)
(GGGAAAAAAGAGAGAAAAGAAGACUAAGAAGAAAUAUAAGAGCCACC); 5'UTR-012
(Upstream UTR) (SEQ ID NO. 50)
(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAUAUAUAAGAGCCACC); 5'UTR-013
(Upstream UTR) (SEQ ID NO. 51)
(GGGAAAUAAGAGACAAAACAAGAGUAAGAAGAAAUAUAAGAGCCACC); 5'UTR-014
(Upstream UTR) (SEQ ID NO. 52)
(GGGAAAUUAGAGAGUAAAGAACAGUAAGUAGAAUUAAAAGAGCCACC); 5'UTR-15
(Upstream UTR) (SEQ ID NO. 53)
(GGGAAAUAAGAGAGAAUAGAAGAGUAAGAAGAAAUAUAAGAGCCACC); 5'UTR-016
(Upstream UTR) (SEQ ID NO. 54)
(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAAUUAAGAGCCACC); 5'UTR-017
(Upstream UTR) (SEQ ID NO. 55)
(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUUUAAGAGCCACC); or 5'UTR-018
(Upstream UTR) (SEQ ID NO. 56)
(UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGA
AAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC).
[0696] In some embodiments, the 3'UTR comprises:
TABLE-US-00004 142-3p 3'UTR (UTR including miR142-3p binding site)
(SEQ ID NO. 57) (UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCC
AUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC
CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3'UTR (UTR
including miR142-3p binding site) (SEQ ID NO. 58)
(UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACAC
AUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC
CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3'UTR (UTR
including miR142-3p binding site) (SEQ ID NO. 59)
(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAAA
GUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCAC
CCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3'UTR (UTR
including miR142-3p binding site) (SEQ ID NO. 60)
(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUC
CCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGCAC
CCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3'UTR (UTR
including miR142-3p binding site) (SEQ ID NO. 61)
(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUC
CCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGCAC
CCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3'UTR (UTR
including miR142-3p binding site) (SEQ ID NO. 62)
(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUC
CCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGA
AACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3'UTR (UTR
including miR142-3p binding site) (SEQ ID NO. 63)
(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUC
CCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUA
AAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC); 3'UTR-001 (Creatine
Kinase UTR) (See SEQ ID NO. 64); 3'UTR-002 (Myoglobin UTR) (See SEQ
ID NO. 65); 3'UTR-003 (.alpha.-actin UTR) (See SEQ ID NO. 66);
3'UTR-004 (Albumin UTR) (See SEQ ID NO. 67); 3'UTR-005
(.alpha.-globin UTR) (See SEQ ID NO. 68); 3'UTR-006 (G-CSF UTR)
(See SEQ ID NO. 69); 3'UTR-007 (Col1a2; collagen, type I, alpha 2
UTR) (See SEQ ID NO. 70); 3'UTR-008 (Col6a2; collagen, type VI,
alpha 2 UTR) (See SEQ ID NO. 71); 3'UTR-009 (RPN1; ribophorin I
UTR) (See SEQ ID NO. 72); 3'UTR-010 (LRP1; low density lipoprotein
receptor-related protein 1 UTR) (See SEQ ID NO. 73); 3'UTR-011
(Nnt1; cardiotrophin-like cytokine factor 1 UTR) (See SEQ ID NO.
74); 3'UTR-012 (Col6a1; collagen, type VI, alpha 1 UTR) (See SEQ ID
NO. 75); 3'UTR-013 (Calr; calreticulin UTR) (See SEQ ID NO. 76);
3'UTR-014 (Col1a1; collagen, type I, alpha 1 UTR) (See SEQ ID NO.
77); 3'UTR-015 (Plod1; procollagen-lysine, 2-oxoglutarate
5-dioxygenase 1 UTR) (See SEQ ID NO. 78); 3'UTR-016 (Nucb1;
nucleobindin 1 UTR) (See SEQ ID NO. 79); 3'UTR-017 (.alpha.-globin)
(See SEQ ID NO. 80); 3'UTR-018 (See SEQ ID NO. 81); 3'UTR (miR
142-3p and miR 126-3p binding sites variant 1) (SEQ ID NO. 149)
UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCC
AUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC
CGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC;
3'UTR (miR 142-3p and miR 126-3p binding sites variant 2) (SEQ ID
NO. 150) UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCC
UAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC
CGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC; or
3'UTR (miR 142-3p binding site variant 3) (SEQ ID NO. 151)
UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC
CCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAA
ACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC.
[0697] In certain embodiments, the 5'UTR and/or 3'UTR sequence of
the invention comprises a nucleotide sequence at least about 60%,
at least about 70%, at least about 80%, at least about 90%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99%, or about 100% identical to a
sequence selected from the group consisting of 5'UTR sequences
comprising any of SEQ ID NOs: 39 to 56, 83, 189 to 191 and/or 3'UTR
sequences comprises any of SEQ ID NOs: 57 to 81, 84, 149 to 151,
161 to 172, 192 to 199, and any combination thereof.
[0698] The polynucleotides of the invention can comprise
combinations of features. For example, the ORF can be flanked by a
5'UTR that comprises a strong Kozak translational initiation signal
and/or a 3'UTR comprising an oligo(dT) sequence for templated
addition of a poly-A tail. A 5'UTR can comprise a first
polynucleotide fragment and a second polynucleotide fragment from
the same and/or different UTRs (see, e.g., US2010/0293625, herein
incorporated by reference in its entirety).
[0699] Other non-UTR sequences can be used as regions or subregions
within the polynucleotides of the invention. For example, introns
or portions of intron sequences can be incorporated into the
polynucleotides of the invention. Incorporation of intronic
sequences can increase protein production as well as polynucleotide
expression levels. In some embodiments, the polynucleotide of the
invention comprises an internal ribosome entry site (IRES) instead
of or in addition to a UTR (see, e.g., Yakubov et al., Biochem.
Biophys. Res. Commun. 2010 394(1):189-193, the contents of which
are incorporated herein by reference in their entirety). In some
embodiments, the polynucleotide comprises an IRES instead of a
5'UTR sequence. In some embodiments, the polynucleotide comprises
an ORF and a viral capsid sequence. In some embodiments, the
polynucleotide comprises a synthetic 5'UTR in combination with a
non-synthetic 3'UTR.
[0700] In some embodiments, the UTR can also include at least one
translation enhancer polynucleotide, translation enhancer element,
or translational enhancer elements (collectively, "TEE," which
refers to nucleic acid sequences that increase the amount of
polypeptide or protein produced from a polynucleotide. As a
non-limiting example, the TEE can include those described in
US2009/0226470, incorporated herein by reference in its entirety,
and others known in the art. As a non-limiting example, the TEE can
be located between the transcription promoter and the start codon.
In some embodiments, the 5'UTR comprises a TEE.
[0701] In one aspect, a TEE is a conserved element in a UTR that
can promote translational activity of a nucleic acid such as, but
not limited to, cap-dependent or cap-independent translation.
[0702] In one non-limiting example, the TEE comprises the TEE
sequence in the 5'-leader of the Gtx homeodomain protein. See
Chappell et al., PNAS 2004 101:9590-9594, incorporated herein by
reference in its entirety.
[0703] In some embodiments, the polynucleotide of the invention
comprises one or multiple copies of a TEE. The TEE in a
translational enhancer polynucleotide can be organized in one or
more sequence segments. A sequence segment can harbor one or more
of the TEEs provided herein, with each TEE being present in one or
more copies. When multiple sequence segments are present in a
translational enhancer polynucleotide, they can be homogenous or
heterogeneous. Thus, the multiple sequence segments in a
translational enhancer polynucleotide can harbor identical or
different types of the TEE provided herein, identical or different
number of copies of each of the TEE, and/or identical or different
organization of the TEE within each sequence segment. In one
embodiment, the polynucleotide of the invention comprises a
translational enhancer polynucleotide sequence. Non-limiting
examples of TEE sequences are described in U.S. Publication
2014/0200261, the contents of which are incorporated herein by
reference in their entirety.
12. MICRORNA (MIRNA) BINDING SITES
[0704] Polynucleotides of the invention can include regulatory
elements, for example, microRNA (miRNA) binding sites,
transcription factor binding sites, structured mRNA sequences
and/or motifs, artificial binding sites engineered to act as
pseudo-receptors for endogenous nucleic acid binding molecules, and
combinations thereof. In some embodiments, polynucleotides
including such regulatory elements are referred to as including
"sensor sequences". Non-limiting examples of sensor sequences are
described in U.S. Publication 2014/0200261, the contents of which
are incorporated herein by reference in their entirety.
[0705] In some embodiments, a polynucleotide (e.g., a ribonucleic
acid (RNA), e.g., a messenger RNA (mRNA)) of the invention
comprises an open reading frame (ORF) encoding a polypeptide of
interest and further comprises one or more miRNA binding site(s).
Inclusion or incorporation of miRNA binding site(s) provides for
regulation of polynucleotides of the invention, and in turn, of the
polypeptides encoded therefrom, based on tissue-specific and/or
cell-type specific expression of naturally-occurring miRNAs.
[0706] The present invention also provides pharmaceutical
compositions and formulations that comprise any of the
polynucleotides described above. In some embodiments, the
composition or formulation further comprises a delivery agent.
[0707] In some embodiments, the composition or formulation can
contain a polynucleotide comprising a sequence optimized nucleic
acid sequence disclosed herein which encodes a polypeptide. In some
embodiments, the composition or formulation can contain a
polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a
polynucleotide (e.g., an ORF) having significant sequence identity
to a sequence optimized nucleic acid sequence disclosed herein
which encodes a polypeptide. In some embodiments, the
polynucleotide further comprises a miRNA binding site, e.g., a
miRNA binding site that binds
[0708] A miRNA, e.g., a natural-occurring miRNA, is a 19-25
nucleotide long noncoding RNA that binds to a polynucleotide and
down-regulates gene expression either by reducing stability or by
inhibiting translation of the polynucleotide. A miRNA sequence
comprises a "seed" region, i.e., a sequence in the region of
positions 2-8 of the mature miRNA. A miRNA seed can comprise
positions 2-8 or 2-7 of the mature miRNA. In some embodiments, a
miRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of the
mature miRNA), wherein the seed-complementary site in the
corresponding miRNA binding site is flanked by an adenosine (A)
opposed to miRNA position 1. In some embodiments, a miRNA seed can
comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA),
wherein the seed-complementary site in the corresponding miRNA
binding site is flanked by an adenosine (A) opposed to miRNA
position 1. See, for example, Grimson A, Farh K K, Johnston W K,
Garrett-Engele P, Lim L P, Bartel D P; Mol Cell. 2007 Jul. 6;
27(1):91-105. miRNA profiling of the target cells or tissues can be
conducted to determine the presence or absence of miRNA in the
cells or tissues. In some embodiments, a polynucleotide (e.g., a
ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the
invention comprises one or more microRNA binding sites, microRNA
target sequences, microRNA complementary sequences, or microRNA
seed complementary sequences. Such sequences can correspond to,
e.g., have complementarity to, any known microRNA such as those
taught in US Publication US2005/0261218 and US Publication
US2005/0059005, the contents of each of which are incorporated
herein by reference in their entirety.
[0709] microRNAs derive enzymatically from regions of RNA
transcripts that fold back on themselves to form short hairpin
structures often termed a pre-miRNA (precursor-miRNA). A pre-miRNA
typically has a two-nucleotide overhang at its 3' end, and has 3'
hydroxyl and 5' phosphate groups. This precursor-mRNA is processed
in the nucleus and subsequently transported to the cytoplasm where
it is further processed by DICER (a RNase III enzyme), to form a
mature microRNA of approximately 22 nucleotides. The mature
microRNA is then incorporated into a ribonuclear particle to form
the RNA-induced silencing complex, RISC, which mediates gene
silencing. Art-recognized nomenclature for mature miRNAs typically
designates the arm of the pre-miRNA from which the mature miRNA
derives; "5p" means the microRNA is from the 5 prime arm of the
pre-miRNA hairpin and "3p" means the microRNA is from the 3 prime
end of the pre-miRNA hairpin. A miR referred to by number herein
can refer to either of the two mature microRNAs originating from
opposite arms of the same pre-miRNA (e.g., either the 3p or 5p
microRNA). All miRs referred to herein are intended to include both
the 3p and 5p arms/sequences, unless particularly specified by the
3p or 5p designation.
[0710] As used herein, the term "microRNA (miRNA or miR) binding
site" refers to a sequence within a polynucleotide, e.g., within a
DNA or within an RNA transcript, including in the 5'UTR and/or
3'UTR, that has sufficient complementarity to all or a region of a
miRNA to interact with, associate with or bind to the miRNA. In
some embodiments, a polynucleotide of the invention comprising an
ORF encoding a polypeptide of interest and further comprises one or
more miRNA binding site(s). In exemplary embodiments, a 5'UTR
and/or 3'UTR of the polynucleotide (e.g., a ribonucleic acid (RNA),
e.g., a messenger RNA (mRNA)) comprises the one or more miRNA
binding site(s).
[0711] A miRNA binding site having sufficient complementarity to a
miRNA refers to a degree of complementarity sufficient to
facilitate miRNA-mediated regulation of a polynucleotide, e.g.,
miRNA-mediated translational repression or degradation of the
polynucleotide. In exemplary aspects of the invention, a miRNA
binding site having sufficient complementarity to the miRNA refers
to a degree of complementarity sufficient to facilitate
miRNA-mediated degradation of the polynucleotide, e.g.,
miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage
of mRNA. The miRNA binding site can have complementarity to, for
example, a 19-25 nucleotide long miRNA sequence, to a long 19-23
nucleotide miRNA sequence, or to a long 22 nucleotide miRNA
sequence. A miRNA binding site can be complementary to only a
portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4
nucleotides of the full length of a naturally-occurring miRNA
sequence, or to a portion less than 1, 2, 3, or 4 nucleotides
shorter than a naturally-occurring miRNA sequence. Full or complete
complementarity (e.g., full complementarity or complete
complementarity over all or a significant portion of the length of
a naturally-occurring miRNA) is preferred when the desired
regulation is mRNA degradation.
[0712] In some embodiments, a miRNA binding site includes a
sequence that has complementarity (e.g., partial or complete
complementarity) with an miRNA seed sequence. In some embodiments,
the miRNA binding site includes a sequence that has complete
complementarity with a miRNA seed sequence. In some embodiments, a
miRNA binding site includes a sequence that has complementarity
(e.g., partial or complete complementarity) with an miRNA sequence.
In some embodiments, the miRNA binding site includes a sequence
that has complete complementarity with a miRNA sequence. In some
embodiments, a miRNA binding site has complete complementarity with
a miRNA sequence but for 1, 2, or 3 nucleotide substitutions,
terminal additions, and/or truncations.
[0713] In some embodiments, the miRNA binding site is the same
length as the corresponding miRNA. In other embodiments, the miRNA
binding site is one, two, three, four, five, six, seven, eight,
nine, ten, eleven or twelve nucleotide(s) shorter than the
corresponding miRNA at the 5' terminus, the 3' terminus, or both.
In still other embodiments, the microRNA binding site is two
nucleotides shorter than the corresponding microRNA at the 5'
terminus, the 3' terminus, or both. The miRNA binding sites that
are shorter than the corresponding miRNAs are still capable of
degrading the mRNA incorporating one or more of the miRNA binding
sites or preventing the mRNA from translation.
[0714] In some embodiments, the miRNA binding site binds the
corresponding mature miRNA that is part of an active RISC
containing Dicer. In another embodiment, binding of the miRNA
binding site to the corresponding miRNA in RISC degrades the mRNA
containing the miRNA binding site or prevents the mRNA from being
translated. In some embodiments, the miRNA binding site has
sufficient complementarity to miRNA so that a RISC complex
comprising the miRNA cleaves the polynucleotide comprising the
miRNA binding site. In other embodiments, the miRNA binding site
has imperfect complementarity so that a RISC complex comprising the
miRNA induces instability in the polynucleotide comprising the
miRNA binding site. In another embodiment, the miRNA binding site
has imperfect complementarity so that a RISC complex comprising the
miRNA represses transcription of the polynucleotide comprising the
miRNA binding site.
[0715] In some embodiments, the miRNA binding site has one, two,
three, four, five, six, seven, eight, nine, ten, eleven or twelve
mismatch(es) from the corresponding miRNA.
[0716] In some embodiments, the miRNA binding site has at least
about ten, at least about eleven, at least about twelve, at least
about thirteen, at least about fourteen, at least about fifteen, at
least about sixteen, at least about seventeen, at least about
eighteen, at least about nineteen, at least about twenty, or at
least about twenty-one contiguous nucleotides complementary to at
least about ten, at least about eleven, at least about twelve, at
least about thirteen, at least about fourteen, at least about
fifteen, at least about sixteen, at least about seventeen, at least
about eighteen, at least about nineteen, at least about twenty, or
at least about twenty-one, respectively, contiguous nucleotides of
the corresponding miRNA.
[0717] By engineering one or more miRNA binding sites into a
polynucleotide of the invention, the polynucleotide can be targeted
for degradation or reduced translation, provided the miRNA in
question is available. This can reduce off-target effects upon
delivery of the polynucleotide. For example, if a polynucleotide of
the invention is not intended to be delivered to a tissue or cell
but ends up is said tissue or cell, then a miRNA abundant in the
tissue or cell can inhibit the expression of the gene of interest
if one or multiple binding sites of the miRNA are engineered into
the 5'UTR and/or 3'UTR of the polynucleotide. Thus, in some
embodiments, incorporation of one or more miRNA binding sites into
an mRNA of the disclosure may reduce the hazard of off-target
effects upon nucleic acid molecule delivery and/or enable
tissue-specific regulation of expression of a polypeptide encoded
by the mRNA. In yet other embodiments, incorporation of one or more
miRNA binding sites into an mRNA of the disclosure can modulate
immune responses upon nucleic acid delivery in vivo. In further
embodiments, incorporation of one or more miRNA binding sites into
an mRNA of the disclosure can modulate accelerated blood clearance
(ABC) of lipid-comprising compounds and compositions described
herein.
[0718] Conversely, miRNA binding sites can be removed from
polynucleotide sequences in which they naturally occur in order to
increase protein expression in specific tissues. For example, a
binding site for a specific miRNA can be removed from a
polynucleotide to improve protein expression in tissues or cells
containing the miRNA.
[0719] Regulation of expression in multiple tissues can be
accomplished through introduction or removal of one or more miRNA
binding sites, e.g., one or more distinct miRNA binding sites. The
decision whether to remove or insert a miRNA binding site can be
made based on miRNA expression patterns and/or their profilings in
tissues and/or cells in development and/or disease. Identification
of MiRNAs, miRNA binding sites, and their expression patterns and
role in biology have been reported (e.g., Bonauer et al., Curr Drug
Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011
18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec.
20. doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233;
Landgraf et al, Cell, 2007 129:1401-1414; Gentner and Naldini,
Tissue Antigens. 2012 80:393-403 and all references therein; each
of which is incorporated herein by reference in its entirety).
[0720] miRNAs and miRNA binding sites can correspond to any known
sequence, including non-limiting examples described in U.S.
Publication Nos. 2014/0200261, 2005/0261218, and 2005/0059005, each
of which are incorporated herein by reference in their
entirety.
[0721] Examples of tissues where miRNA are known to regulate mRNA,
and thereby protein expression, include, but are not limited to,
liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial
cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p,
miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7,
miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194,
miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
[0722] Specifically, miRNAs are known to be differentially
expressed in immune cells (also called hematopoietic cells), such
as antigen presenting cells (APCs) (e.g., dendritic cells and
macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes,
granulocytes, natural killer cells, etc. Immune cell specific
miRNAs are involved in immunogenicity, autoimmunity, the
immune-response to infection, inflammation, as well as unwanted
immune response after gene therapy and tissue/organ
transplantation. Immune cells specific miRNAs also regulate many
aspects of development, proliferation, differentiation and
apoptosis of hematopoietic cells (immune cells). For example,
miR-142 and miR-146 are exclusively expressed in immune cells,
particularly abundant in myeloid dendritic cells. It has been
demonstrated that the immune response to a polynucleotide can be
shut-off by adding miR-142 binding sites to the 3'-UTR of the
polynucleotide, enabling more stable gene transfer in tissues and
cells. miR-142 efficiently degrades exogenous polynucleotides in
antigen presenting cells and suppresses cytotoxic elimination of
transduced cells (e.g., Annoni A et al., blood, 2009, 114,
5152-5161; Brown B D, et al., Nat med. 2006, 12(5), 585-591; Brown
B D, et al., blood, 2007, 110(13): 4144-4152, each of which is
incorporated herein by reference in its entirety).
[0723] An antigen-mediated immune response can refer to an immune
response triggered by foreign antigens, which, when entering an
organism, are processed by the antigen presenting cells and
displayed on the surface of the antigen presenting cells. T cells
can recognize the presented antigen and induce a cytotoxic
elimination of cells that express the antigen.
[0724] Introducing a miR-142 binding site into the 5'UTR and/or
3'UTR of a polynucleotide of the invention can selectively repress
gene expression in antigen presenting cells through miR-142
mediated degradation, limiting antigen presentation in antigen
presenting cells (e.g., dendritic cells) and thereby preventing
antigen-mediated immune response after the delivery of the
polynucleotide. The polynucleotide is then stably expressed in
target tissues or cells without triggering cytotoxic
elimination.
[0725] In one embodiment, binding sites for miRNAs that are known
to be expressed in immune cells, in particular, antigen presenting
cells, can be engineered into a polynucleotide of the invention to
suppress the expression of the polynucleotide in antigen presenting
cells through miRNA mediated RNA degradation, subduing the
antigen-mediated immune response. Expression of the polynucleotide
is maintained in non-immune cells where the immune cell specific
miRNAs are not expressed. For example, in some embodiments, to
prevent an immunogenic reaction against a liver specific protein,
any miR-122 binding site can be removed and a miR-142 (and/or
mirR-146) binding site can be engineered into the 5'UTR and/or
3'UTR of a polynucleotide of the invention.
[0726] To further drive the selective degradation and suppression
in APCs and macrophage, a polynucleotide of the invention can
include a further negative regulatory element in the 5'UTR and/or
3'UTR, either alone or in combination with miR-142 and/or miR-146
binding sites. As a non-limiting example, the further negative
regulatory element is a Constitutive Decay Element (CDE).
[0727] Immune cell specific miRNAs include, but are not limited to,
hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c,
hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p,
hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184,
hsa-let-7f-1-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p,
miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p,
miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-143-3p,
miR-143-5p, miR-146a-3p, miR-146a-5p, miR-146b-3p, miR-146b-5p,
miR-147a, miR-147b, miR-148a-5p, miR-148a-3p, miR-150-3p,
miR-150-5p, miR-151b, miR-155-3p, miR-155-5p, miR-15a-3p,
miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p, miR-16-2-3p,
miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p, miR-181a-2-3p,
miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p, miR-21-5p,
miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,
miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,
miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,
miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p,
miR-27b-5p, miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p,
miR-29b-1-5p, miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p,
miR-30e-5p, miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p,
miR-345-5p, miR-346, miR-34a-3p, miR-34a-5p, miR-363-3p,
miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR-493-3p,
miR-493-5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j,
miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p,
miR-99a-5p, miR-99b-3p, and miR-99b-5p. Furthermore, novel miRNAs
can be identified in immune cell through micro-array hybridization
and microtome analysis (e.g., Jima D D et al, Blood, 2010,
116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11, 288, the
content of each of which is incorporated herein by reference in its
entirety.)
[0728] miRNAs that are known to be expressed in the liver include,
but are not limited to, miR-107, miR-122-3p, miR-122-5p,
miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303,
miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p,
miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p,
miR-557, miR-581, miR-939-3p, and miR-939-5p. MiRNA binding sites
from any liver specific miRNA can be introduced to or removed from
a polynucleotide of the invention to regulate expression of the
polynucleotide in the liver. Liver specific miRNA binding sites can
be engineered alone or further in combination with immune cell
(e.g., APC) miRNA binding sites in a polynucleotide of the
invention.
[0729] miRNAs that are known to be expressed in the lung include,
but are not limited to, let-7a-2-3p, let-7a-3p, let-7a-5p,
miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p,
miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134,
miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p,
miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p,
miR-337-3p, miR-337-5p, miR-381-3p, and miR-381-5p. miRNA binding
sites from any lung specific miRNA can be introduced to or removed
from a polynucleotide of the invention to regulate expression of
the polynucleotide in the lung. Lung specific miRNA binding sites
can be engineered alone or further in combination with immune cell
(e.g., APC) miRNA binding sites in a polynucleotide of the
invention.
[0730] miRNAs that are known to be expressed in the heart include,
but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p,
miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210,
miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p,
miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and
miR-92b-5p. mMiRNA binding sites from any heart specific microRNA
can be introduced to or removed from a polynucleotide of the
invention to regulate expression of the polynucleotide in the
heart. Heart specific miRNA binding sites can be engineered alone
or further in combination with immune cell (e.g., APC) miRNA
binding sites in a polynucleotide of the invention.
[0731] miRNAs that are known to be expressed in the nervous system
include, but are not limited to, miR-124-5p, miR-125a-3p,
miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p,
miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p,
miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p,
miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,
miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b,
miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p,
miR-23a-5p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p,
miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329,
miR-342-3p, miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383,
miR-410, miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483,
miR-510, miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571,
miR-7-1-3p, miR-7-2-3p, miR-7-5p, miR-802, miR-922, miR-9-3p, and
miR-9-5p. miRNAs enriched in the nervous system further include
those specifically expressed in neurons, including, but not limited
to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p,
miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e,
miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328,
miR-922 and those specifically expressed in glial cells, including,
but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,
miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,
miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. miRNA
binding sites from any CNS specific miRNA can be introduced to or
removed from a polynucleotide of the invention to regulate
expression of the polynucleotide in the nervous system. Nervous
system specific miRNA binding sites can be engineered alone or
further in combination with immune cell (e.g., APC) miRNA binding
sites in a polynucleotide of the invention.
[0732] miRNAs that are known to be expressed in the pancreas
include, but are not limited to, miR-105-3p, miR-105-5p, miR-184,
miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p,
miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p,
miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p,
miR-493-5p, and miR-944. MiRNA binding sites from any pancreas
specific miRNA can be introduced to or removed from a
polynucleotide of the invention to regulate expression of the
polynucleotide in the pancreas. Pancreas specific miRNA binding
sites can be engineered alone or further in combination with immune
cell (e.g. APC) miRNA binding sites in a polynucleotide of the
invention.
[0733] miRNAs that are known to be expressed in the kidney include,
but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p,
miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p,
miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p,
miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p,
miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p, miR-324-3p,
miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562. miRNA
binding sites from any kidney specific miRNA can be introduced to
or removed from a polynucleotide of the invention to regulate
expression of the polynucleotide in the kidney. Kidney specific
miRNA binding sites can be engineered alone or further in
combination with immune cell (e.g., APC) miRNA binding sites in a
polynucleotide of the invention.
[0734] miRNAs that are known to be expressed in the muscle include,
but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286,
miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p,
miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b,
miR-25-3p, and miR-25-5p. MiRNA binding sites from any muscle
specific miRNA can be introduced to or removed from a
polynucleotide of the invention to regulate expression of the
polynucleotide in the muscle. Muscle specific miRNA binding sites
can be engineered alone or further in combination with immune cell
(e.g., APC) miRNA binding sites in a polynucleotide of the
invention.
[0735] miRNAs are also differentially expressed in different types
of cells, such as, but not limited to, endothelial cells,
epithelial cells, and adipocytes.
[0736] miRNAs that are known to be expressed in endothelial cells
include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p,
miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p,
miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p,
miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p,
miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p,
miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p,
miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p,
miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p,
miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p,
and miR-92b-5p. Many novel miRNAs are discovered in endothelial
cells from deep-sequencing analysis (e.g., Voellenkle C et al.,
RNA, 2012, 18, 472-484, herein incorporated by reference in its
entirety). miRNA binding sites from any endothelial cell specific
miRNA can be introduced to or removed from a polynucleotide of the
invention to regulate expression of the polynucleotide in the
endothelial cells.
[0737] miRNAs that are known to be expressed in epithelial cells
include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246,
miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p,
miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494,
miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p,
miR-449b-5p specific in respiratory ciliated epithelial cells,
let-7 family, miR-133a, miR-133b, miR-126 specific in lung
epithelial cells, miR-382-3p, miR-382-5p specific in renal
epithelial cells, and miR-762 specific in corneal epithelial cells.
miRNA binding sites from any epithelial cell specific miRNA can be
introduced to or removed from a polynucleotide of the invention to
regulate expression of the polynucleotide in the epithelial
cells.
[0738] In addition, a large group of miRNAs are enriched in
embryonic stem cells, controlling stem cell self-renewal as well as
the development and/or differentiation of various cell lineages,
such as neural cells, cardiac, hematopoietic cells, skin cells,
osteogenic cells and muscle cells (e.g., Kuppusamy K T et al.,
Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A,
Semin Cancer Biol. 2012, 22(5-6), 428-436; Goff L A et al., PLoS
One, 2009, 4:e7192; Morin R D et al., Genome Res, 2008, 18,
610-621; Yoo J K et al., Stem Cells Dev. 2012, 21(11), 2049-2057,
each of which is herein incorporated by reference in its entirety).
MiRNAs abundant in embryonic stem cells include, but are not
limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,
miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,
miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,
miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p,
miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p,
miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e,
miR-367-3p, miR-367-5p, miR-369-3p, miR-369-5p, miR-370, miR-371,
miR-373, miR-380-5p, miR-423-3p, miR-423-5p, miR-486-5p,
miR-520c-3p, miR-548e, miR-548f, miR-548g-3p, miR-548g-5p,
miR-548i, miR-548k, miR-548l, miR-548m, miR-548n, miR-548o-3p,
miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p, miR-664b-3p,
miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p, miR-885-5p,
miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p, miR-99b-3p and
miR-99b-5p. Many predicted novel miRNAs are discovered by deep
sequencing in human embryonic stem cells (e.g., Morin R D et al.,
Genome Res, 2008, 18, 610-621; Goff L A et al., PLoS One, 2009,
4:e7192; Bar M et al., Stem cells, 2008, 26, 2496-2505, the content
of each of which is incorporated herein by reference in its
entirety).
[0739] In one embodiment, the binding sites of embryonic stem cell
specific miRNAs can be included in or removed from the 3'UTR of a
polynucleotide of the invention to modulate the development and/or
differentiation of embryonic stem cells, to inhibit the senescence
of stem cells in a degenerative condition (e.g. degenerative
diseases), or to stimulate the senescence and apoptosis of stem
cells in a disease condition (e.g. cancer stem cells).
[0740] In some embodiments, miRNAs are selected based on expression
and abundance in immune cells of the hematopoietic lineage, such as
B cells, T cells, macrophages, dendritic cells, and cells that are
known to express TLR7/TLR8 and/or able to secrete cytokines such as
endothelial cells and platelets. In some embodiments, the miRNA set
thus includes miRs that may be responsible in part for the
immunogenicity of these cells, and such that a corresponding
miR-site incorporation in polynucleotides of the present invention
(e.g., mRNAs) could lead to destabilization of the mRNA and/or
suppression of translation from these mRNAs in the specific cell
type. Non-limiting representative examples include miR-142,
miR-144, miR-150, miR-155 and miR-223, which are specific for many
of the hematopoietic cells; miR-142, miR150, miR-16 and miR-223,
which are expressed in B cells; miR-223, miR-451, miR-26a, miR-16,
which are expressed in progenitor hematopoietic cells; and miR-126,
which is expressed in plasmacytoid dendritic cells, platelets and
endothelial cells. For further discussion of tissue expression of
miRs see e.g., Teruel-Montoya, R. et al. (2014) PLoS One 9:e102259;
Landgraf, P. et al. (2007) Cell 129:1401-1414; Bissels, U. et al.
(2009) RNA 15:2375-2384. Any one miR-site incorporation in the
3'UTR and/or 5' UTR may mediate such effects in multiple cell types
of interest (e.g., miR-142 is abundant in both B cells and
dendritic cells).
[0741] In some embodiments, it may be beneficial to target the same
cell type with multiple miRs and to incorporate binding sites to
each of the 3p and 5p arm if both are abundant (e.g., both
miR-142-3p and miR142-5p are abundant in hematopoietic stem cells).
Thus, in certain embodiments, polynucleotides of the invention
contain two or more (e.g., two, three, four or more) miR bindings
sites from: (i) the group consisting of miR-142, miR-144, miR-150,
miR-155 and miR-223 (which are expressed in many hematopoietic
cells); or (ii) the group consisting of miR-142, miR150, miR-16 and
miR-223 (which are expressed in B cells); or the group consisting
of miR-223, miR-451, miR-26a, miR-16 (which are expressed in
progenitor hematopoietic cells).
[0742] In some embodiments, it may also be beneficial to combine
various miRs such that multiple cell types of interest are targeted
at the same time (e.g., miR-142 and miR-126 to target many cells of
the hematopoietic lineage and endothelial cells). Thus, for
example, in certain embodiments, polynucleotides of the invention
comprise two or more (e.g., two, three, four or more) miRNA
bindings sites, wherein: (i) at least one of the miRs targets cells
of the hematopoietic lineage (e.g., miR-142, miR-144, miR-150,
miR-155 or miR-223) and at least one of the miRs targets
plasmacytoid dendritic cells, platelets or endothelial cells (e.g.,
miR-126); or (ii) at least one of the miRs targets B cells (e.g.,
miR-142, miR150, miR-16 or miR-223) and at least one of the miRs
targets plasmacytoid dendritic cells, platelets or endothelial
cells (e.g., miR-126); or (iii) at least one of the miRs targets
progenitor hematopoietic cells (e.g., miR-223, miR-451, miR-26a or
miR-16) and at least one of the miRs targets plasmacytoid dendritic
cells, platelets or endothelial cells (e.g., miR-126); or (iv) at
least one of the miRs targets cells of the hematopoietic lineage
(e.g., miR-142, miR-144, miR-150, miR-155 or miR-223), at least one
of the miRs targets B cells (e.g., miR-142, miR150, miR-16 or
miR-223) and at least one of the miRs targets plasmacytoid
dendritic cells, platelets or endothelial cells (e.g., miR-126); or
any other possible combination of the foregoing four classes of miR
binding sites (i.e., those targeting the hematopoietic lineage,
those targeting B cells, those targeting progenitor hematopoietic
cells and/or those targeting plamacytoid dendritic
cells/platelets/endothelial cells).
[0743] In one embodiment, to modulate immune responses,
polynucleotides of the present invention can comprise one or more
miRNA binding sequences that bind to one or more miRs that are
expressed in conventional immune cells or any cell that expresses
TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or
chemokines (e.g., in immune cells of peripheral lymphoid organs
and/or splenocytes and/or endothelial cells). It has now been
discovered that incorporation into an mRNA of one or more miRs that
are expressed in conventional immune cells or any cell that
expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines
and/or chemokines (e.g., in immune cells of peripheral lymphoid
organs and/or splenocytes and/or endothelial cells) reduces or
inhibits immune cell activation (e.g., B cell activation, as
measured by frequency of activated B cells) and/or cytokine
production (e.g., production of IL-6, IFN-.gamma. and/or
TNF.alpha.). Furthermore, it has now been discovered that
incorporation into an mRNA of one or more miRs that are expressed
in conventional immune cells or any cell that expresses TLR7 and/or
TLR8 and secrete pro-inflammatory cytokines and/or chemokines
(e.g., in immune cells of peripheral lymphoid organs and/or
splenocytes and/or endothelial cells) can reduce or inhibit an
anti-drug antibody (ADA) response against a protein of interest
encoded by the mRNA.
[0744] In another embodiment, to modulate accelerated blood
clearance of an polynucleotide delivered in a lipid-comprising
compound or composition, polynucleotides of the invention can
comprise one or more miR binding sequences that bind to one or more
miRNAs expressed in conventional immune cells or any cell that
expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines
and/or chemokines (e.g., in immune cells of peripheral lymphoid
organs and/or splenocytes and/or endothelial cells). It has now
been discovered that incorporation into an mRNA of one or more miR
binding sites reduces or inhibits accelerated blood clearance (ABC)
of the lipid-comprising compound or composition for use in
delivering the mRNA. Furthermore, it has now been discovered that
incorporation of one or more miR binding sites into an mRNA reduces
serum levels of anti-PEG anti-IgM (e.g, reduces or inhibits the
acute production of IgMs that recognize polyethylene glycol (PEG)
by B cells) and/or reduces or inhibits proliferation and/or
activation of plasmacytoid dendritic cells following administration
of a lipid-comprising compound or composition comprising the
mRNA.
[0745] In some embodiments, miR sequences may correspond to any
known microRNA expressed in immune cells, including but not limited
to those taught in US Publication US2005/0261218 and US Publication
US2005/0059005, the contents of which are incorporated herein by
reference in their entirety. Non-limiting examples of miRs
expressed in immune cells include those expressed in spleen cells,
myeloid cells, dendritic cells, plasmacytoid dendritic cells, B
cells, T cells and/or macrophages. For example, miR-142-3p,
miR-142-5p, miR-16, miR-21, miR-223, miR-24 and miR-27 are
expressed in myeloid cells, miR-155 is expressed in dendritic
cells, B cells and T cells, miR-146 is upregulated in macrophages
upon TLR stimulation and miR-126 is expressed in plasmacytoid
dendritic cells. In certain embodiments, the miR(s) is expressed
abundantly or preferentially in immune cells. For example, miR-142
(miR-142-3p and/or miR-142-5p), miR-126 (miR-126-3p and/or
miR-126-5p), miR-146 (miR-146-3p and/or miR-146-5p) and miR-155
(miR-155-3p and/or miR155-5p) are expressed abundantly in immune
cells. These microRNA sequences are known in the art and, thus, one
of ordinary skill in the art can readily design binding sequences
or target sequences to which these microRNAs will bind based upon
Watson-Crick complementarity.
[0746] Accordingly, in various embodiments, polynucleotides of the
present invention comprise at least one microRNA binding site for a
miR selected from the group consisting of miR-142, miR-146,
miR-155, miR-126, miR-16, miR-21, miR-223, miR-24 and miR-27. In
another embodiment, the mRNA comprises at least two miR binding
sites for microRNAs expressed in immune cells. In various
embodiments, the polynucleotide of the invention comprises 1-4,
one, two, three or four miR binding sites for microRNAs expressed
in immune cells. In another embodiment, the polynucleotide of the
invention comprises three miR binding sites. These miR binding
sites can be for microRNAs selected from the group consisting of
miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223,
miR-24, miR-27, and combinations thereof. In one embodiment, the
polynucleotide of the invention comprises two or more (e.g., two,
three, four) copies of the same miR binding site expressed in
immune cells, e.g., two or more copies of a miR binding site
selected from the group of miRs consisting of miR-142, miR-146,
miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, miR-27.
[0747] In one embodiment, the polynucleotide of the invention
comprises three copies of the same miR binding site. In certain
embodiments, use of three copies of the same miR binding site can
exhibit beneficial properties as compared to use of a single miR
binding site. Non-limiting examples of sequences for 3' UTRs
containing three miR bindings sites are shown in SEQ ID NO: 165
(three miR-142-3p binding sites), and SEQ ID NO: 167 (three
miR-142-5p binding sites).
[0748] In another embodiment, the polynucleotide of the invention
comprises two or more (e.g., two, three, four) copies of at least
two different miR binding sites expressed in immune cells.
Non-limiting examples of sequences of 3' UTRs containing two or
more different miR binding sites are shown in SEQ ID NO: 164 (one
miR-142-3p binding site and one miR-126-3p binding site), SEQ ID
NO: 168 (two miR-142-5p binding sites and one miR-142-3p binding
sites) and SEQ ID NO: 171 (two miR-155-5p binding sites and one
miR-142-3p binding sites).
[0749] In another embodiment, the polynucleotide of the invention
comprises at least two miR binding sites for microRNAs expressed in
immune cells, wherein one of the miR binding sites is for
miR-142-3p. In various embodiments, the polynucleotide of the
invention comprises binding sites for miR-142-3p and miR-155
(miR-155-3p or miR-155-5p), miR-142-3p and miR-146 (miR-146-3 or
miR-146-5p), or miR-142-3p and miR-126 (miR-126-3p or
miR-126-5p).
[0750] In another embodiment, the polynucleotide of the invention
comprises at least two miR binding sites for microRNAs expressed in
immune cells, wherein one of the miR binding sites is for
miR-126-3p. In various embodiments, the polynucleotide of the
invention comprises binding sites for miR-126-3p and miR-155
(miR-155-3p or miR-155-5p), miR-126-3p and miR-146 (miR-146-3p or
miR-146-5p), or miR-126-3p and miR-142 (miR-142-3p or
miR-142-5p).
[0751] In another embodiment, the polynucleotide of the invention
comprises at least two miR binding sites for microRNAs expressed in
immune cells, wherein one of the miR binding sites is for
miR-142-5p. In various embodiments, the polynucleotide of the
invention comprises binding sites for miR-142-5p and miR-155
(miR-155-3p or miR-155-5p), miR-142-5p and miR-146 (miR-146-3 or
miR-146-5p), or miR-142-5p and miR-126 (miR-126-3p or
miR-126-5p).
[0752] In yet another embodiment, the polynucleotide of the
invention comprises at least two miR binding sites for microRNAs
expressed in immune cells, wherein one of the miR binding sites is
for miR-155-5p. In various embodiments, the polynucleotide of the
invention comprises binding sites for miR-155-5p and miR-142
(miR-142-3p or miR-142-5p), miR-155-5p and miR-146 (miR-146-3 or
miR-146-5p), or miR-155-5p and miR-126 (miR-126-3p or
miR-126-5p).
[0753] miRNA can also regulate complex biological processes such as
angiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol
2011 18:171-176). In the polynucleotides of the invention, miRNA
binding sites that are involved in such processes can be removed or
introduced, in order to tailor the expression of the
polynucleotides to biologically relevant cell types or relevant
biological processes. In this context, the polynucleotides of the
invention are defined as auxotrophic polynucleotides.
[0754] In some embodiments, a polynucleotide of the invention
comprises a miRNA binding site, wherein the miRNA binding site
comprises one or more nucleotide sequences selected from TABLE 3,
including one or more copies of any one or more of the miRNA
binding site sequences. In some embodiments, a polynucleotide of
the invention further comprises at least one, two, three, four,
five, six, seven, eight, nine, ten, or more of the same or
different miRNA binding sites selected from TABLE 3, including any
combination thereof.
[0755] In some embodiments, the miRNA binding site binds to miR-142
or is complementary to miR-142. In some embodiments, the miR-142
comprises SEQ ID NO:34. In some embodiments, the miRNA binding site
binds to miR-142-3p or miR-142-5p. In some embodiments, the
miR-142-3p binding site comprises SEQ ID NO:35. In some
embodiments, the miR-142-5p binding site comprises SEQ ID NO:37. In
some embodiments, the miRNA binding site comprises a nucleotide
sequence at least 80%, at least 85%, at least 90%, at least 95%, or
100% identical to SEQ ID NO:36 or SEQ ID NO:38.
[0756] In some embodiments, the miRNA binding site binds to miR-126
or is complementary to miR-126. In some embodiments, the miR-126
comprises SEQ ID NO: 156. In some embodiments, the miRNA binding
site binds to miR-126-3p or miR-126-5p. In some embodiments, the
miR-126-3p binding site comprises SEQ ID NO: 158. In some
embodiments, the miR-126-5p binding site comprises SEQ ID NO: 160.
In some embodiments, the miRNA binding site comprises a nucleotide
sequence at least 80%, at least 85%, at least 90%, at least 95%, or
100% identical to SEQ ID NO: 158 or SEQ ID NO: 160.
[0757] In one embodiment, the 3' UTR comprises two miRNA binding
sites, wherein a first miRNA binding site binds to miR-142 and a
second miRNA binding site binds to miR-126. In a specific
embodiment, the 3' UTR binding to miR-142 and miR-126 comprises,
consists, or consists essentially of the sequence of SEQ ID NO: 149
or 150.
TABLE-US-00005 TABLE 3 miR-142, miR-126, and miR-142 and miR-126
binding sites SEQ ID NO. Description Sequence 34 miR-142
GACAGUGCAGUCACCCAUAAAGUAGAAAGC ACUACUAACAGCACUGGAGGGUGUAGUGUU
UCCUACUUUAUGGAUGAGUGUACUGUG 35 miR-142- UGUAGUGUUUCCUACUUUAUGGA 3p
36 miR-142- UCCAUAAAGUAGGAAACACUACA 3p binding site 37 miR-142-
CAUAAAGUAGAAAGCACUACU 5p 38 miR-142- AGUAGUGCUUUCUACUUUAUG 5p
binding site 156 miR-126 CGCUGGCGACGGGACAUUAUUACUUUUGGU
ACGCGCUGUGACACUUCAAACUCGUACCGU GAGUAAUAAUGCGCCGUCCACGGCA 157
miR-126- UCGUACCGUGAGUAAUAAUGCG 3p 158 miR-126-
CGCAUUAUUACUCACGGUACGA 3p binding site 159 miR-126-
CAUUAUUACUUUUGGUACGCG 5p 160 miR-126- CGCGUACCAAAAGUAAUAAUG 5p
binding site
[0758] In some embodiments, a miRNA binding site is inserted in the
polynucleotide of the invention in any position of the
polynucleotide (e.g., the 5'UTR and/or 3'UTR). In some embodiments,
the 5'UTR comprises a miRNA binding site. In some embodiments, the
3'UTR comprises a miRNA binding site. In some embodiments, the
5'UTR and the 3'UTR comprise a miRNA binding site. The insertion
site in the polynucleotide can be anywhere in the polynucleotide as
long as the insertion of the miRNA binding site in the
polynucleotide does not interfere with the translation of a
functional polypeptide in the absence of the corresponding miRNA;
and in the presence of the miRNA, the insertion of the miRNA
binding site in the polynucleotide and the binding of the miRNA
binding site to the corresponding miRNA are capable of degrading
the polynucleotide or preventing the translation of the
polynucleotide.
[0759] In some embodiments, a miRNA binding site is inserted in at
least about 30 nucleotides downstream from the stop codon of an ORF
in a polynucleotide of the invention comprising the ORF. In some
embodiments, a miRNA binding site is inserted in at least about 10
nucleotides, at least about 15 nucleotides, at least about 20
nucleotides, at least about 25 nucleotides, at least about 30
nucleotides, at least about 35 nucleotides, at least about 40
nucleotides, at least about 45 nucleotides, at least about 50
nucleotides, at least about 55 nucleotides, at least about 60
nucleotides, at least about 65 nucleotides, at least about 70
nucleotides, at least about 75 nucleotides, at least about 80
nucleotides, at least about 85 nucleotides, at least about 90
nucleotides, at least about 95 nucleotides, or at least about 100
nucleotides downstream from the stop codon of an ORF in a
polynucleotide of the invention. In some embodiments, a miRNA
binding site is inserted in about 10 nucleotides to about 100
nucleotides, about 20 nucleotides to about 90 nucleotides, about 30
nucleotides to about 80 nucleotides, about 40 nucleotides to about
70 nucleotides, about 50 nucleotides to about 60 nucleotides, about
45 nucleotides to about 65 nucleotides downstream from the stop
codon of an ORF in a polynucleotide of the invention.
[0760] In some embodiments, a miRNA binding site is inserted within
the 3' UTR immediately following the stop codon of the coding
region within the polynucleotide of the invention, e.g., mRNA. In
some embodiments, if there are multiple copies of a stop codon in
the construct, a miRNA binding site is inserted immediately
following the final stop codon. in some embodiments, a miRNA
binding site is inserted further downstream of the stop codon, in
which case there are 3' UTR bases between the stop codon and the
miR binding site(s). In some embodiments, three non-limiting
examples of possible insertion sites for a miR in a 3' UTR are
shown in SEQ ID NOs: 57, 58, and 172, which show a 3' UTR sequence
with a miR-142-3p site inserted in one of three different possible
insertion sites, respectively, within the 3' UTR.
[0761] In some embodiments, one or more miRNA binding sites can be
positioned within the 5' UTR at one or more possible insertion
sites. For example, three non-limiting examples of possible
insertion sites for a miR in a 5' UTR are shown in SEQ ID NOs: 189,
190, and 191, which show a 5' UTR sequence with a miR-142-3p site
inserted into one of three different possible insertion sites,
respectively, within the 5' UTR.
[0762] In one embodiment, a codon optimized open reading frame
encoding a polypeptide of interest comprises a stop codon and the
at least one microRNA binding site is located within the 3' UTR
1-100 nucleotides after the stop codon. In one embodiment, the
codon optimized open reading frame encoding the polypeptide of
interest comprises a stop codon and the at least one microRNA
binding site for a miR expressed in immune cells is located within
the 3' UTR 30-50 nucleotides after the stop codon. In another
embodiment, the codon optimized open reading frame encoding the
polypeptide of interest comprises a stop codon and the at least one
microRNA binding site for a miR expressed in immune cells is
located within the 3' UTR at least 50 nucleotides after the stop
codon. In other embodiments, the codon optimized open reading frame
encoding the polypeptide of interest comprises a stop codon and the
at least one microRNA binding site for a miR expressed in immune
cells is located within the 3' UTR immediately after the stop
codon, or within the 3' UTR 15-20 nucleotides after the stop codon
or within the 3' UTR 70-80 nucleotides after the stop codon. In
other embodiments, the 3'UTR comprises more than one miRNA
bindingsite (e.g., 2-4 miRNA binding sites), wherein there can be a
spacer region (e.g., of 10-100, 20-70 or 30-50 nucleotides in
length) between each miRNA bindingsite. In another embodiment, the
3' UTR comprises a spacer region between the end of the miRNA
bindingsite(s) and the poly A tail nucleotides. For example, a
spacer region of 10-100, 20-70 or 30-50 nucleotides in length can
be situated between the end of the miRNA bindingsite(s) and the
beginning of the poly A tail.
[0763] In one embodiment, a codon optimized open reading frame
encoding a polypeptide of interest comprises a start codon and the
at least one microRNA binding site is located within the 5' UTR
1-100 nucleotides before (upstream of) the start codon. In one
embodiment, the codon optimized open reading frame encoding the
polypeptide of interest comprises a start codon and the at least
one microRNA binding site for a miR expressed in immune cells is
located within the 5' UTR 10-50 nucleotides before (upstream of)
the start codon. In another embodiment, the codon optimized open
reading frame encoding the polypeptide of interest comprises a
start codon and the at least one microRNA binding site for a miR
expressed in immune cells is located within the 5' UTR at least 25
nucleotides before (upstream of) the start codon. In other
embodiments, the codon optimized open reading frame encoding the
polypeptide of interest comprises a start codon and the at least
one microRNA binding site for a miR expressed in immune cells is
located within the 5' UTR immediately before the start codon, or
within the 5' UTR 15-20 nucleotides before the start codon or
within the 5' UTR 70-80 nucleotides before the start codon. In
other embodiments, the 5'UTR comprises more than one miRNA
bindingsite (e.g., 2-4 miRNA binding sites), wherein there can be a
spacer region (e.g., of 10-100, 20-70 or 30-50 nucleotides in
length) between each miRNA bindingsite.
[0764] In one embodiment, the 3' UTR comprises more than one stop
codon, wherein at least one miRNA bindingsite is positioned
downstream of the stop codons. For example, a 3' UTR can comprise
1, 2 or 3 stop codons. Non-limiting examples of triple stop codons
that can be used include: UGAUAAUAG, UGAUAGUAA, UAAUGAUAG,
UGAUAAUAA, UGAUAGUAG, UAAUGAUGA, UAAUAGUAG, UGAUGAUGA, UAAUAAUAA
and UAGUAGUAG. Within a 3' UTR, for example, 1, 2, 3 or 4 miRNA
binding sites, e.g., miR-142-3p binding sites, can be positioned
immediately adjacent to the stop codon(s) or at any number of
nucleotides downstream of the final stop codon. When the 3' UTR
comprises multiple miRNA binding sites, these binding sites can be
positioned directly next to each other in the construct (i.e., one
after the other) or, alternatively, spacer nucleotides can be
positioned between each binding site.
[0765] In one embodiment, the 3' UTR comprises three stop codons
with a single miR-142-3p binding site located downstream of the 3rd
stop codon. Non-limiting examples of sequences of 3' UTR having
three stop codons and a single miR-142-3p binding site located at
different positions downstream of the final stop codon are shown in
SEQ ID NOs: 57, 58, 62, and 172.
TABLE-US-00006 TABLE 4 3'UTRs, miR sequences, and miR binding sites
SEQ ID NO: Sequence 34
GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGU
GUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (miR-142) 35
UGUAGUGUUUCCUACUUUAUGGA (miR 142-3p sequence) 36
UCCAUAAAGUAGGAAACACUACA (miR 142-3p binding site) 37
CAUAAAGUAGAAAGCACUACU (miR 142-5p sequence) 38
AGUAGUGCUUUCUACUUUAUG (miR-142-5p binding site) 157
UCGUACCGUGAGUAAUAAUGCG (miR 126-3p sequence) 158
CGCAUUAUUACUCACGGUACGA (miR 126-3p binding site) 159
CAUUAUUACUUUUGGUACGCG (miR 126-5p sequence) 173
CCUCUGAAAUUCAGUUCUUCAG (miR 146-3p sequence) 174
UGAGAACUGAAUUCCAUGGGUU (miR 146-5p sequence) 175
CUCCUACAUAUUAGCAUUAACA (miR 155-3p sequence) 176
UUAAUGCUAAUCGUGAUAGGGGU (miR 155-5p sequence) 177
CCAGUAUUAACUGUGCUGCUGA (miR 16-3p sequence) 178
UAGCAGCACGUAAAUAUUGGCG (miR 16-5p sequence) 179
CAACACCAGUCGAUGGGCUGU (miR 21-3p sequence) 180
UAGCUUAUCAGACUGAUGUUGA (miR 21-5p sequence) 181
UGUCAGUUUGUCAAAUACCCCA (miR 223-3p sequence) 182
CGUGUAUUUGACAAGCUGAGUU (miR 223-5p sequence) 183
UGGCUCAGUUCAGCAGGAACAG (miR 24-3p sequence) 184
UGCCUACUGAGCUGAUAUCAGU (miR 24-5p sequence) 185
UUCACAGUGGCUAAGUUCCGC (miR 27-3p sequence) 186
AGGGCUUAGCUGCUUGUGAGCA (miR 27-5p sequence) 187
UUAAUGCUAAUUGUGAUAGGGGU (miR 155-5p sequence) 188 AC
CCCUAUCACAAUUAGCAUUAA (miR 155-5p binding site) 39
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (5' UTR) 189
GGGAAAUAAGAGUCCAUAAAGUAGGAAACACUACAAGAAAAGAAGAGUAAGA
AGAAAUAUAAGAGCCACC (5' UTR with miR142-3p binding site at position
p1) 190 GGGAAAUAAGAGAGAAAAGAAGAGUAAUCCAUAAAGUAGGAAACACUACAGA
AGAAAUAUAAGAGCCACC (5' UTR with miR142-3p binding site at position
p2) 191 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAUCCAUAAAGUAGG
AAACACUACAGAGCCACC (5' UTR with miR142-3p binding site at position
p3) 57 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAU
GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site, P1 insertion) 58
UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACAU
GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site, P2 insertion) 59
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAAAGU
AGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR including miR142-3p
binding site) 60
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR including miR142-3p
binding site) 61
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR including including
miR142-3p binding site) 62
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAA
CACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site) 63
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA
GUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC (3' UTR including including
miR142-3p binding site) 81
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA
GUCUGAGUGGGCGGC (3' UTR, no miR binding sites) 149
UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAU
GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG UACCCCC
GUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC (3' UTR with miR 142-3p and miR
126-3p binding sites) 150
UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUA
GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG UACCCCC
GUGGUCUUUGAAUAAAGUCUGAG UGGGCGGC (3' UTR with miR 142-3p and miR
126-3p binding sites variant 2) 151
UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAA
CACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site variant 2) 161
GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCC
UCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGU
GGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p binding site)
162 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC
GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 126-3p binding
site) 163 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA
GUCUGAGUGGGCGGC (3' UTR, no miR binding sites variant 2) 164
UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC
GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 126-3p binding
site variant 3) 165
UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAU
GCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGC
CCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUAC
AGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with 3 miR 142-3p binding
sites) 166 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC
GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-5p binding
site) 167 UGAUAAUAG GCUGGAGCCUCGGUGGCCAUGC UUCUUGCCCCUUGGGCC
UCCCCCCAGCCCCU CCUCCCCUUCCUGCACCCGUACCCCC GUGGU
CUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with 3 miR 142-5p binding sites)
168 UGAUAAUAG GCUGGAGCCUCGGUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCC
CUCCUCCCCUUCCUGCACCCGUACCCCC GUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC (3'
UTR with 2 miR 142-5p binding sites and 1 miR 142-3p binding site)
169 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUA
GCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 155-5p
binding site) 170
UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCAU
GCUUCUUGCCCCUUGGGCCACCCCUAUCACAAUUAGCAUUAAUCCCCCCAGC
CCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUA
AGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with 3 miR 155-5p binding
sites) 171 UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCAU
GCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGC
CCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUA
AGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with 2 miR 155-5p binding
sites and 1 miR 142-3p binding site) 172
UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCA
UAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site, P3 insertion) 192
UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUA
GCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGC
CCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUAC
AGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with 3 miR 142-3p binding
sites variant 2) 193
UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUA
GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site, P1 insertion variant 2) 194
UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACUA
GCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site, P2 insertion variant 2) 195 UGAUAAUAG
GCUGGAGCCUCGGUGGCCAUGC UUCUUGCCCCUUGGGCC UCCCCCCAGCCCCU
CUCCCCUUCCUGCACCCGUACCCCC GUGGUC UUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR
with 3 miR 142-5p binding sites) 196
UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCA
UAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG
UACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 142-3p
binding site, P3 insertion variant 2) 197
UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCC
CCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUA
GCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with miR 155-5p
binding site variant 2) 198
UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCUA
GCUUCUUGCCCCUUGGGCCACCCCUAUCACAAUUAGCAUUAAUCCCCCCAGC
CCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUA
AGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with 3 miR 155-5p binding
sites variant 2) 199
UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCUA
GCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGC
CCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUA
AGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3' UTR with 2 miR 155-5p binding
sites and 1 miR 142-3p binding site variant 2) Stop codon = bold
miR 142-3p binding site = underline miR 126-3p binding site = bold
underline miR 155-5p binding site = italicized miR 142-5p binding
site = italicized and bold underline
[0766] In one embodiment, the polynucleotide of the invention
comprises a 5' UTR, a codon optimized open reading frame encoding a
polypeptide of interest, a 3' UTR comprising the at least one miRNA
binding site for a miR expressed in immune cells, and a 3' tailing
region of linked nucleosides. In various embodiments, the 3' UTR
comprises 1-4, at least two, one, two, three or four miRNA binding
sites for miRs expressed in immune cells, preferably abundantly or
preferentially expressed in immune cells.
[0767] In one embodiment, the at least one miRNA expressed in
immune cells is a miR-142-3p microRNA binding site. In one
embodiment, the miR-142-3p microRNA binding site comprises the
sequence shown in SEQ ID NO: 36. In one embodiment, the 3' UTR of
the mRNA comprising the miR-142-3p microRNA binding site comprises
the sequence shown in SEQ ID NO: 161.
[0768] In one embodiment, the at least one miRNA expressed in
immune cells is a miR-126 microRNA binding site. In one embodiment,
the miR-126 binding site is a miR-126-3p binding site. In one
embodiment, the miR-126-3p microRNA binding site comprises the
sequence shown in SEQ ID NO: 158. In one embodiment, the 3' UTR of
the mRNA of the invention comprising the miR-126-3p microRNA
binding site comprises the sequence shown in SEQ ID NO: 162.
[0769] Non-limiting exemplary sequences for miRs to which a
microRNA binding site(s) of the disclosure can bind include the
following: miR-142-3p (SEQ ID NO: 35), miR-142-5p (SEQ ID NO: 37),
miR-146-3p (SEQ ID NO: 173), miR-146-5p (SEQ ID NO: 174),
miR-155-3p (SEQ ID NO: 175), miR-155-5p (SEQ ID NO: 176),
miR-126-3p (SEQ ID NO: 157), miR-126-5p (SEQ ID NO: 159), miR-16-3p
(SEQ ID NO: 177), miR-16-5p (SEQ ID NO: 178), miR-21-3p (SEQ ID NO:
179), miR-21-5p (SEQ ID NO: 180), miR-223-3p (SEQ ID NO: 181),
miR-223-5p (SEQ ID NO: 182), miR-24-3p (SEQ ID NO: 183), miR-24-5p
(SEQ ID NO: 184), miR-27-3p (SEQ ID NO: 185) and miR-27-5p (SEQ ID
NO: 186). Other suitable miR sequences expressed in immune cells
(e.g., abundantly or preferentially expressed in immune cells) are
known and available in the art, for example at the University of
Manchester's microRNA database, miRBase. Sites that bind any of the
aforementioned miRs can be designed based on Watson-Crick
complementarity to the miR, typically 100% complementarity to the
miR, and inserted into an mRNA construct of the disclosure as
described herein.
[0770] In another embodiment, a polynucleotide of the present
invention (e.g., and mRNA, e.g., the 3' UTR thereof) can comprise
at least one miRNA bindingsite to thereby reduce or inhibit
accelerated blood clearance, for example by reducing or inhibiting
production of IgMs, e.g., against PEG, by B cells and/or reducing
or inhibiting proliferation and/or activation of pDCs, and can
comprise at least one miRNA bindingsite for modulating tissue
expression of an encoded protein of interest.
[0771] miRNA gene regulation can be influenced by the sequence
surrounding the miRNA such as, but not limited to, the species of
the surrounding sequence, the type of sequence (e.g., heterologous,
homologous, exogenous, endogenous, or artificial), regulatory
elements in the surrounding sequence and/or structural elements in
the surrounding sequence. The miRNA can be influenced by the 5'UTR
and/or 3'UTR. As a non-limiting example, a non-human 3'UTR can
increase the regulatory effect of the miRNA sequence on the
expression of a polypeptide of interest compared to a human 3'UTR
of the same sequence type.
[0772] In one embodiment, other regulatory elements and/or
structural elements of the 5'UTR can influence miRNA mediated gene
regulation. One example of a regulatory element and/or structural
element is a structured IRES (Internal Ribosome Entry Site) in the
5'UTR, which is necessary for the binding of translational
elongation factors to initiate protein translation. EIF4A.sub.2
binding to this secondarily structured element in the 5'-UTR is
necessary for miRNA mediated gene expression (Meijer H A et al.,
Science, 2013, 340, 82-85, herein incorporated by reference in its
entirety). The polynucleotides of the invention can further include
this structured 5'UTR in order to enhance microRNA mediated gene
regulation.
[0773] At least one miRNA binding site can be engineered into the
3'UTR of a polynucleotide of the invention. In this context, at
least two, at least three, at least four, at least five, at least
six, at least seven, at least eight, at least nine, at least ten,
or more miRNA binding sites can be engineered into a 3'UTR of a
polynucleotide of the invention. For example, 1 to 10, 1 to 9, 1 to
8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding
sites can be engineered into the 3'UTR of a polynucleotide of the
invention. In one embodiment, miRNA binding sites incorporated into
a polynucleotide of the invention can be the same or can be
different miRNA sites. A combination of different miRNA binding
sites incorporated into a polynucleotide of the invention can
include combinations in which more than one copy of any of the
different miRNA sites are incorporated. In another embodiment,
miRNA binding sites incorporated into a polynucleotide of the
invention can target the same or different tissues in the body. As
a non-limiting example, through the introduction of tissue-,
cell-type-, or disease-specific miRNA binding sites in the 3'-UTR
of a polynucleotide of the invention, the degree of expression in
specific cell types (e.g., myeloid cells, endothelial cells, etc.)
can be reduced.
[0774] In one embodiment, a miRNA binding site can be engineered
near the 5' terminus of the 3'UTR, about halfway between the 5'
terminus and 3' terminus of the 3'UTR and/or near the 3' terminus
of the 3'UTR in a polynucleotide of the invention. As a
non-limiting example, a miRNA binding site can be engineered near
the 5' terminus of the 3'UTR and about halfway between the 5'
terminus and 3' terminus of the 3'UTR. As another non-limiting
example, a miRNA binding site can be engineered near the 3'
terminus of the 3'UTR and about halfway between the 5' terminus and
3' terminus of the 3'UTR. As yet another non-limiting example, a
miRNA binding site can be engineered near the 5' terminus of the
3'UTR and near the 3' terminus of the 3'UTR.
[0775] In another embodiment, a 3'UTR can comprise 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 miRNA binding sites. The miRNA binding sites can
be complementary to a miRNA, miRNA seed sequence, and/or miRNA
sequences flanking the seed sequence.
[0776] In some embodiments, the expression of a polynucleotide of
the invention can be controlled by incorporating at least one
sensor sequence in the polynucleotide and formulating the
polynucleotide for administration. As a non-limiting example, a
polynucleotide of the invention can be targeted to a tissue or cell
by incorporating a miRNA binding site and formulating the
polynucleotide in a lipid nanoparticle comprising a ionizable
lipid, including any of the lipids described herein.
[0777] A polynucleotide of the invention can be engineered for more
targeted expression in specific tissues, cell types, or biological
conditions based on the expression patterns of miRNAs in the
different tissues, cell types, or biological conditions. Through
introduction of tissue-specific miRNA binding sites, a
polynucleotide of the invention can be designed for optimal protein
expression in a tissue or cell, or in the context of a biological
condition.
[0778] In some embodiments, a polynucleotide of the invention can
be designed to incorporate miRNA binding sites that either have
100% identity to known miRNA seed sequences or have less than 100%
identity to miRNA seed sequences. In some embodiments, a
polynucleotide of the invention can be designed to incorporate
miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed
sequences. The miRNA seed sequence can be partially mutated to
decrease miRNA binding affinity and as such result in reduced
downmodulation of the polynucleotide. In essence, the degree of
match or mis-match between the miRNA binding site and the miRNA
seed can act as a rheostat to more finely tune the ability of the
miRNA to modulate protein expression. In addition, mutation in the
non-seed region of a miRNA binding site can also impact the ability
of a miRNA to modulate protein expression.
[0779] In one embodiment, a miRNA sequence can be incorporated into
the loop of a stem loop.
[0780] In another embodiment, a miRNA seed sequence can be
incorporated in the loop of a stem loop and a miRNA binding site
can be incorporated into the 5' or 3' stem of the stem loop.
[0781] In one embodiment, a translation enhancer element (TEE) can
be incorporated on the 5'end of the stem of a stem loop and a miRNA
seed can be incorporated into the stem of the stem loop. In another
embodiment, a TEE can be incorporated on the 5' end of the stem of
a stem loop, a miRNA seed can be incorporated into the stem of the
stem loop and a miRNA binding site can be incorporated into the 3'
end of the stem or the sequence after the stem loop. The miRNA seed
and the miRNA binding site can be for the same and/or different
miRNA sequences.
[0782] In one embodiment, the incorporation of a miRNA sequence
and/or a TEE sequence changes the shape of the stem loop region
which can increase and/or decrease translation. (see e.g, Kedde et
al., "A Pumilio-induced RNA structure switch in p27-3'UTR controls
miR-221 and miR-22 accessibility." Nature Cell Biology. 2010,
incorporated herein by reference in its entirety).
[0783] In one embodiment, the 5'-UTR of a polynucleotide of the
invention can comprise at least one miRNA sequence. The miRNA
sequence can be, but is not limited to, a 19 or 22 nucleotide
sequence and/or a miRNA sequence without the seed.
[0784] In one embodiment the miRNA sequence in the 5'UTR can be
used to stabilize a polynucleotide of the invention described
herein.
[0785] In another embodiment, a miRNA sequence in the 5'UTR of a
polynucleotide of the invention can be used to decrease the
accessibility of the site of translation initiation such as, but
not limited to a start codon. See, e.g., Matsuda et al., PLoS One.
2010 11(5):e15057; incorporated herein by reference in its
entirety, which used antisense locked nucleic acid (LNA)
oligonucleotides and exon-junction complexes (EJCs) around a start
codon (-4 to +37 where the A of the AUG codons is +1) in order to
decrease the accessibility to the first start codon (AUG). Matsuda
showed that altering the sequence around the start codon with an
LNA or EJC affected the efficiency, length and structural stability
of a polynucleotide. A polynucleotide of the invention can comprise
a miRNA sequence, instead of the LNA or EJC sequence described by
Matsuda et al, near the site of translation initiation in order to
decrease the accessibility to the site of translation initiation.
The site of translation initiation can be prior to, after or within
the miRNA sequence. As a non-limiting example, the site of
translation initiation can be located within a miRNA sequence such
as a seed sequence or binding site.
[0786] In some embodiments, a polynucleotide of the invention can
include at least one miRNA in order to dampen the antigen
presentation by antigen presenting cells. The miRNA can be the
complete miRNA sequence, the miRNA seed sequence, the miRNA
sequence without the seed, or a combination thereof. As a
non-limiting example, a miRNA incorporated into a polynucleotide of
the invention can be specific to the hematopoietic system. As
another non-limiting example, a miRNA incorporated into a
polynucleotide of the invention to dampen antigen presentation is
miR-142-3p.
[0787] In some embodiments, a polynucleotide of the invention can
include at least one miRNA in order to dampen expression of the
encoded polypeptide in a tissue or cell of interest. As a
non-limiting example a polynucleotide of the invention can include
at least one miR-142-3p binding site, miR-142-3p seed sequence,
miR-142-3p binding site without the seed, miR-142-5p binding site,
miR-142-5p seed sequence, miR-142-5p binding site without the seed,
miR-146 binding site, miR-146 seed sequence and/or miR-146 binding
site without the seed sequence.
[0788] In some embodiments, a polynucleotide of the invention can
comprise at least one miRNA binding site in the 3'UTR in order to
selectively degrade mRNA therapeutics in the immune cells to subdue
unwanted immunogenic reactions caused by therapeutic delivery. As a
non-limiting example, the miRNA binding site can make a
polynucleotide of the invention more unstable in antigen presenting
cells. Non-limiting examples of these miRNAs include mir-142-5p,
mir-142-3p, mir-146a-5p, and mir-146-3p.
[0789] In one embodiment, a polynucleotide of the invention
comprises at least one miRNA sequence in a region of the
polynucleotide that can interact with a RNA binding protein.
[0790] In some embodiments, the polynucleotide of the invention
(e.g., a RNA, e.g., an mRNA) comprising (i) a sequence-optimized
nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide
(e.g., the wild-type sequence, functional fragment, or variant
thereof) and (ii) a miRNA binding site (e.g., a miRNA binding site
that binds to miR-142).
[0791] In some embodiments, the polynucleotide of the invention
comprises a uracil-modified sequence encoding a PBGD polypeptide
disclosed herein and a miRNA binding site disclosed herein, e.g., a
miRNA binding site that binds to miR-142 and/or a miRNA binding
site that binds to miR-126. In some embodiments, the polynucleotide
of the invention comprises a uracil-modified sequence encoding a
polypeptide disclosed herein and a miRNA binding site disclosed
herein, e.g., a miRNA binding site that binds to miR-142miR-126,
miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21,
miR-223, miR-24, miR-27 or miR-26a. In some embodiments, the miRNA
binding site binds to miR126-3p, miR-142-3p, miR-142-5p, or
miR-155. In some embodiments, the polynucleotide of the invention
comprises a uracil-modified sequence encoding a polypeptide
disclosed herein and at least two different microRNA binding sites,
wherein the microRNA is expressed in an immune cell of
hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and
secretes pro-inflammatory cytokines and/or chemokines, and wherein
the polynucleotide comprises one or more modified nucleobases. In
some embodiments, the uracil-modified sequence encoding a PBGD
polypeptide comprises at least one chemically modified nucleobase,
e.g., 5-methoxyuracil. In some embodiments, at least 95% of a type
of nucleobase (e.g., uracil) in a uracil-modified sequence encoding
a PBGD polypeptide of the invention are modified nucleobases. In
some embodiments, at least 95% of uricil in a uracil-modified
sequence encoding a PBGD polypeptide is 5-methoxyuridine. In some
embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA)
disclosed herein, e.g., comprising an miRNA binding site, is
formulated with a delivery agent comprising, e.g., a compound
having the Formula (I), e.g., any of Compounds 1-232, e.g.,
Compound 18; a compound having the Formula (III), (IV), (V), or
(VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a
compound having the Formula (VIII), e.g., any of Compounds 419-428,
e.g., Compound 428, or any combination thereof. In some
embodiments, the delivery agent comprises Compound 18, DSPC,
Cholesterol, and Compound 428, e.g., with a mole ratio of about
50:10:38.5:1.5.
13. 3' UTRs
[0792] In certain embodiments, a polynucleotide of the present
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide of the invention) further comprises a
3' UTR.
[0793] 3'-UTR is the section of mRNA that immediately follows the
translation termination codon and often contains regulatory regions
that post-transcriptionally influence gene expression. Regulatory
regions within the 3'-UTR can influence polyadenylation,
translation efficiency, localization, and stability of the mRNA. In
one embodiment, the 3'-UTR useful for the invention comprises a
binding site for regulatory proteins or microRNAs.
[0794] In certain embodiments, the 3' UTR useful for the
polynucleotides of the invention comprises a 3'UTR selected from
the group consisting of SEQ ID NO: 57 to 81, 84, 149 to 151, 161 to
172, 192 to 199, or any combination thereof. In some embodiments,
the 3' UTR comprises a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 149 to 151, or any combination
thereof. In some embodiments, the 3' UTR comprises a nucleic acid
sequence of SEQ ID NO: 150. In some embodiments, the 3' UTR
comprises a nucleic acid sequence of SEQ ID NO: 151.
[0795] In certain embodiments, the 3' UTR sequence useful for the
invention comprises a nucleotide sequence at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, or about 100% identical to a sequence
selected from the group consisting of SEQ ID NO: 57 to 81, 84, 149
to 151, 161 to 172, 192 to 199, or any combination thereof.
14. REGIONS HAVING A 5' CAP
[0796] The invention also includes a polynucleotide that comprises
both a 5' Cap and a polynucleotide of the present invention (e.g.,
a polynucleotide comprising a nucleotide sequence encoding a PBGD
polypeptide).
[0797] The 5' cap structure of a natural mRNA is involved in
nuclear export, increasing mRNA stability and binds the mRNA Cap
Binding Protein (CBP), which is responsible for mRNA stability in
the cell and translation competency through the association of CBP
with poly(A) binding protein to form the mature cyclic mRNA
species. The cap further assists the removal of 5' proximal introns
during mRNA splicing.
[0798] Endogenous mRNA molecules can be 5'-end capped generating a
5'-ppp-5'-triphosphate linkage between a terminal guanosine cap
residue and the 5'-terminal transcribed sense nucleotide of the
mRNA molecule. This 5'-guanylate cap can then be methylated to
generate an N7-methyl-guanylate residue. The ribose sugars of the
terminal and/or anteterminal transcribed nucleotides of the 5' end
of the mRNA can optionally also be 2'-O-methylated. 5'-decapping
through hydrolysis and cleavage of the guanylate cap structure can
target a nucleic acid molecule, such as an mRNA molecule, for
degradation.
[0799] In some embodiments, the polynucleotides of the present
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide) incorporate a cap moiety.
[0800] In some embodiments, polynucleotides of the present
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide) comprise a non-hydrolyzable cap
structure preventing decapping and thus increasing mRNA half-life.
Because cap structure hydrolysis requires cleavage of 5'-ppp-5'
phosphorodiester linkages, modified nucleotides can be used during
the capping reaction. For example, a Vaccinia Capping Enzyme from
New England Biolabs (Ipswich, Mass.) can be used with
.alpha.-thio-guanosine nucleotides according to the manufacturer's
instructions to create a phosphorothioate linkage in the 5'-ppp-5'
cap. Additional modified guanosine nucleotides can be used such as
a-methyl-phosphonate and seleno-phosphate nucleotides.
[0801] Additional modifications include, but are not limited to,
2'-O-methylation of the ribose sugars of 5'-terminal and/or
5'-anteterminal nucleotides of the polynucleotide (as mentioned
above) on the 2'-hydroxyl group of the sugar ring. Multiple
distinct 5'-cap structures can be used to generate the 5'-cap of a
nucleic acid molecule, such as a polynucleotide that functions as
an mRNA molecule. Cap analogs, which herein are also referred to as
synthetic cap analogs, chemical caps, chemical cap analogs, or
structural or functional cap analogs, differ from natural (i.e.,
endogenous, wild-type or physiological) 5'-caps in their chemical
structure, while retaining cap function. Cap analogs can be
chemically (i.e., non-enzymatically) or enzymatically synthesized
and/or linked to the polynucleotides of the invention.
[0802] For example, the Anti-Reverse Cap Analog (ARCA) cap contains
two guanines linked by a 5'-5'-triphosphate group, wherein one
guanine contains an N7 methyl group as well as a 3'-O-methyl group
(i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine
(m.sup.7G-3'mppp-G; which can equivalently be designated 3'
O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified,
guanine becomes linked to the 5'-terminal nucleotide of the capped
polynucleotide. The N7- and 3'-O-methylated guanine provides the
terminal moiety of the capped polynucleotide.
[0803] Another exemplary cap is mCAP, which is similar to ARCA but
has a 2'-O-methyl group on guanosine (i.e.,
N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine,
m.sup.7Gm-ppp-G).
[0804] In some embodiments, the cap is a dinucleotide cap analog.
As a non-limiting example, the dinucleotide cap analog can be
modified at different phosphate positions with a boranophosphate
group or a phosphoroselenoate group such as the dinucleotide cap
analogs described in U.S. Pat. No. 8,519,110, the contents of which
are herein incorporated by reference in its entirety.
[0805] In another embodiment, the cap is a cap analog is a
N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap
analog known in the art and/or described herein. Non-limiting
examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide
form of a cap analog include a
N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and a
N7-(4-chlorophenoxyethyl)-m.sup.3'-OG(5')ppp(5')G cap analog (See,
e.g., the various cap analogs and the methods of synthesizing cap
analogs described in Kore et al. Bioorganic & Medicinal
Chemistry 2013 21:4570-4574; the contents of which are herein
incorporated by reference in its entirety). In another embodiment,
a cap analog of the present invention is a
4-chloro/bromophenoxyethyl analog.
[0806] While cap analogs allow for the concomitant capping of a
polynucleotide or a region thereof, in an in vitro transcription
reaction, up to 20% of transcripts can remain uncapped. This, as
well as the structural differences of a cap analog from an
endogenous 5'-cap structures of nucleic acids produced by the
endogenous, cellular transcription machinery, can lead to reduced
translational competency and reduced cellular stability.
[0807] Polynucleotides of the invention (e.g., a polynucleotide
comprising a nucleotide sequence encoding a PBGD polypeptide) can
also be capped post-manufacture (whether IVT or chemical
synthesis), using enzymes, in order to generate more authentic
5'-cap structures. As used herein, the phrase "more authentic"
refers to a feature that closely mirrors or mimics, either
structurally or functionally, an endogenous or wild type feature.
That is, a "more authentic" feature is better representative of an
endogenous, wild-type, natural or physiological cellular function
and/or structure as compared to synthetic features or analogs,
etc., of the prior art, or which outperforms the corresponding
endogenous, wild-type, natural or physiological feature in one or
more respects. Non-limiting examples of more authentic 5'cap
structures of the present invention are those that, among other
things, have enhanced binding of cap binding proteins, increased
half-life, reduced susceptibility to 5' endonucleases and/or
reduced 5'decapping, as compared to synthetic 5'cap structures
known in the art (or to a wild-type, natural or physiological 5'cap
structure). For example, recombinant Vaccinia Virus Capping Enzyme
and recombinant 2'-O-methyltransferase enzyme can create a
canonical 5'-5'-triphosphate linkage between the 5'-terminal
nucleotide of a polynucleotide and a guanine cap nucleotide wherein
the cap guanine contains an N7 methylation and the 5'-terminal
nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is
termed the Cap1 structure. This cap results in a higher
translational-competency and cellular stability and a reduced
activation of cellular pro-inflammatory cytokines, as compared,
e.g., to other 5'cap analog structures known in the art. Cap
structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p
(cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-ppp(5')NlmpN2mp
(cap 2).
[0808] As a non-limiting example, capping chimeric polynucleotides
post-manufacture can be more efficient as nearly 100% of the
chimeric polynucleotides can be capped. This is in contrast to
.about.80% when a cap analog is linked to a chimeric polynucleotide
in the course of an in vitro transcription reaction.
[0809] According to the present invention, 5' terminal caps can
include endogenous caps or cap analogs. According to the present
invention, a 5' terminal cap can comprise a guanine analog. Useful
guanine analogs include, but are not limited to, inosine,
N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine,
8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and
2-azido-guanosine.
15. POLY-A TAILS
[0810] In some embodiments, the polynucleotides of the present
disclosure (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide) further comprise a poly-A tail. In
further embodiments, terminal groups on the poly-A tail can be
incorporated for stabilization. In other embodiments, a poly-A tail
comprises des-3' hydroxyl tails.
[0811] During RNA processing, a long chain of adenine nucleotides
(poly-A tail) can be added to a polynucleotide such as an mRNA
molecule in order to increase stability. Immediately after
transcription, the 3' end of the transcript can be cleaved to free
a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine
nucleotides to the RNA. The process, called polyadenylation, adds a
poly-A tail that can be between, for example, approximately 80 to
approximately 250 residues long, including approximately 80, 90,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
230, 240 or 250 residues long.
[0812] PolyA tails can also be added after the construct is
exported from the nucleus.
[0813] According to the present invention, terminal groups on the
poly A tail can be incorporated for stabilization. Polynucleotides
of the present invention can include des-3' hydroxyl tails. They
can also include structural moieties or 2'-Omethyl modifications as
taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507,
Aug. 23, 2005, the contents of which are incorporated herein by
reference in its entirety).
[0814] The polynucleotides of the present invention can be designed
to encode transcripts with alternative polyA tail structures
including histone mRNA. According to Norbury, "Terminal uridylation
has also been detected on human replication-dependent histone
mRNAs. The turnover of these mRNAs is thought to be important for
the prevention of potentially toxic histone accumulation following
the completion or inhibition of chromosomal DNA replication. These
mRNAs are distinguished by their lack of a 3' poly(A) tail, the
function of which is instead assumed by a stable stem-loop
structure and its cognate stem-loop binding protein (SLBP); the
latter carries out the same functions as those of PABP on
polyadenylated mRNAs" (Norbury, "Cytoplasmic RNA: a case of the
tail wagging the dog," Nature Reviews Molecular Cell Biology; AOP,
published online 29 Aug. 2013; doi: 10.1038/nrm3645) the contents
of which are incorporated herein by reference in its entirety.
[0815] Unique poly-A tail lengths provide certain advantages to the
polynucleotides of the present invention. Generally, the length of
a poly-A tail, when present, is greater than 30 nucleotides in
length. In another embodiment, the poly-A tail is greater than 35
nucleotides in length (e.g., at least or greater than about 35, 40,
45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300,
350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,
1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000
nucleotides).
[0816] In some embodiments, the polynucleotide or region thereof
includes from about 30 to about 3,000 nucleotides (e.g., from 30 to
50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750,
from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to
2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to
750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50
to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from
100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to
2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from
500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to
3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to
2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to
2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to
2,500, and from 2,500 to 3,000).
[0817] In some embodiments, the poly-A tail is designed relative to
the length of the overall polynucleotide or the length of a
particular region of the polynucleotide. This design can be based
on the length of a coding region, the length of a particular
feature or region or based on the length of the ultimate product
expressed from the polynucleotides.
[0818] In this context, the poly-A tail can be 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100% greater in length than the polynucleotide
or feature thereof. The poly-A tail can also be designed as a
fraction of the polynucleotides to which it belongs. In this
context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or
90% or more of the total length of the construct, a construct
region or the total length of the construct minus the poly-A tail.
Further, engineered binding sites and conjugation of
polynucleotides for Poly-A binding protein can enhance
expression.
[0819] Additionally, multiple distinct polynucleotides can be
linked together via the PABP (Poly-A binding protein) through the
3'-end using modified nucleotides at the 3'-terminus of the poly-A
tail. Transfection experiments can be conducted in relevant cell
lines at and protein production can be assayed by ELISA at 12 hr,
24 hr, 48 hr, 72 hr and day 7 post-transfection.
[0820] In some embodiments, the polynucleotides of the present
invention are designed to include a polyA-G Quartet region. The
G-quartet is a cyclic hydrogen bonded array of four guanine
nucleotides that can be formed by G-rich sequences in both DNA and
RNA. In this embodiment, the G-quartet is incorporated at the end
of the poly-A tail. The resultant polynucleotide is assayed for
stability, protein production and other parameters including
half-life at various time points. It has been discovered that the
polyA-G quartet results in protein production from an mRNA
equivalent to at least 75% of that seen using a poly-A tail of 120
nucleotides alone.
16. START CODON REGION
[0821] The invention also includes a polynucleotide that comprises
both a start codon region and the polynucleotide described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide). In some embodiments, the polynucleotides of the
present invention can have regions that are analogous to or
function like a start codon region.
[0822] In some embodiments, the translation of a polynucleotide can
initiate on a codon that is not the start codon AUG. Translation of
the polynucleotide can initiate on an alternative start codon such
as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA,
ATT/AUU, TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003)
169-178 and Matsuda and Mauro PLoS ONE, 2010 5:11; the contents of
each of which are herein incorporated by reference in its
entirety).
[0823] As a non-limiting example, the translation of a
polynucleotide begins on the alternative start codon ACG. As
another non-limiting example, polynucleotide translation begins on
the alternative start codon CTG or CUG. As yet another non-limiting
example, the translation of a polynucleotide begins on the
alternative start codon GTG or GUG.
[0824] Nucleotides flanking a codon that initiates translation such
as, but not limited to, a start codon or an alternative start
codon, are known to affect the translation efficiency, the length
and/or the structure of the polynucleotide. (See, e.g., Matsuda and
Mauro PLoS ONE, 2010 5:11; the contents of which are herein
incorporated by reference in its entirety). Masking any of the
nucleotides flanking a codon that initiates translation can be used
to alter the position of translation initiation, translation
efficiency, length and/or structure of a polynucleotide.
[0825] In some embodiments, a masking agent can be used near the
start codon or alternative start codon in order to mask or hide the
codon to reduce the probability of translation initiation at the
masked start codon or alternative start codon. Non-limiting
examples of masking agents include antisense locked nucleic acids
(LNA) polynucleotides and exon-junction complexes (EJCs) (See,
e.g., Matsuda and Mauro describing masking agents LNA
polynucleotides and EJCs (PLoS ONE, 2010 5:11); the contents of
which are herein incorporated by reference in its entirety).
[0826] In another embodiment, a masking agent can be used to mask a
start codon of a polynucleotide in order to increase the likelihood
that translation will initiate on an alternative start codon. In
some embodiments, a masking agent can be used to mask a first start
codon or alternative start codon in order to increase the chance
that translation will initiate on a start codon or alternative
start codon downstream to the masked start codon or alternative
start codon.
[0827] In some embodiments, a start codon or alternative start
codon can be located within a perfect complement for a miRNA
binding site. The perfect complement of a miRNA binding site can
help control the translation, length and/or structure of the
polynucleotide similar to a masking agent. As a non-limiting
example, the start codon or alternative start codon can be located
in the middle of a perfect complement for a miRNA binding site. The
start codon or alternative start codon can be located after the
first nucleotide, second nucleotide, third nucleotide, fourth
nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide,
eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh
nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth
nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth
nucleotide, eighteenth nucleotide, nineteenth nucleotide, twentieth
nucleotide or twenty-first nucleotide.
[0828] In another embodiment, the start codon of a polynucleotide
can be removed from the polynucleotide sequence in order to have
the translation of the polynucleotide begin on a codon that is not
the start codon. Translation of the polynucleotide can begin on the
codon following the removed start codon or on a downstream start
codon or an alternative start codon. In a non-limiting example, the
start codon ATG or AUG is removed as the first 3 nucleotides of the
polynucleotide sequence in order to have translation initiate on a
downstream start codon or alternative start codon. The
polynucleotide sequence where the start codon was removed can
further comprise at least one masking agent for the downstream
start codon and/or alternative start codons in order to control or
attempt to control the initiation of translation, the length of the
polynucleotide and/or the structure of the polynucleotide.
17. STOP CODON REGION
[0829] The invention also includes a polynucleotide that comprises
both a stop codon region and the polynucleotide described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide). In some embodiments, the polynucleotides of the
present invention can include at least two stop codons before the
3' untranslated region (UTR). The stop codon can be selected from
TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in
the case of RNA. In some embodiments, the polynucleotides of the
present invention include the stop codon TGA in the case or DNA, or
the stop codon UGA in the case of RNA, and one additional stop
codon. In a further embodiment the addition stop codon can be TAA
or UAA. In another embodiment, the polynucleotides of the present
invention include three consecutive stop codons, four stop codons,
or more.
18. INSERTIONS AND SUBSTITUTIONS
[0830] The invention also includes a polynucleotide of the present
disclosure that further comprises insertions and/or
substitutions.
[0831] In some embodiments, the 5'UTR of the polynucleotide can be
replaced by the insertion of at least one region and/or string of
nucleosides of the same base. The region and/or string of
nucleotides can include, but is not limited to, at least 3, at
least 4, at least 5, at least 6, at least 7 or at least 8
nucleotides and the nucleotides can be natural and/or unnatural. As
a non-limiting example, the group of nucleotides can include 5-8
adenine, cytosine, thymine, a string of any of the other
nucleotides disclosed herein and/or combinations thereof.
[0832] In some embodiments, the 5'UTR of the polynucleotide can be
replaced by the insertion of at least two regions and/or strings of
nucleotides of two different bases such as, but not limited to,
adenine, cytosine, thymine, any of the other nucleotides disclosed
herein and/or combinations thereof. For example, the 5'UTR can be
replaced by inserting 5-8 adenine bases followed by the insertion
of 5-8 cytosine bases. In another example, the 5'UTR can be
replaced by inserting 5-8 cytosine bases followed by the insertion
of 5-8 adenine bases.
[0833] In some embodiments, the polynucleotide can include at least
one substitution and/or insertion downstream of the transcription
start site that can be recognized by an RNA polymerase. As a
non-limiting example, at least one substitution and/or insertion
can occur downstream of the transcription start site by
substituting at least one nucleic acid in the region just
downstream of the transcription start site (such as, but not
limited to, +1 to +6). Changes to region of nucleotides just
downstream of the transcription start site can affect initiation
rates, increase apparent nucleotide triphosphate (NTP) reaction
constant values, and increase the dissociation of short transcripts
from the transcription complex curing initial transcription (Brieba
et al, Biochemistry (2002) 41: 5144-5149; herein incorporated by
reference in its entirety). The modification, substitution and/or
insertion of at least one nucleoside can cause a silent mutation of
the sequence or can cause a mutation in the amino acid
sequence.
[0834] In some embodiments, the polynucleotide can include the
substitution of at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 11, at least 12 or at least 13 guanine bases
downstream of the transcription start site.
[0835] In some embodiments, the polynucleotide can include the
substitution of at least 1, at least 2, at least 3, at least 4, at
least 5 or at least 6 guanine bases in the region just downstream
of the transcription start site. As a non-limiting example, if the
nucleotides in the region are GGGAGA, the guanine bases can be
substituted by at least 1, at least 2, at least 3 or at least 4
adenine nucleotides. In another non-limiting example, if the
nucleotides in the region are GGGAGA the guanine bases can be
substituted by at least 1, at least 2, at least 3 or at least 4
cytosine bases. In another non-limiting example, if the nucleotides
in the region are GGGAGA the guanine bases can be substituted by at
least 1, at least 2, at least 3 or at least 4 thymine, and/or any
of the nucleotides described herein.
[0836] In some embodiments, the polynucleotide can include at least
one substitution and/or insertion upstream of the start codon. For
the purpose of clarity, one of skill in the art would appreciate
that the start codon is the first codon of the protein coding
region whereas the transcription start site is the site where
transcription begins. The polynucleotide can include, but is not
limited to, at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7 or at least 8 substitutions and/or
insertions of nucleotide bases. The nucleotide bases can be
inserted or substituted at 1, at least 1, at least 2, at least 3,
at least 4 or at least 5 locations upstream of the start codon. The
nucleotides inserted and/or substituted can be the same base (e.g.,
all A or all C or all T or all G), two different bases (e.g., A and
C, A and T, or C and T), three different bases (e.g., A, C and T or
A, C and T) or at least four different bases.
[0837] As a non-limiting example, the guanine base upstream of the
coding region in the polynucleotide can be substituted with
adenine, cytosine, thymine, or any of the nucleotides described
herein. In another non-limiting example the substitution of guanine
bases in the polynucleotide can be designed so as to leave one
guanine base in the region downstream of the transcription start
site and before the start codon (see Esvelt et al. Nature (2011)
472(7344):499-503; the contents of which is herein incorporated by
reference in its entirety). As a non-limiting example, at least 5
nucleotides can be inserted at 1 location downstream of the
transcription start site but upstream of the start codon and the at
least 5 nucleotides can be the same base type.
19. POLYNUCLEOTIDE COMPRISING AN MRNA ENCODING A PBGD
POLYPEPTIDE
[0838] In certain embodiments, a polynucleotide of the present
disclosure, for example a polynucleotide comprising an mRNA
nucleotide sequence encoding a PBGD polypeptide, comprises from 5'
to 3' end:
[0839] (i) a 5' cap provided above;
[0840] (ii) a 5' UTR, such as the sequences provided above;
[0841] (iii) an open reading frame encoding a PBGD polypeptide,
e.g., a sequence optimized nucleic acid sequence encoding PBGD
disclosed herein;
[0842] (iv) at least one stop codon;
[0843] (v) a 3' UTR, such as the sequences provided above; and
[0844] (vi) a poly-A tail provided above.
[0845] In some embodiments, the polynucleotide further comprises a
miRNA binding site, e.g, a miRNA binding site that binds to
miRNA-142. In some embodiments, the 5'UTR comprises the miRNA
binding site.
[0846] In some embodiments, a polynucleotide of the present
disclosure comprises a nucleotide sequence encoding a polypeptide
sequence at least 70%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical to the
protein sequence of a wild type PBGD (e.g, isoform 1, 2, 3, or
4).
[0847] In some embodiments, a polynucleotide of the present
disclosure, for example a polynucleotide comprising an mRNA
nucleotide sequence encoding a PBGD polypeptide, comprises (1) a 5'
cap provided above, for example, CAP1, (2) a nucleotide sequence
selected form the group consisting of SEQ ID NO: 133, 141, 144, and
145, and (3) a poly-A tail provided above, for example, a poly A
tail of .about.100 residues, wherein SEQ ID NO: 133 comprises from
5' to 3' end: 5' UTR of SEQ ID NO:39, PBGD polypeptide ORF of SEQ
ID NO: 104, and 3'UTR of SEQ ID NO: 149; SEQ ID NO: 141 comprises
from 5' to 3' end: 5' UTR of SEQ ID NO:39, PBGD polypeptide ORF of
SEQ ID NO: 112, and 3'UTR of SEQ ID NO: 149; SEQ ID NO: 144
comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39, PBGD
polypeptide ORF of SEQ ID NO: 112, and 3'UTR of SEQ ID NO: 150; and
SEQ ID NO: 145 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 112, and 3'UTR of SEQ ID NO:
151.
[0848] In some embodiments, a polynucleotide of the present
disclosure, for example a polynucleotide comprising an mRNA
nucleotide sequence encoding a PBGD polypeptide, comprises (1) a 5'
cap provided above, for example, CAP1, (2) a nucleotide sequence
selected form the group consisting of SEQ ID NO: 118-132, 134-140,
142, 143, and 146-148, and (3) a poly-A tail provided above, for
example, a poly A tail of .about.100 residues, wherein SEQ ID NO:
118 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39, PBGD
polypeptide ORF of SEQ ID NO: 89, and 3'UTR of SEQ ID NO: 149; SEQ
ID NO: 119 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 90, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 120 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 91, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 121 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 92, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 122 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 93, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 123 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 94, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 124 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 95, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 125 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 96, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 126 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 97, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 127 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 98, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 128 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 99, and 3'UTR of SEQ ID NO: 149;
SEQ ID NO: 129 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 100, and 3'UTR of SEQ ID NO:
149; SEQ ID NO: 130 comprises from 5' to 3' end: 5' UTR of SEQ ID
NO:39, PBGD polypeptide ORF of SEQ ID NO: 101, and 3'UTR of SEQ ID
NO: 149; SEQ ID NO: 131 comprises from 5' to 3' end: 5' UTR of SEQ
ID NO:39, PBGD polypeptide ORF of SEQ ID NO: 102, and 3'UTR of SEQ
ID NO: 149; SEQ ID NO: 132 comprises from 5' to 3' end: 5' UTR of
SEQ ID NO:39, PBGD polypeptide ORF of SEQ ID NO: 103, and 3'UTR of
SEQ ID NO: 149; SEQ ID NO: 134 comprises from 5' to 3' end: 5' UTR
of SEQ ID NO:39, PBGD polypeptide ORF of SEQ ID NO: 105, and 3'UTR
of SEQ ID NO: 149; SEQ ID NO: 135 comprises from 5' to 3' end: 5'
UTR of SEQ ID NO:39, PBGD polypeptide ORF of SEQ ID NO: 106, and
3'UTR of SEQ ID NO: 149; SEQ ID NO: 136 comprises from 5' to 3'
end: 5' UTR of SEQ ID NO:39, PBGD polypeptide ORF of SEQ ID NO:
107, and 3'UTR of SEQ ID NO: 149; SEQ ID NO: 137 comprises from 5'
to 3' end: 5' UTR of SEQ ID NO:39, PBGD polypeptide ORF of SEQ ID
NO: 108, and 3'UTR of SEQ ID NO: 149; SEQ ID NO: 138 comprises from
5' to 3' end: 5' UTR of SEQ ID NO:39, PBGD polypeptide ORF of SEQ
ID NO: 109, and 3'UTR of SEQ ID NO: 149; SEQ ID NO: 139 comprises
from 5' to 3' end: 5' UTR of SEQ ID NO:39, PBGD polypeptide ORF of
SEQ ID NO: 110, and 3'UTR of SEQ ID NO: 149; SEQ ID NO: 140
comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39, PBGD
polypeptide ORF of SEQ ID NO: 111, and 3'UTR of SEQ ID NO: 149; SEQ
ID NO: 142 comprises from 5' to 3' end: 5' UTR of SEQ ID NO:39,
PBGD polypeptide ORF of SEQ ID NO: 113, and 3'UTR of SEQ ID NO:
149; SEQ ID NO: 143 comprises from 5' to 3' end: 5' UTR of SEQ ID
NO:39, PBGD polypeptide ORF of SEQ ID NO: 114, and 3'UTR of SEQ ID
NO: 149; SEQ ID NO: 146 comprises from 5' to 3' end: 5' UTR of SEQ
ID NO:39, PBGD polypeptide ORF of SEQ ID NO: 115, and 3'UTR of SEQ
ID NO: 149; SEQ ID NO: 147 comprises from 5' to 3' end: 5' UTR of
SEQ ID NO:39, PBGD polypeptide ORF of SEQ ID NO: 116, and 3'UTR of
SEQ ID NO: 149; and SEQ ID NO: 148 comprises from 5' to 3' end: 5'
UTR of SEQ ID NO:39, PBGD polypeptide ORF of SEQ ID NO: 117, and
3'UTR of SEQ ID NO: 149.
TABLE-US-00007 TABLE 5 mRNA Constructs SEQ ID Con- Sequence (5' UTR
= bold underline; NO struct 3' UTR comprising a stop codon = bold
italics) 133 #16
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCCCCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCCCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAGACCCUGC
CCGAGAAGAGCGUGGUGGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCCCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCCGUGGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUGGACCUGGUGGGCGUGCUGCACG
ACCCCGAGACCCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UGGAGGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCCAGCACGAGGACG
GCCCCGAGGACGACCCCCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCCC
GGGGCCCCCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGAGCAAGGGCGCCAAGAACAUCCUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 141
#24 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCGCCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCGCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAGACCCUGC
CCGAGAAGAGCGUGGUGGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCUCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCCGUGGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUGGACCUGGUGGGCGUGCUGCACG
ACCCCGAGACCCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UGGAGGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCCAGCACGAGGACG
GCCCCGAGGACGACCCUCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCAC
GGGGCCCUCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGAGCAAGGGCGCCAAGAACAUCCUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 144
#27 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCGCCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCGCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAGACCCUGC
CCGAGAAGAGCGUGGUGGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCUCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCCGUGGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUGGACCUGGUGGGCGUGCUGCACG
ACCCCGAGACCCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UGGAGGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCCAGCACGAGGACG
GCCCCGAGGACGACCCUCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCAC
GGGGCCCUCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGAGCAAGGGCGCCAAGAACAUCCUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 145
#28 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCGCCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCGCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAGACCCUGC
CCGAGAAGAGCGUGGUGGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCUCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCCGUGGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUGGACCUGGUGGGCGUGCUGCACG
ACCCCGAGACCCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UGGAGGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCCAGCACGAGGACG
GCCCCGAGGACGACCCUCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCAC
GGGGCCCUCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGAGCAAGGGCGCCAAGAACAUCCUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 118 #1
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCAGCCACCGCCGAGGAAAACAGCCCCAAGAUGC
GGGUGAUCAGAGUGGGCACCCGGAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCUCCUACCCCGGCCUGCAGUUCGAGA
UCAUUGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGCCUGUUCACAAAAGAGCUGGAACACGCCCUGGAAA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCCCCUGGCUUCACCAUCGGCGCCAUCUGCAAGAGAGAGAACCCCCACG
ACGCCGUGGUGUUCCACCCUAAGUUCGUGGGCAAGACACUGGAAACCCUGC
CCGAGAAGUCCGUGGUGGGCACCAGCAGCCUGCGGAGAGCCGCCCAGCUGC
AGCGGAAGUUCCCCCACCUGGAAUUUCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAAUUUUCCGCUAUCAUCCUGG
CCACAGCCGGACUGCAGCGGAUGGGCUGGCACAACAGAGUGGGCCAGAUCC
UGCACCCCGAGGAAUGCAUGUACGCCGUGGGCCAGGGAGCCCUGGGCGUGG
AAGUGCGGGCCAAGGACCAGGACAUCCUGGAUCUGGUGGGCGUGCUGCAUG
ACCCCGAGACACUGCUGCGGUGUAUCGCCGAGCGGGCCUUCCUGCGGCACC
UGGAAGGCGGCUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GACAGCUGUACCUGACAGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACCAUGCAGGCCACCAUCCACGUGCCCGCCCAGCACGAGGACG
GCCCCGAGGACGACCCUCAGCUGGUCGGCAUCACCGCCCGGAACAUCCCCA
GAGGCCCCCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGUCCAAGGGCGCCAAGAACAUCCUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 119 #2
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCUACCGCCGAAGAGAACAGCCCAAAGAUGC
GCGUGAUCAGGGUCGGCACGCGCAAGUCCCAGCUCGCCCGGAUCCAAACCG
AUAGCGUGGUGGCCACGCUCAAGGCGAGCUAUCCGGGCUUACAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGAUAAGAUACUGGACACCGCCCUGUCCA
AGAUCGGCGAAAAGAGCCUGUUCACCAAGGAACUGGAGCACGCGCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCGACCGUGC
UGCCGCCGGGAUUCACCAUCGGCGCCAUCUGCAAGAGGGAGAAUCCGCACG
AUGCCGUGGUGUUCCACCCAAAGUUCGUGGGCAAGACCUUGGAAACCCUGC
CAGAGAAGUCUGUGGUCGGCACCUCCAGCCUGCGGCGAGCCGCCCAGCUGC
AGCGAAAGUUCCCGCACCUGGAGUUCAGGUCCAUCCGCGGAAAUCUGAACA
CCAGGCUGCGCAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAUCCUGG
CCACCGCAGGCCUCCAAAGAAUGGGCUGGCAUAACCGAGUCGGCCAGAUCC
UCCACCCGGAGGAGUGCAUGUACGCAGUGGGCCAAGGCGCCCUGGGCGUCG
AGGUGCGUGCCAAGGACCAGGACAUCCUGGACCUGGUGGGCGUGCUCCACG
AUCCAGAGACACUGCUGAGAUGCAUCGCGGAGCGCGCCUUCCUGCGCCAUC
UGGAGGGAGGCUGCUCCGUCCCGGUGGCCGUACAUACCGCCAUGAAGGACG
GUCAGCUGUACCUCACCGGCGGCGUAUGGUCCCUCGACGGUAGCGACAGCA
UACAGGAGACGAUGCAGGCCACCAUCCACGUGCCGGCGCAGCACGAGGAUG
GACCAGAGGACGACCCGCAGCUGGUGGGUAUCACCGCCAGGAAUAUCCCGC
GGGGACCUCAGCUGGCCGCCCAGAACCUGGGCAUCUCCCUCGCCAACCUCC
UGCUGAGCAAGGGCGCCAAGAACAUCCUGGACGUGGCCAGGCAGCUCAACG AUGCCCAU 120 #3
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAAAACAGCCCGAAGAUGC
GGGUGAUCAGGGUGGGCACCAGGAAGUCCCAGCUCGCCCGGAUCCAGACCG
ACAGCGUGGUCGCCACCUUGAAGGCCUCCUACCCGGGCCUCCAGUUCGAGA
UCAUCGCCAUGUCCACAACCGGCGACAAGAUCCUGGAUACCGCCCUCAGCA
AGAUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAAUGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCUACCGUGC
UGCCACCAGGCUUCACAAUCGGCGCCAUCUGCAAGAGAGAGAACCCGCACG
ACGCCGUGGUGUUCCAUCCGAAGUUCGUGGGCAAGACCCUGGAAACCCUGC
CGGAGAAGUCCGUAGUGGGAACCUCAAGCCUGAGGCGCGCCGCCCAGCUCC
AGAGGAAGUUCCCUCACCUGGAAUUCCGGUCCAUCAGGGGCAACCUGAACA
CGCGCCUGCGGAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAUCCUGG
CCACAGCCGGCCUUCAGCGCAUGGGCUGGCACAACAGGGUGGGCCAGAUCC
UGCACCCGGAAGAAUGCAUGUACGCCGUGGGCCAAGGCGCCCUCGGCGUGG
AAGUGCGUGCCAAGGACCAGGACAUCCUGGACCUGGUGGGCGUGCUGCACG
ACCCUGAGACGCUGCUCAGGUGCAUCGCCGAACGCGCGUUCCUGCGGCACC
UGGAGGGAGGCUGCAGCGUCCCGGUGGCCGUCCACACCGCCAUGAAGGACG
GCCAGCUCUACCUGACUGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UUCAGGAAACCAUGCAGGCCACCAUCCACGUGCCUGCCCAGCACGAGGACG
GCCCGGAGGACGACCCUCAACUGGUGGGCAUUACUGCGCGAAACAUCCCGC
GCGGACCUCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UCCUGUCCAAGGGCGCCAAGAACAUCCUCGACGUGGCCAGGCAGCUGAACG ACGCGCAC 121 #4
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGAAACGCCGCCGCGACCGCGGAGGAGAACUCGCCUAAGAUGA
GAGUGAUAAGGGUAGGCACCCGGAAGUCUCAACUCGCCAGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUCAAGGCCAGCUAUCCAGGACUCCAGUUCGAAA
UCAUCGCCAUGUCCACCACAGGCGAUAAGAUCCUGGACACCGCCCUGUCCA
AGAUCGGCGAGAAGUCCCUCUUCACCAAGGAACUGGAGCACGCCCUGGAGA
AGAACGAGGUCGAUCUGGUCGUGCACAGCCUGAAGGAUCUGCCUACCGUGC
UCCCGCCGGGCUUCACCAUCGGCGCCAUCUGCAAGAGGGAGAAUCCUCACG
ACGCCGUGGUGUUCCACCCGAAGUUCGUGGGCAAGACCCUGGAGACACUGC
CAGAAAAGUCGGUGGUGGGCACCAGCAGCCUGCGGCGGGCGGCCCAGCUGC
AGCGGAAGUUCCCACACCUGGAGUUCAGGUCCAUCCGUGGCAAUCUGAACA
CCCGGCUGCGUAAGCUGGACGAGCAGCAGGAAUUCAGCGCGAUCAUCCUGG
CAACCGCCGGUCUGCAAAGGAUGGGCUGGCACAACAGGGUGGGCCAGAUCC
UGCACCCUGAGGAGUGCAUGUACGCCGUGGGCCAGGGAGCCCUGGGCGUGG
AAGUGCGGGCCAAGGACCAGGACAUCCUGGACCUGGUGGGUGUGCUCCACG
ACCCUGAAACCCUGCUGCGGUGCAUCGCCGAAAGGGCCUUCCUGAGGCACC
UCGAGGGCGGCUGCAGCGUGCCGGUCGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGAGGAGUGUGGAGCCUGGACGGCUCCGACUCCA
UCCAGGAGACUAUGCAGGCCACCAUUCAUGUGCCGGCCCAGCAUGAGGACG
GUCCGGAGGACGAUCCACAGCUGGUCGGCAUCACCGCGCGGAACAUCCCAA
GAGGCCCGCAACUGGCCGCUCAGAACCUGGGCAUAUCCCUGGCCAACCUGC
UCCUGAGCAAGGGCGCCAAGAACAUCCUGGACGUGGCCAGGCAGCUGAAUG ACGCCCAC 122 #5
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGU
CCGGCAACGGCAACGCCGCCGCUACCGCCGAGGAGAACUCCCCUAAGAUGC
GGGUCAUCAGGGUGGGCACCCGAAAGUCCCAACUUGCCCGGAUCCAGACCG
ACUCCGUCGUGGCCACCCUCAAGGCUAGCUAUCCAGGCCUCCAGUUCGAAA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUUCUGGACACCGCCCUGUCCA
AGAUCGGCGAGAAGAGUCUGUUCACGAAGGAGCUCGAGCACGCCCUGGAAA
AGAACGAGGUGGACCUGGUGGUGCAUUCCCUGAAGGACCUGCCAACCGUGC
UGCCGCCGGGCUUCACUAUAGGAGCCAUCUGCAAGCGGGAGAACCCGCACG
ACGCGGUGGUGUUCCAUCCGAAGUUCGUGGGCAAGACUCUGGAAACCCUGC
CGGAGAAGUCCGUGGUGGGAACUAGCUCCCUGCGGCGGGCCGCCCAGCUGC
AGAGGAAGUUCCCGCACCUGGAGUUCAGGAGCAUACGCGGCAACCUGAACA
CCCGCCUGCGUAAGCUCGACGAGCAGCAGGAAUUCAGUGCCAUCAUCCUGG
CCACGGCGGGCCUGCAGCGGAUGGGCUGGCACAACAGGGUGGGCCAGAUCC
UCCACCCGGAGGAAUGUAUGUACGCCGUGGGCCAGGGCGCACUGGGCGUGG
AGGUCCGCGCCAAGGACCAAGACAUCCUGGACCUGGUCGGCGUGCUGCACG
ACCCUGAAACCCUGCUGAGGUGCAUUGCCGAGAGAGCCUUCCUGAGGCAUC
UGGAGGGCGGCUGCAGCGUGCCUGUGGCCGUGCACACAGCCAUGAAGGACG
GUCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACUCCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUCCCGGCCCAACACGAGGACG
GACCUGAGGACGAUCCUCAGCUGGUGGGCAUCACCGCCAGGAACAUCCCUC
GGGGCCCGCAGCUGGCCGCCCAGAACCUGGGCAUCUCCCUCGCCAACCUGC
UGCUGUCCAAGGGCGCCAAGAACAUCCUCGACGUGGCCAGACAGCUGAACG ACGCCCAC 123 #6
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCGAAGAUGA
GGGUGAUAAGGGUGGGCACACGGAAGUCCCAGCUCGCCCGCAUCCAAACCG
ACUCCGUGGUGGCCACCCUCAAGGCCAGCUACCCGGGCCUCCAAUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGUCUA
AGAUAGGCGAAAAGAGCCUGUUCACCAAGGAGCUGGAGCAUGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUCCACAGUCUCAAGGACCUGCCAACCGUGC
UGCCGCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGUGAGAACCCGCACG
AUGCUGUGGUGUUCCACCCUAAGUUCGUGGGAAAGACCCUGGAGACGCUGC
CGGAAAAGAGCGUGGUCGGCACCUCCAGCCUGCGGAGGGCCGCCCAACUCC
AGAGGAAGUUCCCGCACCUGGAGUUCAGGAGCAUCCGCGGCAACCUGAACA
CCAGGCUGCGAAAGCUGGACGAGCAGCAGGAAUUCUCGGCCAUCAUCCUCG
CCACCGCCGGCUUGCAAAGAAUGGGCUGGCAUAAUCGCGUGGGCCAGAUCC
UGCACCCUGAGGAGUGCAUGUACGCCGUGGGCCAGGGUGCUCUGGGAGUGG
AGGUGCGGGCCAAGGACCAGGAUAUCCUGGACCUGGUCGGCGUGCUUCAUG
ACCCGGAGACGCUCCUGAGGUGCAUCGCCGAGCGGGCCUUCCUGAGACACC
UGGAGGGCGGCUGCUCCGUGCCAGUGGCCGUGCACACCGCCAUGAAGGACG
GACAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGAAGCGACAGCA
UCCAAGAAACCAUGCAGGCGACCAUUCACGUCCCUGCCCAGCACGAGGAUG
GACCAGAGGACGACCCGCAGCUGGUGGGCAUCACCGCCCGCAACAUCCCUA
GAGGCCCACAGCUGGCCGCCCAGAAUCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGUCUAAGGGAGCCAAGAACAUCCUGGACGUGGCCAGGCAGCUGAACG ACGCCCAU 124 #7
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGU
CCGGCAACGGCAACGCCGCAGCCACCGCCGAGGAGAAUUCCCCGAAGAUGC
GGGUGAUCCGGGUGGGCACCAGAAAGAGCCAGCUCGCCCGCAUCCAAACCG
ACUCCGUGGUGGCCACCCUCAAGGCCUCCUACCCAGGCUUGCAGUUCGAAA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUUGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCAUGCUCUGGAGA
AGAACGAGGUGGACCUCGUGGUGCACUCCCUGAAGGACCUGCCGACUGUGC
UGCCGCCUGGCUUCACGAUCGGCGCCAUAUGCAAGCGGGAAAACCCACACG
ACGCCGUGGUCUUCCACCCAAAGUUCGUGGGCAAGACCCUGGAAACCCUGC
CGGAAAAGAGCGUGGUCGGCACAAGCUCCCUGAGGAGAGCCGCCCAACUGC
AAAGGAAGUUCCCUCACCUCGAGUUCAGGUCCAUCCGGGGCAACCUGAACA
CCAGGCUGAGAAAGCUCGACGAACAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACGGCCGGCCUGCAGAGGAUGGGAUGGCAUAACAGGGUGGGCCAGAUCC
UGCACCCGGAGGAGUGCAUGUACGCCGUGGGCCAGGGAGCCCUCGGCGUGG
AGGUCAGGGCCAAGGAUCAGGAUAUCCUGGACCUGGUGGGCGUGCUGCACG
AUCCUGAGACGCUGCUGAGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UAGAGGGCGGAUGCAGCGUGCCGGUCGCGGUCCACACCGCGAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGUCCCUGGACGGCAGCGAUUCAA
UCCAGGAGACGAUGCAGGCCACCAUCCACGUGCCAGCCCAGCACGAGGAUG
GCCCGGAGGACGACCCGCAGCUGGUGGGCAUUACAGCCAGGAACAUCCCUC
GGGGCCCGCAGCUGGCCGCCCAGAAUCUGGGCAUCAGCCUGGCGAACCUGC
UGCUCAGCAAGGGAGCGAAGAACAUCCUGGACGUGGCCCGCCAGCUGAACG AUGCCCAC 125 #8
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCAAAGAUGC
GGGUGAUCAGGGUGGGCACCCGCAAGAGCCAACUCGCCAGAAUCCAGACCG
ACAGCGUGGUGGCCACCUUGAAGGCCAGCUACCCGGGCCUCCAGUUCGAGA
UCAUCGCUAUGUCCACCACCGGCGACAAGAUCCUGGACACCGCGCUGUCCA
AGAUCGGCGAAAAGAGCCUGUUCACCAAGGAACUGGAGCACGCCCUCGAGA
AGAACGAGGUGGACCUGGUGGUGCACUCCCUGAAGGACCUGCCGACGGUCC
UGCCGCCGGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAAAACCCGCACG
ACGCUGUGGUGUUCCACCCAAAGUUCGUGGGCAAGACCCUGGAAACCCUGC
CAGAAAAGAGCGUGGUGGGCACCAGCAGCCUCAGGAGAGCCGCCCAGCUGC
AGAGGAAGUUCCCGCACCUGGAGUUCAGGAGCAUCAGGGGCAACCUGAACA
CCAGGCUGCGUAAGCUGGACGAGCAGCAGGAGUUCUCCGCCAUCAUCCUCG
CCACAGCCGGCCUCCAGAGGAUGGGUUGGCACAACAGGGUGGGCCAGAUCC
UGCACCCGGAAGAGUGCAUGUACGCAGUGGGCCAGGGCGCCCUUGGCGUGG
AAGUGCGAGCCAAGGAUCAGGAUAUCCUGGACCUGGUGGGCGUGCUGCACG
ACCCGGAAACUCUGCUGCGGUGCAUCGCCGAAAGGGCCUUCCUGCGCCACC
UCGAAGGCGGCUGUAGCGUGCCGGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUCGACGGCAGCGACAGCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCGGCCCAGCAUGAGGAUG
GCCCGGAGGACGACCCUCAGCUGGUGGGCAUCACCGCCCGCAACAUCCCAA
GAGGACCGCAACUGGCCGCCCAGAACCUGGGCAUCUCCCUGGCCAACCUGC
UCCUGAGCAAGGGCGCGAAGAACAUCCUCGACGUCGCACGGCAGCUGAACG ACGCCCAC 126 #9
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCGACGGCCGAGGAAAAUAGCCCGAAGAUGC
GGGUGAUCAGGGUGGGCACCAGGAAGUCCCAGCUCGCCAGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUCAAGGCCUCCUACCCGGGCCUCCAAUUCGAGA
UCAUCGCCAUGUCCACCACCGGCGACAAGAUCCUCGACACCGCCCUGAGCA
AGAUCGGCGAAAAGUCGCUGUUCACCAAGGAGCUGGAGCACGCCCUCGAGA
AGAACGAGGUGGACCUGGUAGUGCACUCCCUAAAGGACCUGCCGACCGUGC
UGCCGCCGGGCUUCACGAUCGGCGCCAUCUGCAAGCGCGAGAACCCGCAUG
AUGCCGUCGUUUUCCACCCUAAGUUCGUGGGCAAGACCCUGGAGACGCUGC
CGGAGAAGUCGGUGGUGGGAACCAGCAGCCUGAGGAGGGCCGCACAACUGC
AGAGGAAGUUCCCGCAUCUGGAGUUCCGCAGCAUUCGAGGCAACCUGAACA
CGCGCCUGAGAAAGCUCGAUGAACAGCAGGAGUUCAGCGCCAUCAUUCUGG
CCACUGCCGGACUGCAGCGGAUGGGCUGGCACAACAGAGUGGGCCAGAUCC
UGCAUCCGGAAGAGUGUAUGUACGCCGUGGGCCAGGGUGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGAUAUACUGGAUCUGGUCGGCGUGCUCCACG
ACCCAGAAACACUCCUGAGGUGCAUCGCUGAGAGAGCCUUCCUCCGGCACC
UCGAGGGCGGCUGUUCCGUGCCGGUGGCCGUCCAUACCGCCAUGAAGGACG
GUCAGCUGUACCUGACCGGAGGCGUUUGGUCCCUGGACGGCAGCGACAGCA
UCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCGGCGCAGCACGAGGACG
GCCCGGAAGACGACCCGCAGCUGGUCGGCAUCACGGCCAGAAACAUCCCGC
GGGGCCCGCAGCUGGCGGCCCAGAACCUGGGAAUCUCCCUGGCCAACCUGC
UGCUGAGCAAGGGCGCGAAGAACAUCCUGGACGUGGCCAGGCAGCUGAACG AUGCCCAC 127
#10 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGUAACGGCAACGCCGCCGCCACCGCCGAGGAGAACUCCCCGAAGAUGC
GCGUGAUUCGGGUCGGCACAAGAAAGUCUCAACUCGCCCGAAUCCAAACGG
ACAGCGUGGUGGCCACCCUCAAGGCGAGCUACCCGGGCCUCCAGUUCGAAA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGUCGA
AGAUUGGCGAAAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAAGUCGACCUGGUCGUGCACAGCCUGAAGGACCUGCCGACCGUUC
UGCCGCCGGGCUUCACCAUCGGAGCCAUCUGCAAGCGGGAGAAUCCGCACG
ACGCCGUGGUCUUCCACCCAAAGUUCGUGGGAAAGACCCUCGAGACACUGC
CGGAGAAGUCCGUGGUGGGAACCUCCUCCCUGCGGAGGGCCGCCCAACUGC
AGCGGAAGUUCCCACACCUGGAAUUCCGGUCCAUCAGAGGCAACCUCAACA
CCAGGCUGAGGAAGCUCGAUGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACAGCCGGACUGCAGCGCAUGGGCUGGCAUAACAGAGUGGGCCAGAUCC
UCCACCCGGAGGAGUGCAUGUACGCCGUGGGACAAGGCGCGCUGGGCGUGG
AAGUUCGGGCCAAGGACCAGGAUAUCCUGGACCUGGUGGGCGUGCUCCACG
ACCCAGAGACGCUGCUGCGGUGCAUCGCCGAGCGCGCCUUCCUGCGGCACC
UCGAGGGCGGCUGCAGCGUGCCGGUCGCUGUGCACACAGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGUCUGGACGGCAGCGACUCCA
UCCAGGAGACUAUGCAAGCCACCAUCCAUGUGCCGGCCCAACAUGAGGACG
GCCCGGAGGACGACCCGCAACUGGUGGGCAUCACCGCCCGGAACAUCCCGA
GGGGCCCGCAGCUGGCCGCCCAGAACCUGGGCAUUAGCCUGGCCAACCUGC
UCCUGAGCAAGGGCGCUAAGAACAUCCUGGACGUCGCCAGACAGCUGAACG ACGCCCAC 128
#11 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGUAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCUCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCCCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAAACCCUGC
CUGAGAAGUCCGUCGUAGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCCCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CUACCGCCGGUCUGCAACGAAUGGGCUGGCACAAUAGGGUGGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCGGUGGGACAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUUGAUCUGGUGGGCGUGCUGCACG
ACCCCGAGACGCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUU
UGGAGGGCGGAUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCUGCUCAGCACGAAGACG
GCCCAGAGGACGACCCCCAGCUGGUAGGCAUCACCGCCCGGAACAUCCCCC
GGGGCCCUCAGCUCGCCGCACAGAACCUUGGAAUCAGCCUGGCCAACCUGC
UGUUGUCAAAGGGCGCCAAGAAUAUCCUCGACGUGGCCCGGCAGCUGAACG ACGCCCAC 129
#12 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCGCCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCCCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACUCUGGAAACCCUGC
CUGAGAAGUCCGUGGUCGGAACAAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCCCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACAGCCGGCCUUCAGAGGAUGGGCUGGCACAAUCGGGUAGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCGGUAGGUCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCUUAGAUCUGGUUGGCGUGCUGCACG
ACCCCGAAACACUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UCGAGGGCGGCUGCAGUGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCAGCUCAGCACGAAGACG
GACCAGAGGACGACCCCCAGUUAGUGGGAAUCACCGCCCGGAACAUCCCCC
GGGGCCCUCAGCUCGCGGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGUCUAAGGGCGCCAAGAACAUCCUAGACGUGGCCCGGCAGCUGAACG ACGCCCAC 130
#13 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCAGGAAGUCACAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCGCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCCCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAAACCCUGC
CAGAGAAGUCUGUGGUCGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCCCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCUGGCCUACAGCGGAUGGGCUGGCACAAUAGAGUUGGUCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCCGUCGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUACUAGACCUCGUGGGCGUGCUGCACG
ACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UGGAAGGCGGUUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCGGCCCAACACGAGGACG
GACCUGAGGACGACCCCCAGCUUGUGGGAAUCACCGCCCGGAACAUCCCCC
GGGGCCCUCAACUGGCAGCCCAGAACUUAGGCAUAAGCCUGGCCAACCUGC
UGCUGUCCAAGGGCGCCAAGAACAUCCUCGAUGUGGCCCGGCAGCUGAACG ACGCCCAC 131
#14 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGCAAGAGUCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCACCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCCCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACACUGGAAACCCUGC
CGGAGAAGUCCGUGGUAGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCCCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CAACAGCCGGCUUACAGCGUAUGGGCUGGCACAACAGGGUGGGACAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCUGUGGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCUUAGAUCUCGUCGGCGUGCUGCACG
ACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUC
UUGAGGGCGGAUGCUCCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACUAUGCAGGCCACCAUCCACGUGCCAGCCCAGCACGAAGACG
GCCCAGAGGACGACCCCCAGCUGGUGGGAAUCACCGCCCGGAACAUCCCCC
GGGGCCCUCAACUGGCCGCACAGAACCUAGGCAUCAGCCUGGCCAACCUGC
UGCUCAGCAAGGGCGCCAAGAAUAUCUUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 132
#15 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGUCUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCUCCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCCCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAGACUCUGC
CAGAGAAGUCUGUAGUGGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCCCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACGGCCGGAUUACAGAGAAUGGGCUGGCACAACCGAGUGGGACAGAUCC
UGCACCCCGAGGAGUGCAUGUAUGCCGUUGGCCAAGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUCGAUCUCGUGGGCGUGCUGCACG
ACCCCGAGACUUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UAGAGGGCGGCUGCUCGGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCAGCCCAGCACGAGGAUG
GCCCUGAAGACGACCCCCAGCUGGUGGGCAUCACCGCCCGGAACAUCCCCC
GGGGCCCACAAUUGGCCGCUCAGAACUUAGGCAUUAGCCUGGCCAACCUGC
UGCUGUCUAAGGGCGCCAAGAACAUACUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 134
#17 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCGAGGAUCCAGACGG
ACAGCGUGGUGGCGACGCUGAAGGCGAGCUACCCGGGGCUGCAGUUCGAGA
UCAUCGCGAUGAGCACGACGGGGGACAAGAUCCUGGACACGGCGCUGAGCA
AGAUCGGGGAGAAGAGCCUGUUCACGAAGGAGCUGGAGCACGCGCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCGACGGUGC
UGCCGCCGGGGUUCACGAUCGGGGCGAUCUGCAAGAGGGAGAACCCGCACG
ACGCGGUGGUGUUCCACCCGAAGUUCGUGGGGAAGACGCUGGAGACGCUGC
CGGAGAAGAGCGUGGUGGGGACGAGCAGCCUGAGGAGGGCGGCGCAGCUGC
AGAGGAAGUUCCCGCACCUGGAGUUCAGGAGCAUCAGGGGGAACCUGAACA
CGAGGCUGAGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCGAUCAUCCUGG
CGACGGCGGGGCUGCAGAGGAUGGGGUGGCACAACAGGGUGGGGCAGAUCC
UGCACCCGGAGGAGUGCAUGUACGCGGUGGGGCAGGGGGCGCUGGGGGUGG
AGGUGAGGGCGAAGGACCAGGACAUCCUGGACCUGGUGGGGGUGCUGCACG
ACCCGGAGACGCUGCUGAGGUGCAUCGCGGAGAGGGCGUUCCUGAGGCACC
UGGAGGGGGGGUGCAGCGUGCCGGUGGCGGUGCACACGGCGAUGAAGGACG
GGCAGCUGUACCUGACGGGGGGGGUGUGGAGCCUGGACGGGAGCGACAGCA
UCCAGGAGACGAUGCAGGCGACGAUCCACGUGCCGGCGCAGCACGAGGACG
GGCCGGAGGACGACCCGCAGCUGGUGGGGAUCACGGCGAGGAACAUCCCGA
GGGGGCCGCAGCUGGCGGCGCAGAACCUGGGGAUCAGCCUGGCGAACCUGC
UGCUGAGCAAGGGGGCGAAGAACAUCCUGGACGUGGCGAGGCAGCUGAACG ACGCGCAC 135
#18 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCCCGCAUCCAGACCG
ACUCCGUCGUCGCCACCCUCAAGGCCUCCUACCCCGGCCUCCAGUUCGAGA
UCAUCGCCAUGUCCACCACCGGCGACAAGAUCCUCGACACCGCCCUCUCCA
AGAUCGGCGAGAAGUCCCUCUUCACCAAGGAGCUCGAGCACGCCCUCGAGA
AGAACGAGGUCGACCUCGUCGUCCACUCCCUCAAGGACCUCCCCACCGUCC
UCCCCCCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGCGAGAACCCCCACG
ACGCCGUCGUCUUCCACCCCAAGUUCGUCGGCAAGACCCUCGAGACCCUCC
CCGAGAAGUCCGUCGUCGGCACCUCCUCCCUCCGCCGCGCCGCCCAGCUCC
AGCGCAAGUUCCCCCACCUCGAGUUCCGCUCCAUCCGCGGCAACCUCAACA
CCCGCCUCCGCAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAUCCUCG
CCACCGCCGGCCUCCAGCGCAUGGGCUGGCACAACCGCGUCGGCCAGAUCC
UCCACCCCGAGGAGUGCAUGUACGCCGUCGGCCAGGGCGCCCUCGGCGUCG
AGGUCCGCGCCAAGGACCAGGACAUCCUCGACCUCGUCGGCGUCCUCCACG
ACCCCGAGACCCUCCUCCGCUGCAUCGCCGAGCGCGCCUUCCUCCGCCACC
UCGAGGGCGGCUGCUCCGUCCCCGUCGCCGUCCACACCGCCAUGAAGGACG
GCCAGCUCUACCUCACCGGCGGCGUCUGGUCCCUCGACGGCUCCGACUCCA
UCCAGGAGACCAUGCAGGCCACCAUCCACGUCCCCGCCCAGCACGAGGACG
GCCCCGAGGACGACCCCCAGCUCGUCGGCAUCACCGCCCGCAACAUCCCCC
GCGGCCCCCAGCUCGCCGCCCAGAACCUCGGCAUCUCCCUCGCCAACCUCC
UCCUCUCCAAGGGCGCCAAGAACAUCCUCGACGUCGCCCGCCAGCUCAACG ACGCCCAC 136
#19 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGUAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCUCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCUCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUGGAAACCCUGC
CUGAGAAGUCCGUCGUAGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCACACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CUACCGCCGGUCUGCAACGAAUGGGCUGGCACAAUAGGGUGGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCGGUGGGACAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUUGAUCUGGUGGGCGUGCUGCACG
ACCCCGAGACGCUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUU
UGGAGGGCGGAUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCUGCUCAGCACGAAGACG
GCCCAGAGGACGACCCUCAGCUGGUAGGCAUCACCGCCCGGAACAUCCCUC
GGGGCCCUCAGCUCGCCGCACAGAACCUUGGAAUCAGCCUGGCCAACCUGC
UGUUGUCAAAGGGCGCCAAGAAUAUCCUCGACGUGGCCCGGCAGCUGAACG ACGCCCAC 137
#20 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCGCCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCGCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACUCUGGAAACCCUGC
CUGAGAAGUCCGUGGUCGGAACAAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCACACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACAGCCGGCCUUCAGAGGAUGGGCUGGCACAAUCGGGUAGGCCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCGGUAGGUCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCUUAGAUCUGGUUGGCGUGCUGCACG
ACCCCGAAACACUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UCGAGGGCGGCUGCAGUGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCAGCUCAGCACGAAGACG
GACCAGAGGACGACCCACAGUUAGUGGGAAUCACCGCCCGGAACAUCCCGC
GGGGCCCUCAGCUCGCGGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGUCUAAGGGCGCCAAGAACAUCCUAGACGUGGCCCGGCAGCUGAACG ACGCCCAC 138
#21 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCAGGAAGUCACAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGUCCCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCGCCAGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCGCACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAAACCCUGC
CAGAGAAGUCUGUGGUCGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCGCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCUGGCCUACAGCGGAUGGGCUGGCACAAUAGAGUUGGUCAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCCGUCGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUACUAGACCUCGUGGGCGUGCUGCACG
ACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UGGAAGGCGGUUGCAGCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCGGCCCAACACGAGGACG
GACCUGAGGACGACCCACAGCUUGUGGGAAUCACCGCCCGGAACAUCCCAC
GGGGCCCUCAACUGGCAGCCCAGAACUUAGGCAUAAGCCUGGCCAACCUGC
UGCUGUCCAAGGGCGCCAAGAACAUCCUCGAUGUGGCCCGGCAGCUGAACG ACGCCCAC 139
#22 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGCAAGAGUCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGAGUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCACCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCACACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACACUGGAAACCCUGC
CGGAGAAGUCCGUGGUAGGCACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCGCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CAACAGCCGGCUUACAGCGUAUGGGCUGGCACAACAGGGUGGGACAGAUCC
UGCACCCCGAGGAGUGCAUGUACGCUGUGGGCCAGGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCUUAGAUCUCGUCGGCGUGCUGCACG
ACCCCGAAACCUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCAUC
UUGAGGGCGGAUGCUCCGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACUAUGCAGGCCACCAUCCACGUGCCAGCCCAGCACGAAGACG
GCCCAGAGGACGACCCACAGCUGGUGGGAAUCACCGCCCGGAACAUCCCGC
GGGGCCCUCAACUGGCCGCACAGAACCUAGGCAUCAGCCUGGCCAACCUGC
UGCUCAGCAAGGGCGCCAAGAAUAUCUUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 140
#23 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCAGAAAGAGCCAGCUGGCCCGGAUCCAGACCG
ACAGCGUGGUGGCCACCCUGAAGGCCAGCUACCCCGGCCUGCAGUUCGAGA
UCAUCGCCAUGAGCACCACCGGCGACAAGAUCCUGGACACCGCCCUGAGCA
AGAUCGGCGAGAAGUCUCUGUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCCACCGUGC
UGCCUCCUGGCUUCACCAUCGGCGCCAUCUGCAAGCGGGAGAACCCACACG
ACGCCGUGGUGUUCCACCCCAAGUUCGUGGGCAAGACCCUUGAGACUCUGC
CAGAGAAGUCUGUAGUGGGAACCAGCAGCCUGCGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCUCACCUGGAGUUCCGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACGGCCGGAUUACAGAGAAUGGGCUGGCACAACCGAGUGGGACAGAUCC
UGCACCCCGAGGAGUGCAUGUAUGCCGUUGGCCAAGGCGCCCUGGGCGUGG
AGGUGCGGGCCAAGGACCAGGACAUCCUCGAUCUCGUGGGCGUGCUGCACG
ACCCCGAGACUUUGCUGCGGUGCAUCGCCGAGCGGGCCUUCCUGCGGCACC
UAGAGGGCGGCUGCUCGGUGCCCGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACAGCA
UCCAGGAGACAAUGCAGGCCACCAUCCACGUGCCAGCCCAGCACGAGGAUG
GCCCUGAAGACGACCCACAGCUGGUGGGCAUCACCGCCCGGAACAUCCCGC
GGGGCCCACAAUUGGCCGCUCAGAACUUAGGCAUUAGCCUGGCCAACCUGC
UGCUGUCUAAGGGCGCCAAGAACAUACUGGACGUGGCCCGGCAGCUGAACG ACGCCCAC 142
#25 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCGAGGAUCCAGACGG
ACAGCGUGGUGGCGACGCUGAAGGCGAGCUACCCGGGGCUGCAGUUCGAGA
UCAUCGCGAUGAGCACGACGGGCGACAAGAUCCUGGACACGGCGCUGAGCA
AGAUCGGGGAGAAGAGCCUGUUCACGAAGGAGCUGGAGCACGCGCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACAGCCUGAAGGACCUGCCGACGGUGC
UGCCGCCGGGGUUCACGAUCGGGGCGAUCUGCAAGAGGGAGAACCCGCACG
ACGCGGUGGUGUUCCACCCGAAGUUCGUGGGGAAGACGCUGGAGACGCUGC
CGGAGAAGAGCGUGGUGGGGACGAGCAGCCUGAGGAGGGCGGCGCAGCUGC
AGAGGAAGUUCCCGCACCUGGAGUUCAGGAGCAUCAGGGGUAACCUGAACA
CGAGGCUGAGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCGAUCAUCCUGG
CGACGGCGGGGCUGCAGAGGAUGGGGUGGCACAACAGGGUGGGGCAGAUCC
UGCACCCGGAGGAGUGCAUGUACGCGGUGGGGCAGGGCGCGCUGGGCGUGG
AGGUGAGGGCGAAGGACCAGGACAUCCUGGACCUGGUGGGCGUGCUGCACG
ACCCGGAGACGCUGCUGAGGUGCAUCGCGGAGAGGGCGUUCCUGAGGCACC
UGGAGGGCGGGUGCAGCGUGCCGGUGGCGGUGCACACGGCGAUGAAGGACG
GGCAGCUGUACCUGACGGGAGGGGUGUGGAGCCUGGACGGGAGCGACAGCA
UCCAGGAGACGAUGCAGGCGACGAUCCACGUGCCGGCGCAGCACGAGGACG
GGCCGGAGGACGACCCGCAGCUGGUGGGGAUCACGGCGAGGAACAUCCCGA
GGGGUCCGCAGCUGGCGGCGCAGAACCUGGGGAUCAGCCUGGCGAACCUGC
UGCUGAGCAAGGGAGCGAAGAACAUCCUGGACGUGGCGAGGCAGCUGAACG ACGCGCAC 143
#26 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCCGAGGAGAACAGCCCCAAGAUGC
GGGUGAUCCGGGUGGGCACCCGGAAGAGCCAGCUGGCCCGCAUCCAGACCG
ACUCCGUCGUCGCCACCCUCAAGGCCUCCUACCCCGGCCUCCAGUUCGAGA
UCAUCGCCAUGUCCACCACCGGCGACAAGAUCCUCGACACCGCCCUCUCCA
AGAUCGGCGAGAAGUCCCUCUUCACCAAGGAGCUCGAGCACGCCCUCGAGA
AGAACGAGGUCGACCUCGUCGUCCACUCCCUCAAGGACCUCCCCACCGUCC
UCCCACCCGGCUUCACCAUCGGCGCCAUCUGCAAGCGCGAGAACCCUCACG
ACGCCGUCGUCUUCCACCCCAAGUUCGUCGGCAAGACCCUCGAGACCCUCC
CCGAGAAGUCCGUCGUCGGCACCUCCUCCCUCCGCCGCGCCGCCCAGCUCC
AGCGCAAGUUCCCACACCUCGAGUUCCGCUCCAUCCGCGGCAACCUCAACA
CCCGCCUCCGCAAGCUCGACGAGCAGCAGGAGUUCUCCGCCAUCAUCCUCG
CCACCGCCGGCCUCCAGCGCAUGGGCUGGCACAACCGCGUCGGCCAGAUCC
UCCACCCCGAGGAGUGCAUGUACGCCGUCGGCCAGGGCGCCCUCGGCGUCG
AGGUCCGCGCCAAGGACCAGGACAUCCUCGACCUCGUCGGCGUCCUCCACG
ACCCCGAGACCCUCCUCCGCUGCAUCGCCGAGCGCGCCUUCCUCCGCCACC
UCGAGGGCGGCUGCUCCGUCCCCGUCGCCGUCCACACCGCCAUGAAGGACG
GCCAGCUCUACCUCACCGGCGGCGUCUGGUCCCUCGACGGCUCCGACUCCA
UCCAGGAGACCAUGCAGGCCACCAUCCACGUCCCCGCCCAGCACGAGGACG
GCCCCGAGGACGACCCUCAGCUCGUCGGCAUCACCGCCCGCAACAUCCCGC
GCGGCCCUCAGCUCGCCGCCCAGAACCUCGGCAUCUCCCUCGCCAACCUCC
UCCUCUCCAAGGGCGCCAAGAACAUCCUCGACGUCGCCCGCCAGCUCAACG ACGCCCAC 146
#29 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGU
CCGGAAACGGAAACGCCGCCGCUACCGCCGAGGAAAAUAGCCCGAAGAUGA
GAGUGAUCCGGGUGGGUACCAGGAAGUCCCAGCUCGCCAGGAUCCAAACGG
ACUCGGUGGUGGCCACCCUCAAGGCUAGCUACCCGGGCCUGCAAUUCGAGA
UCAUUGCUAUGUCCACCACCGGCGACAAGAUCCUGGAUACGGCCCUGUCCA
AGAUCGGCGAAAAGAGCCUCUUCACCAAGGAGCUGGAACACGCGCUCGAGA
AGAACGAGGUGGACCUGGUCGUCCACAGCCUCAAGGACCUGCCUACGGUGC
UGCCGCCGGGAUUCACCAUCGGCGCCAUCUGUAAGCGGGAGAAUCCGCACG
ACGCCGUGGUGUUCCACCCUAAGUUCGUGGGCAAGACCCUCGAGACACUGC
CGGAAAAGUCCGUCGUGGGCACCUCCUCCCUGAGAAGGGCCGCUCAGCUCC
AGAGAAAGUUCCCGCACCUGGAAUUCAGGAGCAUCCGGGGCAACCUGAAUA
CCCGGCUUCGCAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACCGGGUGGGCCAGAUCC
UGCACCCGGAGGAGUGCAUGUAUGCCGUGGGUCAGGGAGCCCUGGGCGUGG
AGGUCCGGGCCAAGGACCAGGACAUCCUGGACCUCGUGGGCGUGCUCCACG
ACCCUGAAACCCUCCUGAGGUGCAUCGCCGAGAGGGCCUUCCUCCGGCACC
UGGAGGGCGGCUGUUCCGUCCCUGUGGCCGUGCAUACCGCCAUGAAGGACG
GACAGCUGUACCUGACCGGCGGCGUGUGGUCCCUGGACGGCUCCGACAGCA
UCCAGGAAACCAUGCAGGCCACUAUCCACGUGCCGGCCCAGCACGAAGACG
GCCCAGAGGAUGACCCGCAACUGGUCGGCAUUACCGCCAGGAACAUACCAA
GGGGCCCGCAGCUGGCCGCCCAGAACCUGGGCAUCUCCCUGGCCAACCUGC
UGCUGUCCAAGGGAGCCAAGAACAUUCUGGACGUGGCCAGGCAGCUCAAUG AUGCCCAC 147
#30 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGA
GCGGCAACGGCAACGCCGCCGCCACCGCAGAGGAGAAUAGCCCGAAGAUGA
GGGUGAUCCGAGUGGGCACCAGGAAGUCCCAGCUUGCGCGAAUUCAGACCG
ACAGCGUGGUGGCCACCCUCAAGGCCUCCUACCCGGGACUCCAGUUCGAGA
UCAUCGCCAUGAGCACCACGGGAGACAAGAUCCUGGACACCGCCCUGUCCA
AGAUCGGCGAAAAGAGCCUCUUCACCAAGGAGCUGGAGCACGCCCUGGAGA
AGAACGAGGUCGAUCUGGUGGUGCACAGCCUGAAGGACCUGCCGACCGUCC
UGCCGCCGGGAUUCACCAUCGGUGCCAUCUGUAAGCGGGAGAACCCGCACG
ACGCCGUGGUGUUCCACCCGAAGUUCGUCGGCAAGACCCUGGAAACCCUGC
CGGAGAAGUCCGUGGUGGGCACCAGCAGCCUGAGGCGGGCCGCCCAGCUGC
AGCGGAAGUUCCCGCACCUGGAAUUCAGGAGCAUCCGGGGCAACCUGAACA
CCCGGCUGCGGAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCAGGCCUCCAGCGCAUGGGAUGGCACAACAGGGUAGGCCAAAUCC
UGCACCCGGAGGAGUGUAUGUACGCCGUGGGCCAGGGAGCCCUGGGCGUGG
AGGUGAGAGCCAAGGACCAGGACAUCCUAGACCUGGUCGGCGUGCUGCACG
ACCCGGAGACACUGCUGAGAUGCAUCGCGGAGAGAGCCUUCCUGCGACACC
UGGAGGGCGGCUGCUCCGUGCCGGUGGCCGUGCACACCGCCAUGAAGGACG
GCCAGCUGUAUCUGACCGGCGGCGUGUGGAGCCUGGACGGCAGCGACUCCA
UCCAAGAAACCAUGCAGGCUACCAUCCACGUGCCGGCCCAGCACGAGGAUG
GACCAGAGGACGAUCCUCAACUGGUGGGCAUCACUGCCAGGAACAUCCCAA
GAGGCCCGCAGCUGGCCGCCCAGAACCUGGGCAUCAGCCUGGCCAACCUGC
UGCUGUCCAAGGGCGCCAAGAACAUUCUCGAUGUGGCCAGGCAGCUGAACG AUGCCCAC 148
#31 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGU
CGGGAAACGGCAACGCCGCCGCCACGGCCGAGGAGAACAGCCCGAAGAUGA
GAGUGAUUAGGGUGGGCACCCGGAAGUCCCAACUCGCGCGGAUCCAGACCG
ACUCCGUGGUGGCCACCCUCAAGGCCAGCUACCCGGGCCUCCAGUUCGAGA
UUAUCGCCAUGUCCACCACAGGCGACAAGAUCCUCGACACCGCACUCUCGA
AGAUCGGCGAGAAGUCCCUGUUCACCAAGGAACUGGAGCACGCCCUGGAGA
AGAACGAGGUGGACCUGGUGGUGCACUCCCUGAAGGACCUGCCGACCGUGC
UCCCACCAGGCUUCACCAUCGGCGCAAUCUGUAAGCGCGAGAAUCCGCACG
ACGCCGUGGUGUUCCACCCAAAGUUCGUGGGCAAGACCCUCGAAACCCUCC
CGGAAAAGAGCGUGGUGGGUACCAGCUCCCUGCGGAGAGCUGCCCAGCUGC
AGAGAAAGUUCCCGCAUCUGGAAUUCAGGAGCAUCAGGGGAAAUCUGAAUA
CCAGACUGCGCAAGCUGGACGAGCAGCAGGAGUUCAGCGCCAUCAUCCUGG
CCACCGCCGGCCUGCAGCGGAUGGGCUGGCACAACAGGGUGGGCCAGAUAC
UGCAUCCGGAGGAGUGUAUGUACGCCGUGGGCCAGGGCGCCCUCGGCGUGG
AGGUGAGAGCCAAGGACCAAGACAUCCUGGACCUAGUGGGCGUGCUGCAUG
ACCCUGAAACCCUGCUCAGGUGCAUCGCCGAGAGGGCCUUCCUGCGGCACC
UGGAGGGCGGCUGCAGCGUGCCGGUGGCCGUCCACACCGCCAUGAAGGACG
GCCAGCUGUACCUGACCGGCGGCGUCUGGAGCCUGGACGGAUCCGACAGCA
UCCAGGAAACCAUGCAGGCCACCAUCCACGUGCCGGCCCAGCACGAGGACG
GCCCUGAGGACGACCCUCAGCUGGUGGGCAUCACCGCUAGGAACAUCCCAA
GGGGCCCGCAGCUGGCCGCCCAGAACCUCGGCAUCAGCCUGGCCAACCUGC
UGCUGUCCAAGGGCGCCAAGAAUAUCCUGGACGUGGCCAGGCAGCUGAACG ACGCCCAC
20. METHODS OF MAKING POLYNUCLEOTIDES
[0849] The present disclosure also provides methods for making a
polynucleotide of the invention (e.g., a polynucleotide comprising
a nucleotide sequence encoding a PBGD polypeptide) or a complement
thereof.
[0850] In some aspects, a polynucleotide (e.g., a RNA, e.g., an
mRNA) disclosed herein, and encoding a PBGD polypeptide, can be
constructed using in vitro transcription. In other aspects, a
polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and
encoding a PBGD polypeptide, can be constructed by chemical
synthesis using an oligonucleotide synthesizer.
[0851] In other aspects, a polynucleotide (e.g., a RNA, e.g., an
mRNA) disclosed herein, and encoding a PBGD polypeptide is made by
using a host cell. In certain aspects, a polynucleotide (e.g., a
RNA, e.g., an mRNA) disclosed herein, and encoding a PBGD
polypeptide is made by one or more combination of the IVT, chemical
synthesis, host cell expression, or any other methods known in the
art.
[0852] Naturally occurring nucleosides, non-naturally occurring
nucleosides, or combinations thereof, can totally or partially
naturally replace occurring nucleosides present in the candidate
nucleotide sequence and can be incorporated into a
sequence-optimized nucleotide sequence (e.g., a RNA, e.g., an mRNA)
encoding a PBGD polypeptide. The resultant polynucleotides, e.g.,
mRNAs, can then be examined for their ability to produce protein
and/or produce a therapeutic outcome.
a. In Vitro Transcription/Enzymatic Synthesis
[0853] The polynucleotides of the present invention disclosed
herein (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide) can be transcribed using an in vitro
transcription (IVT) system. The system typically comprises a
transcription buffer, nucleotide triphosphates (NTPs), an RNase
inhibitor and a polymerase. The NTPs can be selected from, but are
not limited to, those described herein including natural and
unnatural (modified) NTPs. The polymerase can be selected from, but
is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant
polymerases such as, but not limited to, polymerases able to
incorporate polynucleotides disclosed herein. See U.S. Publ. No.
US20130259923, which is herein incorporated by reference in its
entirety.
[0854] Any number of RNA polymerases or variants can be used in the
synthesis of the polynucleotides of the present invention. RNA
polymerases can be modified by inserting or deleting amino acids of
the RNA polymerase sequence. As a non-limiting example, the RNA
polymerase can be modified to exhibit an increased ability to
incorporate a 2'-modified nucleotide triphosphate compared to an
unmodified RNA polymerase (see International Publication
WO2008078180 and U.S. Pat. No. 8,101,385; herein incorporated by
reference in their entireties).
[0855] Variants can be obtained by evolving an RNA polymerase,
optimizing the RNA polymerase amino acid and/or nucleic acid
sequence and/or by using other methods known in the art. As a
non-limiting example, T7 RNA polymerase variants can be evolved
using the continuous directed evolution system set out by Esvelt et
al. (Nature 472:499-503 (2011); herein incorporated by reference in
its entirety) where clones of T7 RNA polymerase can encode at least
one mutation such as, but not limited to, lysine at position 93
substituted for threonine (K93T), 14M, A7T, E63V, V64D, A65E, D66Y,
T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L,
L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M2671,
G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y,
S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523L, H524N,
G542V, E565K, K577E, K577M, N601S, S684Y, L6991, K713E, N748D,
Q754R, E775K, A827V, D851N or L864F. As another non-limiting
example, T7 RNA polymerase variants can encode at least mutation as
described in U.S. Pub. Nos. 20100120024 and 20070117112; herein
incorporated by reference in their entireties. Variants of RNA
polymerase can also include, but are not limited to, substitutional
variants, conservative amino acid substitution, insertional
variants, and/or deletional variants.
[0856] In one aspect, the polynucleotide can be designed to be
recognized by the wild type or variant RNA polymerases. In doing
so, the polynucleotide can be modified to contain sites or regions
of sequence changes from the wild type or parent chimeric
polynucleotide.
[0857] Polynucleotide or nucleic acid synthesis reactions can be
carried out by enzymatic methods utilizing polymerases. Polymerases
catalyze the creation of phosphodiester bonds between nucleotides
in a polynucleotide or nucleic acid chain. Currently known DNA
polymerases can be divided into different families based on amino
acid sequence comparison and crystal structure analysis. DNA
polymerase I (pol I) or A polymerase family, including the Klenow
fragments of E. coli, Bacillus DNA polymerase I, Thermus aquaticus
(Taq) DNA polymerases, and the T7 RNA and DNA polymerases, is among
the best studied of these families. Another large family is DNA
polymerase .alpha. (pol .alpha.) or B polymerase family, including
all eukaryotic replicating DNA polymerases and polymerases from
phages T4 and RB69. Although they employ similar catalytic
mechanism, these families of polymerases differ in substrate
specificity, substrate analog-incorporating efficiency, degree and
rate for primer extension, mode of DNA synthesis, exonuclease
activity, and sensitivity against inhibitors.
[0858] DNA polymerases are also selected based on the optimum
reaction conditions they require, such as reaction temperature, pH,
and template and primer concentrations. Sometimes a combination of
more than one DNA polymerases is employed to achieve the desired
DNA fragment size and synthesis efficiency. For example, Cheng et
al. increase pH, add glycerol and dimethyl sulfoxide, decrease
denaturation times, increase extension times, and utilize a
secondary thermostable DNA polymerase that possesses a 3' to 5'
exonuclease activity to effectively amplify long targets from
cloned inserts and human genomic DNA. (Cheng et al., PNAS
91:5695-5699 (1994), the contents of which are incorporated herein
by reference in their entirety). RNA polymerases from bacteriophage
T3, T7, and SP6 have been widely used to prepare RNAs for
biochemical and biophysical studies. RNA polymerases, capping
enzymes, and poly-A polymerases are disclosed in the co-pending
International Publication No. WO2014028429, the contents of which
are incorporated herein by reference in their entirety.
[0859] In one aspect, the RNA polymerase which can be used in the
synthesis of the polynucleotides of the present invention is a Syn5
RNA polymerase (see Zhu et al. Nucleic Acids Research 2013, doi:
10.1093/nar/gktl 193, which is herein incorporated by reference in
its entirety). The Syn5 RNA polymerase was recently characterized
from marine cyanophage Syn5 by Zhu et al. where they also
identified the promoter sequence (see Zhu et al. Nucleic Acids
Research 2013, the contents of which is herein incorporated by
reference in its entirety). Zhu et al. found that Syn5 RNA
polymerase catalyzed RNA synthesis over a wider range of
temperatures and salinity as compared to T7 RNA polymerase.
Additionally, the requirement for the initiating nucleotide at the
promoter was found to be less stringent for Syn5 RNA polymerase as
compared to the T7 RNA polymerase making Syn5 RNA polymerase
promising for RNA synthesis.
[0860] In one aspect, a Syn5 RNA polymerase can be used in the
synthesis of the polynucleotides described herein. As a
non-limiting example, a Syn5 RNA polymerase can be used in the
synthesis of the polynucleotide requiring a precise
3'-terminus.
[0861] In one aspect, a Syn5 promoter can be used in the synthesis
of the polynucleotides. As a non-limiting example, the Syn5
promoter can be 5'-ATTGGGCACCCGTAAGGG-3' (SEQ ID NO: 82 as
described by Zhu et al. (Nucleic Acids Research 2013).
[0862] In one aspect, a Syn5 RNA polymerase can be used in the
synthesis of polynucleotides comprising at least one chemical
modification described herein and/or known in the art (see e.g.,
the incorporation of pseudo-UTP and 5Me-CTP described in Zhu et al.
Nucleic Acids Research 2013).
[0863] In one aspect, the polynucleotides described herein can be
synthesized using a Syn5 RNA polymerase which has been purified
using modified and improved purification procedure described by Zhu
et al. (Nucleic Acids Research 2013).
[0864] Various tools in genetic engineering are based on the
enzymatic amplification of a target gene which acts as a template.
For the study of sequences of individual genes or specific regions
of interest and other research needs, it is necessary to generate
multiple copies of a target gene from a small sample of
polynucleotides or nucleic acids. Such methods can be applied in
the manufacture of the polynucleotides of the invention.
[0865] For example, polymerase chain reaction (PCR), strand
displacement amplification (SDA), nucleic acid sequence-based
amplification (NASBA), also called transcription mediated
amplification (TMA), and/or rolling-circle amplification (RCA) can
be utilized in the manufacture of one or more regions of the
polynucleotides of the present invention. Assembling
polynucleotides or nucleic acids by a ligase is also widely
used.
b. Chemical Synthesis
[0866] Standard methods can be applied to synthesize an isolated
polynucleotide sequence encoding an isolated polypeptide of
interest, such as a polynucleotide of the invention (e.g., a
polynucleotide comprising a nucleotide sequence encoding a PBGD
polypeptide). For example, a single DNA or RNA oligomer containing
a codon-optimized nucleotide sequence coding for the particular
isolated polypeptide can be synthesized. In other aspects, several
small oligonucleotides coding for portions of the desired
polypeptide can be synthesized and then ligated. In some aspects,
the individual oligonucleotides typically contain 5' or 3'
overhangs for complementary assembly.
[0867] A polynucleotide disclosed herein (e.g., a RNA, e.g., an
mRNA) can be chemically synthesized using chemical synthesis
methods and potential nucleobase substitutions known in the art.
See, for example, International Publication Nos. WO2014093924,
WO2013052523; WO2013039857, WO2012135805, WO2013151671; U.S. Publ.
No. US20130115272; or U.S. Pat. Nos. 8,999,380 or 8,710,200, all of
which are herein incorporated by reference in their entireties.
c. Purification of Polynucleotides Encoding PBGD
[0868] Purification of the polynucleotides described herein (e.g.,
a polynucleotide comprising a nucleotide sequence encoding a PBGD
polypeptide) can include, but is not limited to, polynucleotide
clean-up, quality assurance and quality control. Clean-up can be
performed by methods known in the arts such as, but not limited to,
AGENCOURT.RTM. beads (Beckman Coulter Genomics, Danvers, Mass.),
poly-T beads, LNA.TM. oligo-T capture probes (EXIQON.RTM. Inc.,
Vedbaek, Denmark) or HPLC based purification methods such as, but
not limited to, strong anion exchange HPLC, weak anion exchange
HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction
HPLC (HIC-HPLC).
[0869] The term "purified" when used in relation to a
polynucleotide such as a "purified polynucleotide" refers to one
that is separated from at least one contaminant. As used herein, a
"contaminant" is any substance that makes another unfit, impure or
inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is
present in a form or setting different from that in which it is
found in nature, or a form or setting different from that which
existed prior to subjecting it to a treatment or purification
method.
[0870] In some embodiments, purification of a polynucleotide of the
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide) removes impurities that can reduce or
remove an unwanted immune response, e.g., reducing cytokine
activity.
[0871] In some embodiments, the polynucleotide of the invention
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) is purified prior to administration using column
chromatography (e.g., strong anion exchange HPLC, weak anion
exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic
interaction HPLC (HIC-HPLC), or (LCMS)).
[0872] In some embodiments, the polynucleotide of the invention
(e.g., a polynucleotide comprising a nucleotide sequence a PBGD
polypeptide) purified using column chromatography (e.g., strong
anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC, hydrophobic interaction HPLC (HIC-HPLC), or (LCMS))
presents increased expression of the encoded PBGD protein compared
to the expression level obtained with the same polynucleotide of
the present disclosure purified by a different purification
method.
[0873] In some embodiments, a column chromatography (e.g., strong
anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC), hydrophobic interaction HPLC (HIC-HPLC), or (LCMS))
purified polynucleotide comprises a nucleotide sequence encoding a
PBGD polypeptide comprising one or more of the point mutations
known in the art.
[0874] In some embodiments, the use of RP-HPLC purified
polynucleotide increases PBGD protein expression levels in cells
when introduced into those cells, e.g., by 10-100%, i.e., at least
about 10%, at least about 20%, at least about 25%, at least about
30%, at least about 35%, at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 90%, at least about 95%, or at least about 100%
with respect to the expression levels of PBGD protein in the cells
before the RP-HPLC purified polynucleotide was introduced in the
cells, or after a non-RP-HPLC purified polynucleotide was
introduced in the cells.
[0875] In some embodiments, the use of RP-HPLC purified
polynucleotide increases functional PBGD protein expression levels
in cells when introduced into those cells, e.g., by 10-100%, i.e.,
at least about 10%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95%, or at
least about 100% with respect to the functional expression levels
of PBGD protein in the cells before the RP-HPLC purified
polynucleotide was introduced in the cells, or after a non-RP-HPLC
purified polynucleotide was introduced in the cells.
[0876] In some embodiments, the use of RP-HPLC purified
polynucleotide increases detectable PBGD activity in cells when
introduced into those cells, e.g., by 10-100%, i.e., at least about
10%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 90%, at least about 95%, or at least about 100% with
respect to the activity levels of functional PBGD in the cells
before the RP-HPLC purified polynucleotide was introduced in the
cells, or after a non-RP-HPLC purified polynucleotide was
introduced in the cells.
[0877] In some embodiments, the purified polynucleotide is at least
about 80% pure, at least about 85% pure, at least about 90% pure,
at least about 95% pure, at least about 96% pure, at least about
97% pure, at least about 98% pure, at least about 99% pure, or
about 100% pure.
[0878] A quality assurance and/or quality control check can be
conducted using methods such as, but not limited to, gel
electrophoresis, UV absorbance, or analytical HPLC. In another
embodiment, the polynucleotide can be sequenced by methods
including, but not limited to reverse-transcriptase-PCR.
d. Quantification of Expressed Polynucleotides Encoding PBGD
[0879] In some embodiments, the polynucleotides of the present
invention (e.g., a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide), their expression products, as well as
degradation products and metabolites can be quantified according to
methods known in the art.
[0880] In some embodiments, the polynucleotides of the present
invention can be quantified in exosomes or when derived from one or
more bodily fluid. As used herein "bodily fluids" include
peripheral blood, serum, plasma, ascites, urine, cerebrospinal
fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous
humor, amniotic fluid, cerumen, breast milk, broncheoalveolar
lavage fluid, semen, prostatic fluid, cowper's fluid or
pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst
fluid, pleural and peritoneal fluid, pericardial fluid, lymph,
chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit,
vaginal secretions, mucosal secretion, stool water, pancreatic
juice, lavage fluids from sinus cavities, bronchopulmonary
aspirates, blastocyl cavity fluid, and umbilical cord blood.
Alternatively, exosomes can be retrieved from an organ selected
from the group consisting of lung, heart, pancreas, stomach,
intestine, bladder, kidney, ovary, testis, skin, colon, breast,
prostate, brain, esophagus, liver, and placenta.
[0881] In the exosome quantification method, a sample of not more
than 2 mL is obtained from the subject and the exosomes isolated by
size exclusion chromatography, density gradient centrifugation,
differential centrifugation, nanomembrane ultrafiltration,
immunoabsorbent capture, affinity purification, microfluidic
separation, or combinations thereof. In the analysis, the level or
concentration of a polynucleotide can be an expression level,
presence, absence, truncation or alteration of the administered
construct. It is advantageous to correlate the level with one or
more clinical phenotypes or with an assay for a human disease
biomarker.
[0882] The assay can be performed using construct specific probes,
cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry,
electrophoresis, mass spectrometry, or combinations thereof while
the exosomes can be isolated using immunohistochemical methods such
as enzyme linked immunosorbent assay (ELISA) methods. Exosomes can
also be isolated by size exclusion chromatography, density gradient
centrifugation, differential centrifugation, nanomembrane
ultrafiltration, immunoabsorbent capture, affinity purification,
microfluidic separation, or combinations thereof.
[0883] These methods afford the investigator the ability to
monitor, in real time, the level of polynucleotides remaining or
delivered. This is possible because the polynucleotides of the
present invention differ from the endogenous forms due to the
structural or chemical modifications.
[0884] In some embodiments, the polynucleotide can be quantified
using methods such as, but not limited to, ultraviolet visible
spectroscopy (UV/Vis). A non-limiting example of a UV/Vis
spectrometer is a NANODROP.RTM. spectrometer (ThermoFisher,
Waltham, Mass.). The quantified polynucleotide can be analyzed in
order to determine if the polynucleotide can be of proper size,
check that no degradation of the polynucleotide has occurred.
Degradation of the polynucleotide can be checked by methods such
as, but not limited to, agarose gel electrophoresis, HPLC based
purification methods such as, but not limited to, strong anion
exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid
chromatography-mass spectrometry (LCMS), capillary electrophoresis
(CE) and capillary gel electrophoresis (CGE).
21. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS
[0885] The present invention provides pharmaceutical compositions
and formulations that comprise any of the polynucleotides described
above. In some embodiments, the composition or formulation further
comprises a delivery agent.
[0886] In some embodiments, the composition or formulation can
contain a polynucleotide comprising a sequence optimized nucleic
acid sequence disclosed herein which encodes a PBGD polypeptide. In
some embodiments, the composition or formulation can contain a
polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a
polynucleotide (e.g., an ORF) having significant sequence identity
to a sequence optimized nucleic acid sequence disclosed herein
which encodes a PBGD polypeptide. In some embodiments, the
polynucleotide further comprises a miRNA binding site, e.g., a
miRNA binding site that binds miR-126, miR-142, miR-144, miR-146,
miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27 and
miR-26a.
[0887] Pharmaceutical compositions or formulation can optionally
comprise one or more additional active substances, e.g.,
therapeutically and/or prophylactically active substances.
Pharmaceutical compositions or formulation of the present invention
can be sterile and/or pyrogen-free. General considerations in the
formulation and/or manufacture of pharmaceutical agents can be
found, for example, in Remington: The Science and Practice of
Pharmacy 21.sup.st ed., Lippincott Williams & Wilkins, 2005
(incorporated herein by reference in its entirety). In some
embodiments, compositions are administered to humans, human
patients or subjects. For the purposes of the present disclosure,
the phrase "active ingredient" generally refers to polynucleotides
to be delivered as described herein.
[0888] Formulations and pharmaceutical compositions described
herein can be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of associating the active ingredient with an
excipient and/or one or more other accessory ingredients, and then,
if necessary and/or desirable, dividing, shaping and/or packaging
the product into a desired single- or multi-dose unit.
[0889] A pharmaceutical composition or formulation in accordance
with the present disclosure can be prepared, packaged, and/or sold
in bulk, as a single unit dose, and/or as a plurality of single
unit doses. As used herein, a "unit dose" refers to a discrete
amount of the pharmaceutical composition comprising a predetermined
amount of the active ingredient. The amount of the active
ingredient is generally equal to the dosage of the active
ingredient that would be administered to a subject and/or a
convenient fraction of such a dosage such as, for example, one-half
or one-third of such a dosage.
[0890] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
present disclosure can vary, depending upon the identity, size,
and/or condition of the subject being treated and further depending
upon the route by which the composition is to be administered.
[0891] In some embodiments, the compositions and formulations
described herein can contain at least one polynucleotide of the
invention. As a non-limiting example, the composition or
formulation can contain 1, 2, 3, 4 or 5 polynucleotides of the
invention. In some embodiments, the compositions or formulations
described herein can comprise more than one type of polynucleotide.
In some embodiments, the composition or formulation can comprise a
polynucleotide in linear and circular form. In another embodiment,
the composition or formulation can comprise a circular
polynucleotide and an in vitro transcribed (IVT) polynucleotide. In
yet another embodiment, the composition or formulation can comprise
an IVT polynucleotide, a chimeric polynucleotide and a circular
polynucleotide.
[0892] Although the descriptions of pharmaceutical compositions and
formulations provided herein are principally directed to
pharmaceutical compositions and formulations that are suitable for
administration to humans, it will be understood by the skilled
artisan that such compositions are generally suitable for
administration to any other animal, e.g., to non-human animals,
e.g. non-human mammals.
[0893] The present invention provides pharmaceutical formulations
that comprise a polynucleotide described herein (e.g., a
polynucleotide comprising a nucleotide sequence encoding a PBGD
polypeptide). The polynucleotides described herein can be
formulated using one or more excipients to: (1) increase stability;
(2) increase cell transfection; (3) permit the sustained or delayed
release (e.g., from a depot formulation of the polynucleotide); (4)
alter the biodistribution (e.g., target the polynucleotide to
specific tissues or cell types); (5) increase the translation of
encoded protein in vivo; and/or (6) alter the release profile of
encoded protein in vivo. In some embodiments, the pharmaceutical
formulation disclosed herein further comprises a delivery agent
comprising, e.g., a compound having the Formula (I), e.g., any of
Compounds 1-232, e.g., Compound 18; a compound having the Formula
(III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g.,
Compound 236; or a compound having the Formula (VIII), e.g., any of
Compounds 419-428, e.g., Compound 428, or any combination thereof.
In some embodiments, the delivery agent comprises Compound 18,
DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of
about 50:10:38.5:1.5.
[0894] A pharmaceutically acceptable excipient, as used herein,
includes, but are not limited to, any and all solvents, dispersion
media, or other liquid vehicles, dispersion or suspension aids,
diluents, granulating and/or dispersing agents, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, binders, lubricants or oil, coloring, sweetening or
flavoring agents, stabilizers, antioxidants, antimicrobial or
antifungal agents, osmolality adjusting agents, pH adjusting
agents, buffers, chelants, cyoprotectants, and/or bulking agents,
as suited to the particular dosage form desired. Various excipients
for formulating pharmaceutical compositions and techniques for
preparing the composition are known in the art (see Remington: The
Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro
(Lippincott, Williams & Wilkins, Baltimore, Md., 2006;
incorporated herein by reference in its entirety).
[0895] Exemplary diluents include, but are not limited to, calcium
or sodium carbonate, calcium phosphate, calcium hydrogen phosphate,
sodium phosphate, lactose, sucrose, cellulose, microcrystalline
cellulose, kaolin, mannitol, sorbitol, etc., and/or combinations
thereof.
[0896] Exemplary granulating and/or dispersing agents include, but
are not limited to, starches, pregelatinized starches, or
microcrystalline starch, alginic acid, guar gum, agar,
poly(vinyl-pyrrolidone), providone, cross-linked
poly(vinyl-pyrrolidone) (crospovidone), cellulose, methylcellulose,
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), magnesium aluminum silicate
(VEEGUM.RTM.), sodium lauryl sulfate, etc., and/or combinations
thereof.
[0897] Exemplary surface active agents and/or emulsifiers include,
but are not limited to, natural emulsifiers (e.g., acacia, agar,
alginic acid, sodium alginate, tragacanth, chondrux, cholesterol,
xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol,
wax, and lecithin), sorbitan fatty acid esters (e.g.,
polyoxyethylene sorbitan monooleate [TWEEN.RTM. 80], sorbitan
monopalmitate [SPAN.RTM. 40], glyceryl monooleate, polyoxyethylene
esters, polyethylene glycol fatty acid esters (e.g.,
CREMOPHOR.RTM.), polyoxyethylene ethers (e.g., polyoxyethylene
lauryl ether [BRIJ.RTM. 30]), PLUORINC.RTM. F 68, POLOXAMER.RTM.
188, etc. and/or combinations thereof.
[0898] Exemplary binding agents include, but are not limited to,
starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,
molasses, lactose, lactitol, mannitol), amino acids (e.g.,
glycine), natural and synthetic gums (e.g., acacia, sodium
alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropyl
methylcellulose, etc., and combinations thereof.
[0899] Oxidation is a potential degradation pathway for mRNA,
especially for liquid mRNA formulations. In order to prevent
oxidation, antioxidants can be added to the formulations. Exemplary
antioxidants include, but are not limited to, alpha tocopherol,
ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated
hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene,
monothioglycerol, sodium or potassium metabisulfite, propionic
acid, propyl gallate, sodium ascorbate, etc., and combinations
thereof.
[0900] Exemplary chelating agents include, but are not limited to,
ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,
disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium
edetate, tartaric acid, trisodium edetate, etc., and combinations
thereof.
[0901] Exemplary antimicrobial or antifungal agents include, but
are not limited to, benzalkonium chloride, benzethonium chloride,
methyl paraben, ethyl paraben, propyl paraben, butyl paraben,
benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate,
potassium or sodium sorbate, sodium propionate, sorbic acid, etc.,
and combinations thereof.
[0902] Exemplary preservatives include, but are not limited to,
vitamin A, vitamin C, vitamin E, beta-carotene, citric acid,
ascorbic acid, butylated hydroxyanisol, ethylenediamine, sodium
lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and
combinations thereof.
[0903] In some embodiments, the pH of polynucleotide solutions are
maintained between pH 5 and pH 8 to improve stability. Exemplary
buffers to control pH can include, but are not limited to sodium
phosphate, sodium citrate, sodium succinate, histidine (or
histidine-HCl), sodium malate, sodium carbonate, etc., and/or
combinations thereof.
[0904] Exemplary lubricating agents include, but are not limited
to, magnesium stearate, calcium stearate, stearic acid, silica,
talc, malt, hydrogenated vegetable oils, polyethylene glycol,
sodium benzoate, sodium or magnesium lauryl sulfate, etc., and
combinations thereof.
[0905] The pharmaceutical composition or formulation described here
can contain a cryoprotectant to stabilize a polynucleotide
described herein during freezing. Exemplary cryoprotectants
include, but are not limited to mannitol, sucrose, trehalose,
lactose, glycerol, dextrose, etc., and combinations thereof.
[0906] The pharmaceutical composition or formulation described here
can contain a bulking agent in lyophilized polynucleotide
formulations to yield a "pharmaceutically elegant" cake, stabilize
the lyophilized polynucleotides during long term (e.g., 36 month)
storage. Exemplary bulking agents of the present invention can
include, but are not limited to sucrose, trehalose, mannitol,
glycine, lactose, raffinose, and combinations thereof.
[0907] In some embodiments, the pharmaceutical composition or
formulation further comprises a delivery agent. The delivery agent
of the present disclosure can include, without limitation,
liposomes, lipid nanoparticles, lipidoids, polymers, lipoplexes,
microvesicles, exosomes, peptides, proteins, cells transfected with
polynucleotides, hyaluronidase, nanoparticle mimics, nanotubes,
conjugates, and combinations thereof.
22. DELIVERY AGENTS
[0908] a. Lipid Compound
[0909] The present disclosure provides pharmaceutical compositions
with advantageous properties. The lipid compositions described
herein may be advantageously used in lipid nanoparticle
compositions for the delivery of therapeutic and/or prophylactic
agents, e.g., mRNAs, to mammalian cells or organs. For example, the
lipids described herein have little or no immunogenicity. For
example, the lipid compounds disclosed herein have a lower
immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or
DLinDMA). For example, a formulation comprising a lipid disclosed
herein and a therapeutic or prophylactic agent, e.g., mRNA, has an
increased therapeutic index as compared to a corresponding
formulation which comprises a reference lipid (e.g., MC3, KC2, or
DLinDMA) and the same therapeutic or prophylactic agent.
[0910] In certain embodiments, the present application provides
pharmaceutical compositions comprising:
[0911] (a) a polynucleotide comprising a nucleotide sequence
encoding a PBGD polypeptide; and
[0912] (b) a delivery agent.
[0913] In some embodiments, the delivery agent comprises a lipid
compound having the Formula (I)
##STR00026##
wherein
[0914] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0915] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0916] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --N(R)R.sub.8, --O(CH.sub.2).sub.nOR,
--N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and --C(R)N(R).sub.2C(O)OR, and
each n is independently selected from 1, 2, 3, 4, and 5;
[0917] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0918] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0919] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0920] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0921] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0922] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0923] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0924] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0925] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0926] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0927] each Y is independently a C.sub.3-6 carbocycle;
[0928] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0929] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or
salts or stereoisomers thereof.
[0930] In some embodiments, a subset of compounds of Formula (I)
includes those in which R.sub.1 is selected from the group
consisting of C.sub.5-20 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R';
[0931] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0932] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, and --C(R)N(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5;
[0933] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0934] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0935] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[0936] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0937] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0938] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0939] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0940] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0941] each Y is independently a C.sub.3-6 carbocycle;
[0942] each X is independently selected from the group consisting
of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11,
12, and 13,
[0943] or salts or stereoisomers thereof, wherein alkyl and alkenyl
groups may be linear or branched.
[0944] In some embodiments, a subset of compounds of Formula (I)
includes those in which when R.sub.4 is --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, or --CQ(R).sub.2, then (i) Q is not
--N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or
7-membered heterocycloalkyl when n is 1 or 2.
[0945] In another embodiments, another subset of compounds of
Formula (I) includes those in which R.sub.1 is selected from the
group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R';
[0946] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0947] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, --N(R)R.sub.8,
--O(CH.sub.2).sub.nOR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9) N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and a 5- to 14-membered
heterocycloalkyl having one or more heteroatoms selected from N, O,
and S which is substituted with one or more substituents selected
from oxo (.dbd.O), OH, amino, and C.sub.1-3 alkyl, and each n is
independently selected from 1, 2, 3, 4, and 5;
[0948] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0949] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0950] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0951] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0952] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0953] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0954] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0955] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0956] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0957] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0958] each Y is independently a C.sub.3-6 carbocycle;
[0959] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0960] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[0961] or salts or stereoisomers thereof.
[0962] In another embodiments, another subset of compounds of
Formula (I) includes those in which
[0963] R.sub.1 is selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0964] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0965] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, and a 5- to 14-membered
heterocycloalkyl having one or more heteroatoms selected from N, O,
and S which is substituted with one or more substituents selected
from oxo (.dbd.O), OH, amino, and C.sub.1-3 alkyl, and each n is
independently selected from 1, 2, 3, 4, and 5;
[0966] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0967] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0968] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[0969] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0970] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0971] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0972] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0973] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0974] each Y is independently a C.sub.3-6 carbocycle;
[0975] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0976] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or
salts or stereoisomers thereof.
[0977] In yet another embodiments, another subset of compounds of
Formula (I) includes those in which
[0978] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0979] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0980] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heterocycle having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, --N(R)R.sub.8,
--O(CH.sub.2).sub.nOR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)R,
--C(O)N(R)OR, and --C(.dbd.NR.sub.9)N(R).sub.2, and each n is
independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-
to 14-membered heterocycle and (i) R.sub.4 is --(CH.sub.2).sub.nQ
in which n is 1 or 2, or (ii) R.sub.4 is --(CH.sub.2).sub.nCHQR in
which n is 1, or (iii) R.sub.4 is --CHQR, and --CQ(R).sub.2, then Q
is either a 5- to 14-membered heteroaryl or 8- to 14-membered
heterocycloalkyl;
[0981] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0982] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0983] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0984] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0985] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0986] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0987] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0988] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0989] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0990] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0991] each Y is independently a C.sub.3-6 carbocycle;
[0992] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0993] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[0994] or salts or stereoisomers thereof.
[0995] In yet another embodiments, another subset of compounds of
Formula (I) includes those in which R.sub.1 is selected from the
group consisting of C.sub.5-20 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R';
[0996] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0997] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heterocycle having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-
to 14-membered heterocycle and (i) R.sub.4 is --(CH.sub.2).sub.nQ
in which n is 1 or 2, or (ii) R.sub.4 is --(CH.sub.2).sub.nCHQR in
which n is 1, or (iii) R.sub.4 is --CHQR, and --CQ(R).sub.2, then Q
is either a 5- to 14-membered heteroaryl or 8- to 14-membered
heterocycloalkyl; each R.sub.5 is independently selected from the
group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0998] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0999] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[1000] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1001] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1002] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1003] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1004] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1005] each Y is independently a C.sub.3-6 carbocycle;
[1006] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1007] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[1008] or salts or stereoisomers thereof.
[1009] In still another embodiments, another subset of compounds of
Formula (I) includes those in which
[1010] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1011] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[1012] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, --N(R)R.sub.8,
--O(CH.sub.2).sub.nOR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)R,
--C(O)N(R)OR, and --C(.dbd.NR.sub.9)N(R).sub.2, and each n is
independently selected from 1, 2, 3, 4, and 5;
[1013] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1014] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1015] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[1016] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1017] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[1018] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[1019] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1020] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1021] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1022] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1023] each Y is independently a C.sub.3-6 carbocycle;
[1024] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1025] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[1026] or salts or stereoisomers thereof.
[1027] In still another embodiments, another subset of compounds of
Formula (I) includes those in which
[1028] R.sub.1 is selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1029] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[1030] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5;
[1031] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1032] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1033] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[1034] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1035] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1036] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1037] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1038] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1039] each Y is independently a C.sub.3-6 carbocycle;
[1040] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1041] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[1042] or salts or stereoisomers thereof.
[1043] In yet another embodiments, another subset of compounds of
Formula (I) includes those in which
[1044] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1045] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.2-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[1046] R.sub.4 is --(CH.sub.2).sub.nQ or --(CH.sub.2).sub.nCHQR,
where Q is --N(R).sub.2, and n is selected from 3, 4, and 5;
[1047] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1048] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1049] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[1050] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1051] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1052] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1053] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1054] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.1-12 alkenyl;
[1055] each Y is independently a C.sub.3-6 carbocycle;
[1056] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1057] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[1058] or salts or stereoisomers thereof.
[1059] In yet another embodiments, another subset of compounds of
Formula (I) includes those in which
[1060] R.sub.1 is selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1061] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.2-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[1062] R.sub.4 is --(CH.sub.2).sub.nQ or --(CH.sub.2).sub.nCHQR,
where Q is --N(R).sub.2, and n is selected from 3, 4, and 5;
[1063] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1064] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1065] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[1066] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1067] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1068] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1069] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1070] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.1-12 alkenyl;
[1071] each Y is independently a C.sub.3-6 carbocycle;
[1072] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1073] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[1074] or salts or stereoisomers thereof.
[1075] In still other embodiments, another subset of compounds of
Formula (I) includes those in which
[1076] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1077] R.sub.2 and R.sub.3 are independently selected from the
group consisting of C.sub.1-14 alkyl, C.sub.2-14 alkenyl, --R*YR'',
--YR'', and --R*OR'', or R.sub.2 and R.sub.3, together with the
atom to which they are attached, form a heterocycle or
carbocycle;
[1078] R.sub.4 is selected from the group consisting of
--(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR, and
--CQ(R).sub.2, where Q is --N(R).sub.2, and n is selected from 1,
2, 3, 4, and 5; each R.sub.5 is independently selected from the
group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1079] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1080] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[1081] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1082] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1083] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1084] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1085] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.1-12 alkenyl;
[1086] each Y is independently a C.sub.3-6 carbocycle;
[1087] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1088] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[1089] or salts or stereoisomers thereof.
[1090] In still other embodiments, another subset of compounds of
Formula (I) includes those in which
[1091] R.sub.1 is selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1092] R.sub.2 and R.sub.3 are independently selected from the
group consisting of C.sub.1-14 alkyl, C.sub.2-14 alkenyl, --R*YR'',
--YR'', and --R*OR'', or R.sub.2 and R.sub.3, together with the
atom to which they are attached, form a heterocycle or
carbocycle;
[1093] R.sub.4 is selected from the group consisting of
--(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR, and
--CQ(R).sub.2, where Q is --N(R).sub.2, and n is selected from 1,
2, 3, 4, and 5;
[1094] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1095] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1096] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[1097] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1098] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1099] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1100] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1101] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.1-12 alkenyl;
[1102] each Y is independently a C.sub.3-6 carbocycle;
[1103] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1104] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[1105] or salts or stereoisomers thereof.
[1106] In certain embodiments, a subset of compounds of Formula (I)
includes those of Formula (IA):
##STR00027##
[1107] or a salt or stereoisomer thereof, wherein l is selected
from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9;
M.sub.1 is a bond or M'; R.sub.4 is unsubstituted C.sub.1-3 alkyl,
or --(CH.sub.2).sub.nQ, in which Q is OH, --NHC(S)N(R).sub.2,
--NHC(O)N(R).sub.2, --N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)R.sub.8,
--NHC(.dbd.NR.sub.9)N(R).sub.2, --NHC(.dbd.CHR.sub.9)N(R).sub.2,
--OC(O)N(R).sub.2, --N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M
and M' are independently selected from --C(O)O--, --OC(O)--,
--C(O)N(R')--, --P(O)(OR')O--, --S--S--, an aryl group, and a
heteroaryl group; and
[1108] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[1109] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (IA), or a salt or stereoisomer thereof,
wherein
[1110] l is selected from 1, 2, 3, 4, and 5; m is selected from 5,
6, 7, 8, and 9;
[1111] M.sub.1 is a bond or M';
[1112] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which Q is OH, --NHC(S)N(R).sub.2, or
--NHC(O)N(R).sub.2;
[1113] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, an aryl group, and a
heteroaryl group; and
[1114] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[1115] In certain embodiments, a subset of compounds of Formula (I)
includes those of Formula (II):
##STR00028##
[1116] or a salt or stereoisomer thereof, wherein l is selected
from 1, 2, 3, 4, and 5; M.sub.1 is a bond or M'; R.sub.4 is
unsubstituted C.sub.1-3 alkyl, or --(CH.sub.2).sub.nQ, in which n
is 2, 3, or 4, and Q is OH, --NHC(S)N(R).sub.2, --NHC(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)R.sub.8,
--NHC(.dbd.NR.sub.9)N(R).sub.2, --NHC(.dbd.CHR.sub.9)N(R).sub.2,
--OC(O)N(R).sub.2, --N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M
and M' are independently selected from --C(O)O--, --OC(O)--,
--C(O)N(R')--, --P(O)(OR')O--, --S--S--, an aryl group, and a
heteroaryl group; and
[1117] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[1118] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (II), or a salt or stereoisomer thereof,
wherein
[1119] l is selected from 1, 2, 3, 4, and 5; M.sub.1 is a bond or
M';
[1120] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 2, 3, or 4, and Q is OH,
--NHC(S)N(R).sub.2, or --NHC(O)N(R).sub.2;
[1121] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, an aryl group, and a
heteroaryl group; and
[1122] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[1123] In some embodiments, the compound of Formula (I) is of the
Formula (IIa),
##STR00029##
or a salt thereof, wherein R.sub.4 is as described above.
[1124] In some embodiments, the compound of Formula (I) is of the
Formula (IIb),
##STR00030##
or a salt thereof, wherein R.sub.4 is as described above.
[1125] In some embodiments, the compound of Formula (I) is of the
Formula (IIc),
##STR00031##
or a salt thereof, wherein R.sub.4 is as described above.
[1126] In some embodiments, the compound of Formula (I) is of the
Formula (IIe):
##STR00032##
or a salt thereof, wherein R.sub.4 is as described above.
[1127] In some embodiments, the compound of Formula (IIa), (IIb),
(IIc), or (IIe) comprises an R.sub.4 which is selected from
--(CH.sub.2).sub.nQ and --(CH.sub.2).sub.nCHQR, wherein Q, R and n
are as defined above.
[1128] In some embodiments, Q is selected from the group consisting
of --OR, --OH, --O(CH.sub.2).sub.nN(R).sub.2, --OC(O)R, --CX.sub.3,
--CN, --N(R)C(O)R, --N(H)C(O)R, --N(R)S(O).sub.2R,
--N(H)S(O).sub.2R, --N(R)C(O)N(R).sub.2, --N(H)C(O)N(R).sub.2,
--N(H)C(O)N(H)(R), --N(R)C(S)N(R).sub.2, --N(H)C(S)N(R).sub.2,
--N(H)C(S)N(H)(R), and a heterocycle, wherein R is as defined
above. In some aspects, n is 1 or 2. In some embodiments, Q is OH,
--NHC(S)N(R).sub.2, or --NHC(O)N(R).sub.2.
[1129] In some embodiments, the compound of Formula (I) is of the
Formula (IId),
##STR00033##
or a salt thereof, wherein R.sub.2 and R.sub.3 are independently
selected from the group consisting of C.sub.5-14 alkyl and
C.sub.5-14 alkenyl, n is selected from 2, 3, and 4, and R', R'',
R.sub.5, R.sub.6 and m are as defined above.
[1130] In some aspects of the compound of Formula (IId), R.sub.2 is
C.sub.8 alkyl. In some aspects of the compound of Formula (IId),
R.sub.3 is C.sub.5-C.sub.9 alkyl. In some aspects of the compound
of Formula (IId), m is 5, 7, or 9. In some aspects of the compound
of Formula (IId), each R.sub.5 is H. In some aspects of the
compound of Formula (IId), each R.sub.6 is H.
[1131] In another aspect, the present application provides a lipid
composition (e.g., a lipid nanoparticle (LNP)) comprising: (1) a
compound having the Formula (I); (2) optionally a helper lipid
(e.g. a phospholipid); (3) optionally a structural lipid (e.g. a
sterol); and (4) optionally a lipid conjugate (e.g. a PEG-lipid).
In exemplary embodiments, the lipid composition (e.g., LNP) further
comprises a polynucleotide encoding a PBGD polypeptide, e.g., a
polynucleotide encapsulated therein.
[1132] As used herein, the term "alkyl" or "alkyl group" means a
linear or branched, saturated hydrocarbon including one or more
carbon atoms (e.g., one, two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,
seventeen, eighteen, nineteen, twenty, or more carbon atoms).
[1133] The notation "C.sub.1-14 alkyl" means a linear or branched,
saturated hydrocarbon including 1-14 carbon atoms. An alkyl group
can be optionally substituted.
[1134] As used herein, the term "alkenyl" or "alkenyl group" means
a linear or branched hydrocarbon including two or more carbon atoms
(e.g., two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,
eighteen, nineteen, twenty, or more carbon atoms) and at least one
double bond.
[1135] The notation "C.sub.2-14 alkenyl" means a linear or branched
hydrocarbon including 2-14 carbon atoms and at least one double
bond. An alkenyl group can include one, two, three, four, or more
double bonds. For example, Cis alkenyl can include one or more
double bonds. A Cis alkenyl group including two double bonds can be
a linoleyl group. An alkenyl group can be optionally
substituted.
[1136] As used herein, the term "carbocycle" or "carbocyclic group"
means a mono- or multi-cyclic system including one or more rings of
carbon atoms. Rings can be three, four, five, six, seven, eight,
nine, ten, eleven, twelve, thirteen, fourteen, or fifteen membered
rings.
[1137] The notation "C.sub.3-6 carbocycle" means a carbocycle
including a single ring having 3-6 carbon atoms. Carbocycles can
include one or more double bonds and can be aromatic (e.g., aryl
groups). Examples of carbocycles include cyclopropyl, cyclopentyl,
cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups.
Carbocycles can be optionally substituted.
[1138] As used herein, the term "heterocycle" or "heterocyclic
group" means a mono- or multi-cyclic system including one or more
rings, where at least one ring includes at least one heteroatom.
Heteroatoms can be, for example, nitrogen, oxygen, or sulfur atoms.
Rings can be three, four, five, six, seven, eight, nine, ten,
eleven, or twelve membered rings. Heterocycles can include one or
more double bonds and can be aromatic (e.g., heteroaryl groups).
Examples of heterocycles include imidazolyl, imidazolidinyl,
oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl,
pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl,
isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl,
tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and
isoquinolyl groups. Heterocycles can be optionally substituted.
[1139] As used herein, a "biodegradable group" is a group that can
facilitate faster metabolism of a lipid in a subject. A
biodegradable group can be, but is not limited to, --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group.
[1140] As used herein, an "aryl group" is a carbocyclic group
including one or more aromatic rings. Examples of aryl groups
include phenyl and naphthyl groups.
[1141] As used herein, a "heteroaryl group" is a heterocyclic group
including one or more aromatic rings. Examples of heteroaryl groups
include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and
thiazolyl. Both aryl and heteroaryl groups can be optionally
substituted. For example, M and M' can be selected from the
non-limiting group consisting of optionally substituted phenyl,
oxazole, and thiazole. In the formulas herein, M and M' can be
independently selected from the list of biodegradable groups
above.
[1142] Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and
heterocyclyl) groups can be optionally substituted unless otherwise
specified. Optional substituents can be selected from the group
consisting of, but are not limited to, a halogen atom (e.g., a
chloride, bromide, fluoride, or iodide group), a carboxylic acid
(e.g., --C(O)OH), an alcohol (e.g., a hydroxyl, --OH), an ester
(e.g., --C(O)OR or --OC(O)R), an aldehyde (e.g., --C(O)H), a
carbonyl (e.g., --C(O)R, alternatively represented by C.dbd.O), an
acyl halide (e.g., --C(O)X, in which X is a halide selected from
bromide, fluoride, chloride, and iodide), a carbonate (e.g.,
--OC(O)OR), an alkoxy (e.g., --OR), an acetal (e.g.,
--C(OR).sub.2R'''', in which each OR are alkoxy groups that can be
the same or different and R'''' is an alkyl or alkenyl group), a
phosphate (e.g., P(O).sub.4.sup.3-), a thiol (e.g., --SH), a
sulfoxide (e.g., --S(O)R), a sulfinic acid (e.g., --S(O)OH), a
sulfonic acid (e.g., --S(O).sub.2OH), a thial (e.g., --C(S)H), a
sulfate (e.g., S(O).sub.4.sup.2-), a sulfonyl (e.g.,
--S(O).sub.2--), an amide (e.g., --C(O)NR.sub.2, or --N(R)C(O)R),
an azido (e.g., --N.sub.3), a nitro (e.g., --NO.sub.2), a cyano
(e.g., --CN), an isocyano (e.g., --NC), an acyloxy (e.g.,
--OC(O)R), an amino (e.g., --NR.sub.2, --NRH, or --NH.sub.2), a
carbamoyl (e.g., --OC(O)NR.sub.2, --OC(O)NRH, or --OC(O)NH.sub.2),
a sulfonamide (e.g., --S(O).sub.2NR.sub.2, --S(O).sub.2NRH,
--S(O).sub.2NH.sub.2, --N(R)S(O).sub.2R, --N(H)S(O).sub.2R,
--N(R)S(O).sub.2H, or --N(H)S(O).sub.2H), an alkyl group, an
alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl)
group.
[1143] In any of the preceding, R is an alkyl or alkenyl group, as
defined herein. In some embodiments, the substituent groups
themselves can be further substituted with, for example, one, two,
three, four, five, or six substituents as defined herein. For
example, a C.sub.1-6 alkyl group can be further substituted with
one, two, three, four, five, or six substituents as described
herein.
[1144] The compounds of any one of Formulae (I), (IA), (II), (IIa),
(IIb), (IIc), (IId), and (IIe) include one or more of the following
features when applicable.
[1145] In some embodiments, R.sub.4 is selected from the group
consisting of a C.sub.3-6 carbocycle, --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, and --CQ(R).sub.2, where Q is
selected from a C.sub.3-6 carbocycle, 5- to 14-membered aromatic or
non-aromatic heterocycle having one or more heteroatoms selected
from N, O, S, and P, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR,
--OC(O)R, --CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2,
--C(O)N(R).sub.2, --N(R)C(O)R, --N(R)S(O).sub.2R,
--N(R)C(O)N(R).sub.2, --N(R)C(S)N(R).sub.2, and
--C(R)N(R).sub.2C(O)OR, and each n is independently selected from
1, 2, 3, 4, and 5.
[1146] In another embodiment, R.sub.4 is selected from the group
consisting of a C.sub.3-6 carbocycle, --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, and --CQ(R).sub.2, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --C(R)N(R).sub.2C(O)OR, and a 5- to
14-membered heterocycloalkyl having one or more heteroatoms
selected from N, O, and S which is substituted with one or more
substituents selected from oxo (.dbd.O), OH, amino, and C.sub.1-3
alkyl, and each n is independently selected from 1, 2, 3, 4, and
5.
[1147] In another embodiment, R.sub.4 is selected from the group
consisting of a C.sub.3-6 carbocycle, --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, and --CQ(R).sub.2, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heterocycle having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --C(R)N(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-
to 14-membered heterocycle and (i) R.sub.4 is --(CH.sub.2).sub.nQ
in which n is 1 or 2, or (ii) R.sub.4 is --(CH.sub.2).sub.nCHQR in
which n is 1, or (iii) R.sub.4 is --CHQR, and --CQ(R).sub.2, then Q
is either a 5- to 14-membered heteroaryl or 8- to 14-membered
heterocycloalkyl.
[1148] In another embodiment, R.sub.4 is selected from the group
consisting of a C.sub.3-6 carbocycle, --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, and --CQ(R).sub.2, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --C(R)N(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5.
[1149] In another embodiment, R.sub.4 is unsubstituted C.sub.1-4
alkyl, e.g., unsubstituted methyl.
[1150] In certain embodiments, the disclosure provides a compound
having the Formula (I), wherein R.sub.4 is --(CH.sub.2).sub.nQ or
--(CH.sub.2).sub.nCHQR, where Q is --N(R).sub.2, and n is selected
from 3, 4, and 5.
[1151] In certain embodiments, the disclosure provides a compound
having the Formula (I), wherein R.sub.4 is selected from the group
consisting of --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
and --CQ(R).sub.2, where Q is --N(R).sub.2, and n is selected from
1, 2, 3, 4, and 5.
[1152] In certain embodiments, the disclosure provides a compound
having the Formula (I), wherein R.sub.2 and R.sub.3 are
independently selected from the group consisting of C.sub.2-14
alkyl, C.sub.2-14 alkenyl, --R*YR'', --YR'', and --R*OR'', or
R.sub.2 and R.sub.3, together with the atom to which they are
attached, form a heterocycle or carbocycle, and R.sub.4 is
--(CH.sub.2).sub.nQ or --(CH.sub.2).sub.nCHQR, where Q is
--N(R).sub.2, and n is selected from 3, 4, and 5.
[1153] In certain embodiments, R.sub.2 and R.sub.3 are
independently selected from the group consisting of C.sub.2-14
alkyl, C.sub.2-14 alkenyl, --R*YR'', --YR'', and --R*OR'', or
R.sub.2 and R.sub.3, together with the atom to which they are
attached, form a heterocycle or carbocycle.
[1154] In some embodiments, R.sub.1 is selected from the group
consisting of C.sub.5-20 alkyl and C.sub.5-20 alkenyl.
[1155] In other embodiments, R.sub.1 is selected from the group
consisting of --R*YR'', --YR'', and --R''M'R'.
[1156] In certain embodiments, R.sub.1 is selected from --R*YR''
and --YR''. In some embodiments, Y is a cyclopropyl group. In some
embodiments, R* is C.sub.8 alkyl or C.sub.8 alkenyl. In certain
embodiments, R'' is C.sub.3-12 alkyl. For example, R'' can be
C.sub.3 alkyl. For example, R'' can be C.sub.4-8 alkyl (e.g.,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, or C.sub.8 alkyl).
[1157] In some embodiments, R.sub.1 is C.sub.5-20 alkyl. In some
embodiments, R.sub.1 is C.sub.6 alkyl. In some embodiments, R.sub.1
is C.sub.8 alkyl. In other embodiments, R.sub.1 is C.sub.9 alkyl.
In certain embodiments, R.sub.1 is C.sub.14 alkyl. In other
embodiments, R.sub.1 is C.sub.18 alkyl.
[1158] In some embodiments, R.sub.1 is C.sub.5-20 alkenyl. In
certain embodiments, R.sub.1 is Cis alkenyl. In some embodiments,
R.sub.1 is linoleyl.
[1159] In certain embodiments, R.sub.1 is branched (e.g.,
decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl,
tetradecan-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl,
3-methylundecan-3-yl, 4-methyldodecan-4-yl, or heptadeca-9-yl). In
certain embodiments,
##STR00034##
[1160] R.sub.1 is.
[1161] In certain embodiments, R.sub.1 is unsubstituted C.sub.5-20
alkyl or C.sub.5-20 alkenyl. In certain embodiments, R' is
substituted C.sub.5-20 alkyl or C.sub.5-20 alkenyl (e.g.,
substituted with a C.sub.3-6 carbocycle such as
1-cyclopropylnonyl).
[1162] In other embodiments, R.sub.1 is --R''M'R'.
[1163] In some embodiments, R' is selected from --R*YR'' and
--YR''. In some embodiments, Y is C.sub.3-8 cycloalkyl. In some
embodiments, Y is C.sub.6-10 aryl. In some embodiments, Y is a
cyclopropyl group. In some embodiments, Y is a cyclohexyl group. In
certain embodiments, R* is C.sub.1 alkyl.
[1164] In some embodiments, R'' is selected from the group
consisting of C.sub.3-12 alkyl and C.sub.3-12 alkenyl. In some
embodiments, R'' adjacent to Y is C.sub.1 alkyl. In some
embodiments, R'' adjacent to Y is C.sub.4-9 alkyl (e.g., C.sub.4,
C.sub.5, C.sub.6, C.sub.7 or C.sub.8 or C.sub.9 alkyl).
[1165] In some embodiments, R' is selected from C.sub.4 alkyl and
C.sub.4 alkenyl. In certain embodiments, R' is selected from
C.sub.5 alkyl and C.sub.5 alkenyl. In some embodiments, R' is
selected from C.sub.6 alkyl and C.sub.6 alkenyl. In some
embodiments, R' is selected from C.sub.7 alkyl and C.sub.7 alkenyl.
In some embodiments, R' is selected from C.sub.9 alkyl and C.sub.9
alkenyl.
[1166] In other embodiments, R' is selected from C.sub.11 alkyl and
C.sub.11 alkenyl. In other embodiments, R' is selected from
C.sub.12 alkyl, C.sub.12 alkenyl, C.sub.13 alkyl, C.sub.13 alkenyl,
C.sub.14 alkyl, C.sub.14 alkenyl, C.sub.15 alkyl, C.sub.15 alkenyl,
C.sub.16 alkyl, C.sub.16 alkenyl, C.sub.17 alkyl, C.sub.17 alkenyl,
Cis alkyl, and Cis alkenyl. In certain embodiments, R' is branched
(e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl,
tetradecan-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl,
3-methylundecan-3-yl, 4-methyldodecan-4-yl or heptadeca-9-yl). In
certain embodiments, R' is
##STR00035##
[1167] In certain embodiments, R' is unsubstituted C.sub.1-18
alkyl. In certain embodiments, R' is substituted C.sub.1-18 alkyl
(e.g., C.sub.1-15 alkyl substituted with a C.sub.3-6 carbocycle
such as 1-cyclopropylnonyl).
[1168] In some embodiments, R'' is selected from the group
consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl. In some
embodiments, R'' is C.sub.3 alkyl, C.sub.4 alkyl, C.sub.5 alkyl,
C.sub.6 alkyl, C.sub.7 alkyl, or C.sub.8 alkyl. In some
embodiments, R'' is C.sub.9 alkyl, C.sub.10 alkyl, C.sub.11 alkyl,
C.sub.12 alkyl, C.sub.13 alkyl, or C.sub.14 alkyl.
[1169] In some embodiments, M' is --C(O)O--. In some embodiments,
M' is --OC(O)--.
[1170] In other embodiments, M' is an aryl group or heteroaryl
group. For example, M' can be selected from the group consisting of
phenyl, oxazole, and thiazole.
[1171] In some embodiments, M is --C(O)O-- In some embodiments, M
is --OC(O)--. In some embodiments, M is --C(O)N(R')--. In some
embodiments, M is --P(O)(OR')O--.
[1172] In other embodiments, M is an aryl group or heteroaryl
group. For example, M can be selected from the group consisting of
phenyl, oxazole, and thiazole.
[1173] In some embodiments, M is the same as M'. In other
embodiments, M is different from M'.
[1174] In some embodiments, each R.sub.5 is H. In certain such
embodiments, each R.sub.6 is also H.
[1175] In some embodiments, R.sub.7 is H. In other embodiments,
R.sub.7 is C.sub.1-3 alkyl (e.g., methyl, ethyl, propyl, or
i-propyl).
[1176] In some embodiments, R.sub.2 and R.sub.3 are independently
C.sub.5-14 alkyl or C.sub.5-14 alkenyl.
[1177] In some embodiments, R.sub.2 and R.sub.3 are the same. In
some embodiments, R.sub.2 and R.sub.3 are C.sub.8 alkyl. In certain
embodiments, R.sub.2 and R.sub.3 are C.sub.2 alkyl. In other
embodiments, R.sub.2 and R.sub.3 are C.sub.3 alkyl. In some
embodiments, R.sub.2 and R.sub.3 are C.sub.4 alkyl. In certain
embodiments, R.sub.2 and R.sub.3 are C.sub.5 alkyl. In other
embodiments, R.sub.2 and R.sub.3 are C.sub.6 alkyl. In some
embodiments, R.sub.2 and R.sub.3 are C.sub.7 alkyl.
[1178] In other embodiments, R.sub.2 and R.sub.3 are different. In
certain embodiments, R.sub.2 is C.sub.8 alkyl. In some embodiments,
R.sub.3 is C.sub.1-7 (e.g., C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, or C.sub.7 alkyl) or C.sub.9 alkyl.
[1179] In some embodiments, R.sub.7 and R.sub.3 are H.
[1180] In certain embodiments, R.sub.2 is H.
[1181] In some embodiments, m is 5, 7, or 9.
[1182] In some embodiments, R.sub.4 is selected from
--(CH.sub.2).sub.nQ and --(CH.sub.2).sub.nCHQR.
[1183] In some embodiments, Q is selected from the group consisting
of --OR, --OH, --O(CH.sub.2).sub.nN(R).sub.2, --OC(O)R, --CX.sub.3,
--CN, --N(R)C(O)R, --N(H)C(O)R, --N(R)S(O).sub.2R,
--N(H)S(O).sub.2R, --N(R)C(O)N(R).sub.2, --N(H)C(O)N(R).sub.2,
--N(H)C(O)N(H)(R), --N(R)C(S)N(R).sub.2, --N(H)C(S)N(R).sub.2,
--N(H)C(S)N(H)(R), --C(R)N(R).sub.2C(O)OR, a carbocycle, and a
heterocycle.
[1184] In certain embodiments, Q is --OH.
[1185] In certain embodiments, Q is a substituted or unsubstituted
5- to 10-membered heteroaryl, e.g., Q is an imidazole, a
pyrimidine, a purine, 2-amino-1,9-dihydro-6H-purin-6-one-9-yl (or
guanin-9-yl), adenin-9-yl, cytosin-1-yl, or uracil-1-yl. In certain
embodiments, Q is a substituted 5- to 14-membered heterocycloalkyl,
e.g., substituted with one or more substituents selected from oxo
(.dbd.O), OH, amino, and C.sub.1-3 alkyl. For example, Q is
4-methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, or
isoindolin-2-yl-1,3-dione.
[1186] In certain embodiments, Q is an unsubstituted or substituted
C.sub.6-10 aryl (such as phenyl) or C.sub.3-6 cycloalkyl.
[1187] In some embodiments, n is 1. In other embodiments, n is 2.
In further embodiments, n is 3. In certain other embodiments, n is
4. For example, R.sub.4 can be --(CH.sub.2).sub.2OH. For example,
R.sub.4 can be --(CH.sub.2).sub.3OH. For example, R.sub.4 can be
--(CH.sub.2).sub.4OH. For example, R.sub.4 can be benzyl. For
example, R.sub.4 can be 4-methoxybenzyl.
[1188] In some embodiments, R.sub.4 is a C.sub.3-6 carbocycle. In
some embodiments, R.sub.4 is a C.sub.3-6 cycloalkyl. For example,
R.sub.4 can be cyclohexyl optionally substituted with e.g., OH,
halo, C.sub.1-6 alkyl, etc. For example, R.sub.4 can be
2-hydroxycyclohexyl.
[1189] In some embodiments, R is H.
[1190] In some embodiments, R is unsubstituted C.sub.1-3 alkyl or
unsubstituted C.sub.2-3 alkenyl. For example, R.sub.4 can be
--CH.sub.2CH(OH)CH.sub.3 or --CH.sub.2CH(OH)CH.sub.2CH.sub.3.
[1191] In some embodiments, R is substituted C.sub.1-3 alkyl, e.g.,
CH.sub.2OH. For example, R.sub.4 can be
--CH.sub.2CH(OH)CH.sub.2OH.
[1192] In some embodiments, R.sub.2 and R.sub.3, together with the
atom to which they are attached, form a heterocycle or carbocycle.
In some embodiments, R.sub.2 and R.sub.3, together with the atom to
which they are attached, form a 5- to 14-membered aromatic or
non-aromatic heterocycle having one or more heteroatoms selected
from N, O, S, and P. In some embodiments, R.sub.2 and R.sub.3,
together with the atom to which they are attached, form an
optionally substituted C.sub.3-20 carbocycle (e.g., C.sub.3-18
carbocycle, C.sub.3-15 carbocycle, C.sub.3-12 carbocycle, or
C.sub.3-10 carbocycle), either aromatic or non-aromatic. In some
embodiments, R.sub.2 and R.sub.3, together with the atom to which
they are attached, form a C.sub.3-6 carbocycle. In other
embodiments, R.sub.2 and R.sub.3, together with the atom to which
they are attached, form a C.sub.6 carbocycle, such as a cyclohexyl
or phenyl group. In certain embodiments, the heterocycle or
C.sub.3-6 carbocycle is substituted with one or more alkyl groups
(e.g., at the same ring atom or at adjacent or non-adjacent ring
atoms). For example, R.sub.2 and R.sub.3, together with the atom to
which they are attached, can form a cyclohexyl or phenyl group
bearing one or more C.sub.5 alkyl substitutions. In certain
embodiments, the heterocycle or C.sub.3-6 carbocycle formed by
R.sub.2 and R.sub.3, is substituted with a carbocycle groups. For
example, R.sub.2 and R.sub.3, together with the atom to which they
are attached, can form a cyclohexyl or phenyl group that is
substituted with cyclohexyl. In some embodiments, R.sub.2 and
R.sub.3, together with the atom to which they are attached, form a
C.sub.7-15 carbocycle, such as a cycloheptyl, cyclopentadecanyl, or
naphthyl group.
[1193] In some embodiments, R.sub.4 is selected from
--(CH.sub.2).sub.nQ and --(CH.sub.2).sub.nCHQR. In some
embodiments, Q is selected from the group consisting of --OR, --OH,
--O(CH.sub.2).sub.nN(R).sub.2, --OC(O)R, --CX.sub.3, --CN,
--N(R)C(O)R, --N(H)C(O)R, --N(R)S(O).sub.2R, --N(H)S(O).sub.2R,
--N(R)C(O)N(R).sub.2, --N(H)C(O)N(R).sub.2, --N(H)C(O)N(H)(R),
--N(R)C(S)N(R).sub.2, --N(H)C(S)N(R).sub.2, --N(H)C(S)N(H)(R), and
a heterocycle. In other embodiments, Q is selected from the group
consisting of an imidazole, a pyrimidine, and a purine.
[1194] In some embodiments, R.sub.2 and R.sub.3, together with the
atom to which they are attached, form a heterocycle or carbocycle.
In some embodiments, R.sub.2 and R.sub.3, together with the atom to
which they are attached, form a C.sub.3-6 carbocycle, such as a
phenyl group. In certain embodiments, the heterocycle or C.sub.3-6
carbocycle is substituted with one or more alkyl groups (e.g., at
the same ring atom or at adjacent or non-adjacent ring atoms). For
example, R.sub.2 and R.sub.3, together with the atom to which they
are attached, can form a phenyl group bearing one or more C.sub.5
alkyl substitutions.
[1195] In some embodiments, the pharmaceutical compositions of the
present disclosure, the compound of Formula (I) is selected from
the group consisting of:
##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055##
##STR00056## ##STR00057## ##STR00058## ##STR00059## ##STR00060##
##STR00061## ##STR00062## ##STR00063## ##STR00064## ##STR00065##
##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070##
##STR00071## ##STR00072## ##STR00073## ##STR00074##
and salts or stereoisomers thereof.
[1196] In other embodiments, the compound of Formula (I) is
selected from the group consisting of Compound 1-Compound 147, or
salt or stereoisomers thereof.
[1197] In some embodiments ionizable lipids including a central
piperazine moiety are provided.
[1198] In some embodiments, the delivery agent comprises a lipid
compound having the Formula (III)
##STR00075##
or salts or stereoisomers thereof, wherein
[1199] ring A is or
##STR00076##
[1200] t is 1 or 2;
[1201] A.sub.1 and A.sub.2 are each independently selected from CH
or N;
[1202] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[1203] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
independently selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R''MR', --R*YR'', --YR'', and
--R*OR'';
[1204] each M is independently selected from the group consisting
of --C(O)O--, --OC(O)--, --OC(O)O--, --C(O)N(R')--, --N(R')C(O)--,
--C(O)--, --C(S)--, --C(S)S--, --SC(S)--, --CH(OH)--,
--P(O)(OR')O--, --S(O).sub.2--, an aryl group, and a heteroaryl
group;
[1205] X.sup.1, X.sup.2, and X.sup.3 are independently selected
from the group consisting of a bond, --CH.sub.2--,
--(CH.sub.2).sub.2--, --CHR--, --CHY--, --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--,
--CH.sub.2--OC(O)--, --CH(OH)--, --C(S)--, and --CH(SH--;
[1206] each Y is independently a C.sub.3-6 carbocycle;
[1207] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1208] each R is independently selected from the group consisting
of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle;
[1209] each R' is independently selected from the group consisting
of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H; and
[1210] each R'' is independently selected from the group consisting
of C.sub.3-12 alkyl and C.sub.3-12 alkenyl,
[1211] wherein when ring A is
##STR00077##
then
[1212] i) at least one of X.sup.1, X.sup.2, and X.sup.3 is not
--CH.sub.2--; and/or
[1213] ii) at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 is --R''MR'.
[1214] In some embodiments, the compound is of any of Formulae
(IIIa1)-(IIIa6):
##STR00078##
[1215] The compounds of Formula (III) or any of (IIIa1)-(IIIa6)
include one or more of the following features when applicable.
[1216] In some embodiments, ring A is
##STR00079##
[1217] In some embodiments, ring A is
##STR00080##
[1218] In some embodiments, ring A is
##STR00081##
[1219] In some embodiments, ring A is
##STR00082##
[1220] In some embodiments, ring A is
##STR00083##
[1221] In some embodiments, ring A is or
##STR00084##
wherein ring, in which the N atom is connected with X.sup.2.
[1222] In some embodiments, Z is CH.sub.2.
[1223] In some embodiments, Z is absent.
[1224] In some embodiments, at least one of A.sub.1 and A.sub.2 is
N.
[1225] In some embodiments, each of A.sub.1 and A.sub.2 is N.
[1226] In some embodiments, each of A.sub.1 and A.sub.2 is CH.
[1227] In some embodiments, A.sub.1 is N and A.sub.2 is CH.
[1228] In some embodiments, A.sub.1 is CH and A.sub.2 is N.
[1229] In some embodiments, at least one of X.sup.1, X.sup.2, and
X.sup.3 is not --CH.sub.2--. For example, in certain embodiments,
X.sup.1 is not --CH.sub.2--. In some embodiments, at least one of
X.sup.1, X.sup.2, and X.sup.3 is --C(O)--.
[1230] In some embodiments, X.sup.2 is --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--, or
--CH.sub.2--OC(O)--.
[1231] In some embodiments, X.sup.3 is --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--, or
--CH.sub.2--OC(O)--. In other embodiments, X.sup.3 is
--CH.sub.2--.
[1232] In some embodiments, X.sup.3 is a bond or
--(CH.sub.2).sub.2--.
[1233] In some embodiments, R.sub.1 and R.sub.2 are the same. In
certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are the same. In
some embodiments, R.sub.4 and R.sub.5 are the same. In certain
embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
the same.
[1234] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is --R''MR'. In some embodiments, at
most one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
--R''MR'. For example, at least one of R.sub.1, R.sub.2, and
R.sub.3 may be --R''MR', and/or at least one of R.sub.4 and R.sub.5
is --R''MR'. In certain embodiments, at least one M is --C(O)O--.
In some embodiments, each M is --C(O)O--. In some embodiments, at
least one M is --OC(O)--. In some embodiments, each M is --OC(O)--.
In some embodiments, at least one M is --OC(O)O--. In some
embodiments, each M is --OC(O)O--. In some embodiments, at least
one R'' is C.sub.3 alkyl. In certain embodiments, each R'' is
C.sub.3 alkyl. In some embodiments, at least one R'' is C.sub.8
alkyl. In certain embodiments, each R'' is C.sub.8 alkyl. In some
embodiments, at least one R'' is C.sub.6 alkyl. In certain
embodiments, each R'' is C.sub.6 alkyl. In some embodiments, at
least one R'' is C.sub.7 alkyl. In certain embodiments, each R'' is
C.sub.7 alkyl. In some embodiments, at least one R' is C.sub.8
alkyl. In certain embodiments, each R' is C.sub.8 alkyl. In other
embodiments, at least one R' is C.sub.1 alkyl. In certain
embodiments, each R' is C.sub.1 alkyl. In some embodiments, at
least one R' is C.sub.2 alkyl. In certain embodiments, each R' is
C.sub.2 alkyl.
[1235] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is C.sub.12 alkyl. In certain
embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 are C.sub.12 alkyl.
[1236] In certain embodiments, the compound is selected from the
group consisting of:
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
##STR00100##
[1237] In some embodiments, the delivery agent comprises Compound
236.
[1238] In some embodiments, the delivery agent comprises a compound
having the Formula (IV)
##STR00101##
or salts or stereoisomer thereof, wherein
[1239] A.sub.1 and A.sub.2 are each independently selected from CH
or N and at least one of A.sub.1 and A.sub.2 is N;
[1240] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[1241] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
independently selected from the group consisting of C.sub.6-20
alkyl and C.sub.6-20 alkenyl;
[1242] wherein when ring A is
##STR00102##
then
[1243] i) R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are the
same, wherein R.sub.1 is not C.sub.12 alkyl, C.sub.18 alkyl, or
C.sub.18 alkenyl;
[1244] ii) only one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 is selected from C.sub.6-20 alkenyl;
[1245] iii) at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 have a different number of carbon atoms than at least one
other of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5;
[1246] iv) R.sub.1, R.sub.2, and R.sub.3 are selected from
C.sub.6-20 alkenyl, and R.sub.4 and R.sub.5 are selected from
C.sub.6-20 alkyl; or
[1247] v) R.sub.1, R.sub.2, and R.sub.3 are selected from
C.sub.6-20 alkyl, and R.sub.4 and R.sub.5 are selected from
C.sub.6-20 alkenyl.
[1248] In some embodiments, the compound is of Formula (IVa):
##STR00103##
[1249] The compounds of Formula (IV) or (IVa) include one or more
of the following features when applicable.
[1250] In some embodiments, Z is CH.sub.2.
[1251] In some embodiments, Z is absent.
[1252] In some embodiments, at least one of A.sub.1 and A.sub.2 is
N.
[1253] In some embodiments, each of A.sub.1 and A.sub.2 is N.
[1254] In some embodiments, each of A.sub.1 and A.sub.2 is CH.
[1255] In some embodiments, A.sub.1 is N and A.sub.2 is CH.
[1256] In some embodiments, A.sub.1 is CH and A.sub.2 is N.
[1257] In some embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 are the same, and are not C.sub.12 alkyl, C.sub.18 alkyl,
or C.sub.18 alkenyl. In some embodiments, R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same and are C.sub.9 alkyl or
C.sub.14 alkyl.
[1258] In some embodiments, only one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is selected from C.sub.6-20 alkenyl. In
certain such embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 have the same number of carbon atoms. In some embodiments,
R.sub.4 is selected from C.sub.5-20 alkenyl. For example, R.sub.4
may be C.sub.12 alkenyl or C.sub.18 alkenyl.
[1259] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 have a different number of carbon
atoms than at least one other of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5.
[1260] In certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are
selected from C.sub.6-20 alkenyl, and R.sub.4 and R.sub.5 are
selected from C.sub.6-20 alkyl. In other embodiments, R.sub.1,
R.sub.2, and R.sub.3 are selected from C.sub.6-20 alkyl, and
R.sub.4 and R.sub.5 are selected from C.sub.6-20 alkenyl. In some
embodiments, R.sub.1, R.sub.2, and R.sub.3 have the same number of
carbon atoms, and/or R.sub.4 and R.sub.5 have the same number of
carbon atoms. For example, R.sub.1, R.sub.2, and R.sub.3, or
R.sub.4 and R.sub.5, may have 6, 8, 9, 12, 14, or 18 carbon atoms.
In some embodiments, R.sub.1, R.sub.2, and R.sub.3, or R.sub.4 and
R.sub.5, are Cis alkenyl (e.g., linoleyl). In some embodiments,
R.sub.1, R.sub.2, and R.sub.3, or R.sub.4 and R.sub.5, are alkyl
groups including 6, 8, 9, 12, or 14 carbon atoms.
[1261] In some embodiments, R.sub.1 has a different number of
carbon atoms than R.sub.2, R.sub.3, R.sub.4, and R.sub.5. In other
embodiments, R.sub.3 has a different number of carbon atoms than
R.sub.1, R.sub.2, R.sub.4, and R.sub.5. In further embodiments,
R.sub.4 has a different number of carbon atoms than R.sub.1,
R.sub.2, R.sub.3, and R.sub.5.
[1262] In some embodiments, the compound is selected from the group
consisting of:
##STR00104## ##STR00105## ##STR00106## ##STR00107##
##STR00108##
[1263] In other embodiments, the delivery agent comprises a
compound having the Formula (V)
##STR00109##
or salts or stereoisomers thereof, in which
[1264] A.sub.3 is CH or N;
[1265] A.sub.4 is CH.sub.2 or NH; and at least one of A.sub.3 and
A.sub.4 is N or NH;
[1266] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[1267] R.sub.1, R.sub.2, and R.sub.3 are independently selected
from the group consisting of C.sub.5-20 alkyl, C.sub.5-20 alkenyl,
--R''MR', --R*YR'', --YR'', and --R*OR'';
[1268] each M is independently selected from --C(O)O--, --OC(O)--,
--C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--, --C(S)S--,
--SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--, an aryl
group, and a heteroaryl group;
[1269] X.sup.1 and X.sup.2 are independently selected from the
group consisting of --CH.sub.2--, --(CH.sub.2).sub.2--, --CHR--,
--CHY--, --C(O)--, --C(O)O--, --OC(O)--, --C(O)--CH.sub.2--,
--CH.sub.2--C(O)--, --C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--,
--CH.sub.2--C(O)O--, --CH.sub.2--OC(O)--, --CH(OH)--, --C(S)--, and
--CH(SH)--;
[1270] each Y is independently a C.sub.3-6 carbocycle;
[1271] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1272] each R is independently selected from the group consisting
of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle;
[1273] each R' is independently selected from the group consisting
of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H; and
[1274] each R'' is independently selected from the group consisting
of C.sub.3-12 alkyl and C.sub.3-12 alkenyl.
[1275] In some embodiments, the compound is of Formula (Va):
##STR00110##
[1276] The compounds of Formula (V) or (Va) include one or more of
the following features when applicable.
[1277] In some embodiments, Z is CH.sub.2.
[1278] In some embodiments, Z is absent.
[1279] In some embodiments, at least one of A.sub.3 and A.sub.4 is
N or NH.
[1280] In some embodiments, A.sub.3 is N and A.sub.4 is NH.
[1281] In some embodiments, A.sub.3 is N and A.sub.4 is
CH.sub.2.
[1282] In some embodiments, A.sub.3 is CH and A.sub.4 is NH.
[1283] In some embodiments, at least one of X.sup.1 and X.sup.2 is
not --CH.sub.2--. For example, in certain embodiments, X.sup.1 is
not --CH.sub.2--. In some embodiments, at least one of X.sup.1 and
X.sup.2 is --C(O)--.
[1284] In some embodiments, X.sup.2 is --C(O)--, --C(O)O--,
--OC(O)--, --C(O)--CH.sub.2--, --CH.sub.2--C(O)--,
--C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--, --CH.sub.2--C(O)O--, or
--CH.sub.2--OC(O)--.
[1285] In some embodiments, R.sub.1, R.sub.2, and R.sub.3 are
independently selected from the group consisting of C.sub.5-20
alkyl and C.sub.5-20 alkenyl. In some embodiments, R.sub.1,
R.sub.2, and R.sub.3 are the same. In certain embodiments, R.sub.1,
R.sub.2, and R.sub.3 are C.sub.6, C.sub.9, C.sub.12, or C.sub.14
alkyl. In other embodiments, R.sub.1, R.sub.2, and R.sub.3 are
C.sub.18 alkenyl. For example, R.sub.1, R.sub.2, and R.sub.3 may be
linoleyl.
[1286] In some embodiments, the compound is selected from the group
consisting of:
##STR00111##
[1287] In other embodiments, the delivery agent comprises a
compound having the Formula (VI):
##STR00112##
or salts or stereoisomers thereof, in which
[1288] A.sub.6 and A.sub.7 are each independently selected from CH
or N, wherein at least one of A.sub.6 and A.sub.7 is N;
[1289] Z is CH.sub.2 or absent wherein when Z is CH.sub.2, the
dashed lines (1) and (2) each represent a single bond; and when Z
is absent, the dashed lines (1) and (2) are both absent;
[1290] X.sup.4 and X.sup.5 are independently selected from the
group consisting of --CH.sub.2--, --CH.sub.2).sub.2--, --CHR--,
--CHY--, --C(O)--, --C(O)O--, --OC(O)--, --C(O)--CH.sub.2--,
--CH.sub.2--C(O)--, --C(O)O--CH.sub.2--, --OC(O)--CH.sub.2--,
--CH.sub.2--C(O)O--, --CH.sub.2--OC(O)--, --CH(OH)--, --C(S)--, and
--CH(SH)--;
[1291] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 each are
independently selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R''MR', --R*YR'', --YR'', and
--R*OR'';
[1292] each M is independently selected from the group consisting
of --C(O)O--, --OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--,
--C(S)--, --C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--,
--S(O).sub.2-- an aryl group, and a heteroaryl group;
[1293] each Y is independently a C.sub.3-6 carbocycle;
[1294] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1295] each R is independently selected from the group consisting
of C.sub.1-3 alkyl and a C.sub.3-6 carbocycle;
[1296] each R' is independently selected from the group consisting
of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, and H; and
[1297] each R'' is independently selected from the group consisting
of C.sub.3-12 alkyl and C.sub.3-12 alkenyl.
[1298] In some embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 each are independently selected from the group consisting
of C.sub.6-20 alkyl and C.sub.6-20 alkenyl.
[1299] In some embodiments, R.sub.1 and R.sub.2 are the same. In
certain embodiments, R.sub.1, R.sub.2, and R.sub.3 are the same. In
some embodiments, R.sub.4 and R.sub.5 are the same. In certain
embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
the same.
[1300] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is C.sub.9-12 alkyl. In certain
embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 independently is C.sub.9, C.sub.12 or C.sub.14 alkyl. In
certain embodiments, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
and R.sub.5 is C.sub.9 alkyl.
[1301] In some embodiments, A.sub.6 is N and A.sub.7 is N. In some
embodiments, A.sub.6 is CH and A.sub.7 is N.
[1302] In some embodiments, X.sup.4 is --CH.sub.2-- and X.sup.5 is
--C(O)--. In some embodiments, X.sup.4 and X.sup.5 are
--C(O)--.
[1303] In some embodiments, when A.sub.6 is N and A.sub.7 is N, at
least one of X.sup.4 and X.sup.5 is not --CH.sub.2--, e.g., at
least one of X.sup.4 and X.sup.5 is --C(O)--. In some embodiments,
when A.sub.6 is N and A.sub.7 is N, at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is --R''MR'.
[1304] In some embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is not --R''MR'. In some embodiments,
the compound is
##STR00113##
[1305] In other embodiments, the delivery agent comprises a
compound having the formula:
##STR00114##
[1306] Amine moieties of the lipid compounds disclosed herein can
be protonated under certain conditions. For example, the central
amine moiety of a lipid according to Formula (I) is typically
protonated (i.e., positively charged) at a pH below the pKa of the
amino moiety and is substantially not charged at a pH above the
pKa. Such lipids can be referred to ionizable amino lipids.
[1307] In one specific embodiment, the ionizable amino lipid is
Compound 18. In another embodiment, the ionizable amino lipid is
Compound 236.
[1308] In some embodiments, the amount the ionizable amino lipid,
e.g., compound of Formula (I) ranges from about 1 mol % to 99 mol %
in the lipid composition.
[1309] In one embodiment, the amount of the ionizable amino lipid,
e.g., compound of Formula (I) is at least about 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, or 99 mol % in the lipid
composition.
[1310] In one embodiment, the amount of the ionizable amino lipid,
e.g., the compound of Formula (I) ranges from about 30 mol % to
about 70 mol %, from about 35 mol % to about 65 mol %, from about
40 mol % to about 60 mol %, and from about 45 mol % to about 55 mol
% in the lipid composition.
[1311] In one specific embodiment, the amount of the ionizable
amino lipid, e.g., compound of Formula (I) is about 50 mol % in the
lipid composition.
[1312] In addition to the ionizable amino lipid disclosed herein,
e.g., compound of Formula (I), the lipid composition of the
pharmaceutical compositions disclosed herein can comprise
additional components such as phospholipids, structural lipids,
PEG-lipids, and any combination thereof.
b. Additional Components in the Lipid Composition
[1313] (i) Phospholipids
[1314] The lipid composition of the pharmaceutical composition
disclosed herein can comprise one or more phospholipids, for
example, one or more saturated or (poly)unsaturated phospholipids
or a combination thereof. In general, phospholipids comprise a
phospholipid moiety and one or more fatty acid moieties.
[1315] A phospholipid moiety can be selected, for example, from the
non-limiting group consisting of phosphatidyl choline, phosphatidyl
ethanolamine, phosphatidyl glycerol, phosphatidyl serine,
phosphatidic acid, 2-lysophosphatidyl choline, and a
sphingomyelin.
[1316] A fatty acid moiety can be selected, for example, from the
non-limiting group consisting of lauric acid, myristic acid,
myristoleic acid, palmitic acid, palmitoleic acid, stearic acid,
oleic acid, linoleic acid, alpha-linolenic acid, erucic acid,
phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic
acid, behenic acid, docosapentaenoic acid, and docosahexaenoic
acid.
[1317] Particular phospholipids can facilitate fusion to a
membrane. For example, a cationic phospholipid can interact with
one or more negatively charged phospholipids of a membrane (e.g., a
cellular or intracellular membrane). Fusion of a phospholipid to a
membrane can allow one or more elements (e.g., a therapeutic agent)
of a lipid-containing composition (e.g., LNPs) to pass through the
membrane permitting, e.g., delivery of the one or more elements to
a target tissue.
[1318] Non-natural phospholipid species including natural species
with modifications and substitutions including branching,
oxidation, cyclization, and alkynes are also contemplated. For
example, a phospholipid can be functionalized with or cross-linked
to one or more alkynes (e.g., an alkenyl group in which one or more
double bonds is replaced with a triple bond). Under appropriate
reaction conditions, an alkyne group can undergo a copper-catalyzed
cycloaddition upon exposure to an azide. Such reactions can be
useful in functionalizing a lipid bilayer of a nanoparticle
composition to facilitate membrane permeation or cellular
recognition or in conjugating a nanoparticle composition to a
useful component such as a targeting or imaging moiety (e.g., a
dye).
[1319] Phospholipids include, but are not limited to,
glycerophospholipids such as phosphatidylcholines,
phosphatidylethanolamines, phosphatidylserines,
phosphatidylinositols, phosphatidy glycerols, and phosphatidic
acids. Phospholipids also include phosphosphingolipid, such as
sphingomyelin.
[1320] Examples of phospholipids include, but are not limited to,
the following:
##STR00115## ##STR00116##
[1321] In certain embodiments, a phospholipid useful or potentially
useful in the present invention is an analog or variant of DSPC. In
certain embodiments, a phospholipid useful or potentially useful in
the present invention is a compound of Formula (IX):
##STR00117##
or a salt thereof, wherein:
[1322] each R.sup.1 is independently optionally substituted alkyl;
or optionally two R.sup.1 are joined together with the intervening
atoms to form optionally substituted monocyclic carbocyclyl or
optionally substituted monocyclic heterocyclyl; or optionally three
R.sup.1 are joined together with the intervening atoms to form
optionally substituted bicyclic carbocyclyl or optionally
substitute bicyclic heterocyclyl;
[1323] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
[1324] m is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
[1325] A is of the formula:
##STR00118##
[1326] each instance of L.sup.2 is independently a bond or
optionally substituted C.sub.1-6 alkylene, wherein one methylene
unit of the optionally substituted C.sub.1-6 alkylene is optionally
replaced with --O--, --N(R.sup.N)--, --S--, --C(O)--,
--C(O)N(R.sup.N)--, --NR.sup.NC(O)--, --C(O)O--, --OC(O)--,
--OC(O)O--, --OC(O)N(R.sup.N)--, --NR.sup.NC(O)O--, or
--NR.sup.NC(O)N(R.sup.N)--;
[1327] each instance of R.sup.2 is independently optionally
substituted C.sub.1-30 alkyl, optionally substituted C.sub.1-30
alkenyl, or optionally substituted C.sub.1-30 alkynyl; optionally
wherein one or more methylene units of R.sup.2 are independently
replaced with optionally substituted carbocyclylene, optionally
substituted heterocyclylene, optionally substituted arylene,
optionally substituted heteroarylene, --N(R.sup.N)--, --O--, --S--,
--C(O)--, --C(O)N(R.sup.N)--, --NR.sup.NC(O)--,
--NR.sup.NC(O)N(R.sup.N)--, --C(O)O--, --OC(O)--, --OC(O)O--,
--OC(O)N(R.sup.N)--, --NR.sup.NC(O)O--, --C(O)S--, --SC(O)--,
--C(.dbd.NR.sup.N)--, --C(.dbd.NR.sup.N)N(R.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)N(R.sup.N)--, --C(S)--,
--C(S)N(R.sup.N)--, --NR.sup.NC(S)--, --NR.sup.NC(S)N(R.sup.N)--,
--S(O)--, --OS(O)--, --S(O)O--, --OS(O)O--, --OS(O).sub.2--,
--S(O).sub.2O--, --OS(O).sub.2O--, --N(R.sup.N)S(O)--,
--S(O)N(R.sup.N)--, --N(R.sup.N)S(O)N(R.sup.N)--,
--OS(O)N(R.sup.N)--, --N(R.sup.N) S(O)O--, --S(O).sub.2--,
--N(R.sup.N) S(O).sub.2--, --S(O).sub.2N(R.sup.N)--,
--N(R.sup.N)S(O).sub.2N(R.sup.N)--, --OS(O).sub.2N(R.sup.N)--, or
--N(R.sup.N)S(O).sub.2O--;
[1328] each instance of R.sup.N is independently hydrogen,
optionally substituted alkyl, or a nitrogen protecting group;
[1329] Ring B is optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, or
optionally substituted heteroaryl; and
[1330] p is 1 or 2;
[1331] provided that the compound is not of the formula:
##STR00119##
[1332] wherein each instance of R.sup.2 is independently
unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted
alkynyl.
Phospholipid Head Modifications
[1333] In certain embodiments, a phospholipid useful or potentially
useful in the present invention comprises a modified phospholipid
head (e.g., a modified choline group). In certain embodiments, a
phospholipid with a modified head is DSPC, or analog thereof, with
a modified quaternary amine. For example, in embodiments of Formula
(IX), at least one of R.sup.1 is not methyl. In certain
embodiments, at least one of R.sup.1 is not hydrogen or methyl. In
certain embodiments, the compound of Formula (IX) is of one of the
following formulae:
##STR00120##
or a salt thereof, wherein:
[1334] each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10;
[1335] each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
and
[1336] each v is independently 1, 2, or 3.
[1337] In certain embodiments, the compound of Formula (IX) is of
one of the following formulae:
##STR00121##
or a salt thereof.
[1338] In certain embodiments, a compound of Formula (IX) is one of
the following:
##STR00122## ##STR00123##
or a salt thereof.
[1339] In certain embodiments, a compound of Formula (IX) is of
Formula (IX-a):
##STR00124##
or a salt thereof.
[1340] In certain embodiments, phospholipids useful or potentially
useful in the present invention comprise a modified core. In
certain embodiments, a phospholipid with a modified core described
herein is DSPC, or analog thereof, with a modified core structure.
For example, in certain embodiments of Formula (IX-a), group A is
not of the following formula:
##STR00125##
[1341] In certain embodiments, the compound of Formula (IX-a) is of
one of the following formulae:
##STR00126##
or a salt thereof.
[1342] In certain embodiments, a compound of Formula (IX) is one of
the following:
##STR00127##
or salts thereof.
[1343] In certain embodiments, a phospholipid useful or potentially
useful in the present invention comprises a cyclic moiety in place
of the glyceride moiety. In certain embodiments, a phospholipid
useful in the present invention is DSPC, or analog thereof, with a
cyclic moiety in place of the glyceride moiety. In certain
embodiments, the compound of Formula (IX) is of Formula (IX-b):
##STR00128##
or a salt thereof.
[1344] In certain embodiments, the compound of Formula (IX-b) is of
Formula (IX-b-1):
##STR00129##
or a salt thereof, wherein:
[1345] w is 0, 1, 2, or 3.
[1346] In certain embodiments, the compound of Formula (IX-b) is of
Formula (IX-b-2):
##STR00130##
or a salt thereof.
[1347] In certain embodiments, the compound of Formula (IX-b) is of
Formula (IX-b-3):
##STR00131##
or a salt thereof.
[1348] In certain embodiments, the compound of Formula (IX-b) is of
Formula (IX-b-4):
##STR00132##
or a salt thereof.
[1349] In certain embodiments, the compound of Formula (IX-b) is
one of the following:
##STR00133##
or salts thereof.
Phospholipid Tail Modifications
[1350] In certain embodiments, a phospholipid useful or potentially
useful in the present invention comprises a modified tail. In
certain embodiments, a phospholipid useful or potentially useful in
the present invention is DSPC, or analog thereof, with a modified
tail. As described herein, a "modified tail" may be a tail with
shorter or longer aliphatic chains, aliphatic chains with branching
introduced, aliphatic chains with substituents introduced,
aliphatic chains wherein one or more methylenes are replaced by
cyclic or heteroatom groups, or any combination thereof. For
example, in certain embodiments, the compound of (IX) is of Formula
(IX-a), or a salt thereof, wherein at least one instance of R.sup.2
is each instance of R.sup.2 is optionally substituted C.sub.1-30
alkyl, wherein one or more methylene units of R.sup.2 are
independently replaced with optionally substituted carbocyclylene,
optionally substituted heterocyclylene, optionally substituted
arylene, optionally substituted heteroarylene, --N(R.sup.N)--,
--O--, --S--, --C(O)--, --C(O)N(R.sup.N)--, --NR.sup.NC(O)--,
--NR.sup.NC(O)N(R.sup.N)--, --C(O)O--, --OC(O)--, --OC(O)O--,
--OC(O)N(R.sup.N)--, --NR.sup.NC(O)O--, --C(O)S--, --SC(O)--,
--C(.dbd.NR.sup.N)--, --C(.dbd.NR.sup.N)N(R.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)N(R.sup.N)--, --C(S)--,
--C(S)N(R.sup.N)--, --NR.sup.NC(S)--, --NR.sup.NC(S)N(R.sup.N)--,
--S(O)--, --OS(O)--, --S(O)O--, --OS(O)O--, --OS(O).sub.2--,
--S(O).sub.2O--, --OS(O).sub.2O--, --N(R.sup.N)S(O)--,
--S(O)N(R.sup.N)--, --N(R.sup.N)S(O)N(R.sup.N)--,
--OS(O)N(R.sup.N)--, --N(R.sup.N) S(O)O--, --S(O).sub.2--,
--N(R.sup.N) S(O).sub.2--, --S(O).sub.2N(R.sup.N)--,
--N(R.sup.N)S(O).sub.2N(R.sup.N)--, --OS(O).sub.2N(R.sup.N)--, or
--N(R.sup.N)S(O).sub.2O--.
[1351] In certain embodiments, the compound of Formula (IX) is of
Formula (IX-c):
##STR00134##
or a salt thereof, wherein:
[1352] each x is independently an integer between 0-30, inclusive;
and
[1353] each instance is G is independently selected from the group
consisting of optionally substituted carbocyclylene, optionally
substituted heterocyclylene, optionally substituted arylene,
optionally substituted heteroarylene, --N(R.sup.N)--, --O--, --S--,
--C(O)--, --C(O)N(R.sup.N)--, --NR.sup.NC(O)--,
--NR.sup.NC(O)N(R.sup.N)--, --C(O)O--, --OC(O)--, --OC(O)O--,
--OC(O)N(R.sup.N)--, --NR.sup.NC(O)O--, --C(O)S--, --SC(O)--,
--C(.dbd.NR.sup.N)--, --C(.dbd.NR.sup.N)N(R.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)N(R.sup.N)--, --C(S)--,
--C(S)N(R.sup.N)--, --NR.sup.NC(S)--, --NR.sup.NC(S)N(R'')--,
--S(O)--, --OS(O)--, --S(O)O--, --OS(O)O--, --OS(O).sub.2--,
--S(O).sub.2O--, --OS(O).sub.2O--, --N(R'')S(O)--,
--S(O)N(R.sup.N)--, --N(R.sup.N)S(O)N(R.sup.N)--,
--OS(O)N(R.sup.N)--, --N(R.sup.N)S(O)O--, --S(O).sub.2--,
--N(R.sup.N)S(O).sub.2--, --S(O).sub.2N(R.sup.N)--,
--N(R.sup.N)S(O).sub.2N(R.sup.N)--, --OS(O).sub.2N(R.sup.N)--, or
--N(R.sup.N)S(O).sub.2O--. Each possibility represents a separate
embodiment of the present invention.
[1354] In certain embodiments, the compound of Formula (IX-c) is of
Formula (IX-c-1):
##STR00135##
or salt thereof, wherein:
[1355] each instance of v is independently 1, 2, or 3.
[1356] In certain embodiments, the compound of Formula (IX-c) is of
Formula (IX-c-2):
##STR00136##
or a salt thereof.
[1357] In certain embodiments, the compound of Formula (IX-c) is of
the following formula:
##STR00137##
or a salt thereof.
[1358] In certain embodiments, the compound of Formula (IX-c) is
the following:
##STR00138##
or a salt thereof.
[1359] In certain embodiments, the compound of Formula (IX-c) is of
Formula (IX-c-3):
##STR00139##
or a salt thereof.
[1360] In certain embodiments, the compound of Formula (IX-c) is of
the following formulae:
##STR00140##
or a salt thereof.
[1361] In certain embodiments, the compound of Formula (IX-c) is
the following:
##STR00141##
or a salt thereof.
[1362] In certain embodiments, a phospholipid useful or potentially
useful in the present invention comprises a modified phosphocholine
moiety, wherein the alkyl chain linking the quaternary amine to the
phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in
certain embodiments, a phospholipid useful or potentially useful in
the present invention is a compound of Formula (IX), wherein n is
1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments,
a compound of Formula (IX) is of one of the following formulae:
##STR00142##
or a salt thereof.
[1363] In certain embodiments, a compound of Formula (IX) is one of
the following:
##STR00143## ##STR00144##
or salts thereof.
[1364] (ii) Alternative Lipids
[1365] In certain embodiments, an alternative lipid is used in
place of a phospholipid of the invention. Non-limiting examples of
such alternative lipids include the following:
##STR00145##
[1366] (iii) Structural Lipids
[1367] The lipid composition of a pharmaceutical composition
disclosed herein can comprise one or more structural lipids. As
used herein, the term "structural lipid" refers to sterols and also
to lipids containing sterol moieties.
[1368] Incorporation of structural lipids in the lipid nanoparticle
may help mitigate aggregation of other lipids in the particle.
Structural lipids can be selected from the group including but not
limited to, cholesterol, fecosterol, sitosterol, ergosterol,
campesterol, stigmasterol, brassicasterol, tomatidine, tomatine,
ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids,
and mixtures thereof. In some embodiments, the structural lipid is
a sterol. As defined herein, "sterols" are a subgroup of steroids
consisting of steroid alcohols. In certain embodiments, the
structural lipid is a steroid. In certain embodiments, the
structural lipid is cholesterol. In certain embodiments, the
structural lipid is an analog of cholesterol. In certain
embodiments, the structural lipid is alpha-tocopherol. Examples of
structural lipids include, but are not limited to, the
following:
##STR00146##
[1369] In one embodiment, the amount of the structural lipid (e.g.,
an sterol such as cholesterol) in the lipid composition of a
pharmaceutical composition disclosed herein ranges from about 20
mol % to about 60 mol %, from about 25 mol % to about 55 mol %,
from about 30 mol % to about 50 mol %, or from about 35 mol % to
about 45 mol %.
[1370] In one embodiment, the amount of the structural lipid (e.g.,
an sterol such as cholesterol) in the lipid composition disclosed
herein ranges from about 25 mol % to about 30 mol %, from about 30
mol % to about 35 mol %, or from about 35 mol % to about 40 mol
%.
[1371] In one embodiment, the amount of the structural lipid (e.g.,
a sterol such as cholesterol) in the lipid composition disclosed
herein is about 24 mol %, about 29 mol %, about 34 mol %, or about
39 mol %.
[1372] In some embodiments, the amount of the structural lipid
(e.g., an sterol such as cholesterol) in the lipid composition
disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60
mol %.
[1373] (iv) Polyethylene Glycol (PEG)-Lipids
[1374] The lipid composition of a pharmaceutical composition
disclosed herein can comprise one or more a polyethylene glycol
(PEG) lipid.
[1375] As used herein, the term "PEG-lipid" refers to polyethylene
glycol (PEG)-modified lipids. Non-limiting examples of PEG-lipids
include PEG-modified phosphatidylethanolamine and phosphatidic
acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20),
PEG-modified dialkylamines and PEG-modified
1,2-diacyloxypropan-3-amines. Such lipids are also referred to as
PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG,
PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
[1376] In some embodiments, the PEG-lipid includes, but not limited
to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol
(PEG-DMG),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG),
PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide
(PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or
PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).
[1377] In one embodiment, the PEG-lipid is selected from the group
consisting of a PEG-modified phosphatidylethanolamine, a
PEG-modified phosphatidic acid, a PEG-modified ceramide, a
PEG-modified dialkylamine, a PEG-modified diacylglycerol, a
PEG-modified dialkylglycerol, and mixtures thereof.
[1378] In some embodiments, the lipid moiety of the PEG-lipids
includes those having lengths of from about C.sub.14 to about
C.sub.22, preferably from about C.sub.14 to about C.sub.16. In some
embodiments, a PEG moiety, for example an mPEG-NH.sub.2, has a size
of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one
embodiment, the PEG-lipid is PEG2k-DMG.
[1379] In one embodiment, the lipid nanoparticles described herein
can comprise a PEG lipid which is a non-diffusible PEG.
Non-limiting examples of non-diffusible PEGs include PEG-DSG and
PEG-DSPE.
[1380] PEG-lipids are known in the art, such as those described in
U.S. Pat. No. 8,158,601 and International Publ. No. WO 2015/130584
A.sub.2, which are incorporated herein by reference in their
entirety.
[1381] In general, some of the other lipid components (e.g., PEG
lipids) of various formulae, described herein may be synthesized as
described International Patent Application No. PCT/US2016/000129,
filed Dec. 10, 2016, entitled "Compositions and Methods for
Delivery of Therapeutic Agents," which is incorporated by reference
in its entirety.
[1382] The lipid component of a lipid nanoparticle composition may
include one or more molecules comprising polyethylene glycol, such
as PEG or PEG-modified lipids. Such species may be alternately
referred to as PEGylated lipids. A PEG lipid is a lipid modified
with polyethylene glycol. A PEG lipid may be selected from the
non-limiting group including PEG-modified
phosphatidylethanolamines, PEG-modified phosphatidic acids,
PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified
diacylglycerols, PEG-modified dialkylglycerols, and mixtures
thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG,
PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
[1383] In some embodiments the PEG-modified lipids are a modified
form of PEG DMG. PEG-DMG has the following structure:
##STR00147##
[1384] In one embodiment, PEG lipids useful in the present
invention can be PEGylated lipids described in International
Publication No. WO2012099755, the contents of which is herein
incorporated by reference in its entirety. Any of these exemplary
PEG lipids described herein may be modified to comprise a hydroxyl
group on the PEG chain. In certain embodiments, the PEG lipid is a
PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also
referred to herein as "hydroxy-PEGylated lipid") is a PEGylated
lipid having one or more hydroxyl (--OH) groups on the lipid. In
certain embodiments, the PEG-OH lipid includes one or more hydroxyl
groups on the PEG chain. In certain embodiments, a PEG-OH or
hydroxy-PEGylated lipid comprises an --OH group at the terminus of
the PEG chain. Each possibility represents a separate embodiment of
the present invention.
[1385] In certain embodiments, a PEG lipid useful in the present
invention is a compound of Formula (VII). Provided herein are
compounds of Formula (VII):
##STR00148##
or salts thereof, wherein:
[1386] R.sup.3 is --OR.sup.O;
[1387] R.sup.O is hydrogen, optionally substituted alkyl, or an
oxygen protecting group;
[1388] r is an integer between 1 and 100, inclusive;
[1389] L.sup.1 is optionally substituted C.sub.1-10 alkylene,
wherein at least one methylene of the optionally substituted
C.sub.1-10 alkylene is independently replaced with optionally
substituted carbocyclylene, optionally substituted heterocyclylene,
optionally substituted arylene, optionally substituted
heteroarylene, --O--, --N(R.sup.N)--, --S--, --C(O)--,
--C(O)N(R.sup.N)--, --NR.sup.NC(O)--, --C(O)O--, --OC(O)--,
--OC(O)O--, --OC(O)N(R.sup.N)--, --NR.sup.NC(O)O--, or
--NR.sup.NC(O)N(R.sup.N)--;
[1390] D is a moiety obtained by click chemistry or a moiety
cleavable under physiological conditions;
[1391] m is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
[1392] A is of the formula:
##STR00149##
[1393] each instance of L.sup.2 is independently a bond or
optionally substituted C.sub.1-6 alkylene, wherein one methylene
unit of the optionally substituted C.sub.1-6 alkylene is optionally
replaced with --O--, --N(R.sup.N)--, --S--, --C(O)--,
--C(O)N(R.sup.N)--, --NR.sup.NC(O)--, --C(O)O--, --OC(O)--,
--OC(O)O--, --OC(O)N(R.sup.N)--, --NR.sup.NC(O)O--, or
--NR.sup.NC(O)N(R.sup.N)--;
[1394] each instance of R.sup.2 is independently optionally
substituted C.sub.1-30 alkyl, optionally substituted C.sub.1-30
alkenyl, or optionally substituted C.sub.1-30 alkynyl; optionally
wherein one or more methylene units of R.sup.2 are independently
replaced with optionally substituted carbocyclylene, optionally
substituted heterocyclylene, optionally substituted arylene,
optionally substituted heteroarylene, --N(R.sup.N)--, --O--, --S--,
--C(O)--, --C(O)N(R.sup.N)--, --NR.sup.NC(O)--,
--NR.sup.NC(O)N(R.sup.N)--, --C(O)O--, --OC(O)--, --OC(O)O--,
--OC(O)N(R.sup.N)--, --NR.sup.NC(O)O--, --C(O)S--, --SC(O)--,
--C(.dbd.NR.sup.N)--, --C(.dbd.NR.sup.N)N(R.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)N(R.sup.N)--, --C(S)--,
--C(S)N(R.sup.N)--, --NR.sup.NC(S)--, --NR.sup.NC(S)N(R.sup.N)--,
--S(O)--, --OS(O)--, --S(O)O--, --OS(O)O--, --OS(O).sub.2--,
--S(O).sub.2O--, --OS(O).sub.2O--, --N(R.sup.N) S(O)--,
--S(O)N(R.sup.N)--, --N(R.sup.N) S(O)N(R.sup.N)--,
--OS(O)N(R.sup.N)--, --N(R.sup.N) S(O)O--, --S(O).sub.2--,
--N(R.sup.N) S(O).sub.2--, --S(O).sub.2N(R.sup.N)--,
--N(R.sup.N)S(O).sub.2N(R.sup.N)--, --OS(O).sub.2N(R.sup.N)--, or
--N(R.sup.N)S(O).sub.2O--;
[1395] each instance of R.sup.N is independently hydrogen,
optionally substituted alkyl, or a nitrogen protecting group;
[1396] Ring B is optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, or
optionally substituted heteroaryl; and
[1397] p is 1 or 2.
[1398] In certain embodiments, the compound of Formula (VII) is a
PEG-OH lipid (i.e., R.sup.3 is --OR.sup.O, and R.sup.O is
hydrogen). In certain embodiments, the compound of Formula (VII) is
of Formula (VII-OH):
##STR00150##
or a salt thereof.
[1399] In certain embodiments, D is a moiety obtained by click
chemistry (e.g., triazole). In certain embodiments, the compound of
Formula (VII) is of Formula (VII-a-1) or (VII-a-2):
##STR00151##
(VII-a-1) (VII-a-2),
[1400] or a salt thereof.
[1401] In certain embodiments, the compound of Formula (VII) is of
one of the following formulae:
##STR00152##
or a salt thereof, wherein
[1402] s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[1403] In certain embodiments, the compound of Formula (VII) is of
one of the following formulae:
##STR00153##
or a salt thereof.
[1404] In certain embodiments, a compound of Formula (VII) is of
one of the following formulae:
##STR00154##
or a salt thereof.
[1405] In certain embodiments, a compound of Formula (VII) is of
one of the following formulae:
##STR00155##
or a salt thereof.
[1406] In certain embodiments, D is a moiety cleavable under
physiological conditions (e.g., ester, amide, carbonate, carbamate,
urea). In certain embodiments, a compound of Formula (VII) is of
Formula (VII-b-1) or (VII-b-2):
##STR00156##
or a salt thereof.
[1407] In certain embodiments, a compound of Formula (VII) is of
Formula (VII-b-1-OH) or (VII-b-2-OH):
##STR00157##
or a salt thereof.
[1408] In certain embodiments, the compound of Formula (VII) is of
one of the following formulae:
##STR00158##
or a salt thereof.
[1409] In certain embodiments, a compound of Formula (VII) is of
one of the following formulae:
##STR00159##
or a salt thereof.
[1410] In certain embodiments, a compound of Formula (VII) is of
one of the following formulae:
##STR00160##
or a salt thereof.
[1411] In certain embodiments, a compound of Formula (VII) is of
one of the following formulae:
##STR00161##
or salts thereof.
[1412] In certain embodiments, a PEG lipid useful in the present
invention is a PEGylated fatty acid. In certain embodiments, a PEG
lipid useful in the present invention is a compound of Formula
(VIII). Provided herein are compounds of Formula (VIII):
##STR00162##
or a salts thereof, wherein:
[1413] R.sup.3 is --OR.sup.O;
[1414] R.sup.O is hydrogen, optionally substituted alkyl or an
oxygen protecting group;
[1415] r is an integer between 1 and 100, inclusive;
[1416] R.sup.5 is optionally substituted C.sub.10-40 alkyl,
optionally substituted C.sub.10-40 alkenyl, or optionally
substituted C.sub.10-40 alkynyl; and optionally one or more
methylene groups of R.sup.5 are replaced with optionally
substituted carbocyclylene, optionally substituted heterocyclylene,
optionally substituted arylene, optionally substituted
heteroarylene, --N(R.sup.N)--, --O--, --S--, --C(O)--,
--C(O)N(R.sup.N)--, --NR.sup.NC(O)--, --NR.sup.NC(O)N(R.sup.N)--,
--C(O)O--, --OC(O)--, --OC(O)O--, --OC(O)N(R.sup.N)--,
--NR.sup.NC(O)O--, --C(O)S--, --SC(O)--, --C(.dbd.NR.sup.N)--,
--C(.dbd.NR.sup.N)N(R.sup.N)--, --NR.sup.NC(.dbd.NR.sup.N)--,
--NR.sup.NC(.dbd.NR.sup.N)N(R.sup.N)--, --C(S)--,
--C(S)N(R.sup.N)--, --NR.sup.NC(S)--, --NR.sup.NC(S)N(R.sup.N)--,
--S(O)--, --OS(O)--, --S(O)O--, --OS(O)O--, --OS(O).sub.2--,
--S(O).sub.2O--, --OS(O).sub.2O--, --N(R.sup.N)S(O)--,
--S(O)N(R.sup.N)--, --N(R.sup.N)S(O)N(R.sup.N)--,
--OS(O)N(R.sup.N)--, --N(R.sup.N)S(O)O--, --S(O).sub.2--,
--N(R.sup.N) S(O).sub.2--, --S(O).sub.2N(R.sup.N)--, --N(R.sup.N)
S(O).sub.2N(R.sup.N)-, --OS(O).sub.2N(R.sup.N)--, or
--N(R.sup.N)S(O).sub.2O--; and
[1417] each instance of R.sup.N is independently hydrogen,
optionally substituted alkyl, or a nitrogen protecting group.
[1418] In certain embodiments, the compound of Formula (VIII) is of
Formula (VIII-OH):
##STR00163##
or a salt thereof. In some embodiments, r is 45.
[1419] In certain embodiments, a compound of Formula (VIII) is of
one of the following formulae:
##STR00164##
or a salt thereof. In some embodiments, r is 45.
[1420] In yet other embodiments the compound of Formula (VIII)
is:
##STR00165##
or a salt thereof.
[1421] In one embodiment, the compound of Formula (VIII) is
##STR00166##
[1422] In one embodiment, the amount of PEG-lipid in the lipid
composition of a pharmaceutical composition disclosed herein ranges
from about 0.1 mol % to about 5 mol %, from about 0.5 mol % to
about 5 mol %, from about 1 mol % to about 5 mol %, from about 1.5
mol % to about 5 mol %, from about 2 mol % to about 5 mol % mol %,
from about 0.1 mol % to about 4 mol %, from about 0.5 mol % to
about 4 mol %, from about 1 mol % to about 4 mol %, from about 1.5
mol % to about 4 mol %, from about 2 mol % to about 4 mol %, from
about 0.1 mol % to about 3 mol %, from about 0.5 mol % to about 3
mol %, from about 1 mol % to about 3 mol %, from about 1.5 mol % to
about 3 mol %, from about 2 mol % to about 3 mol %, from about 0.1
mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from
about 1 mol % to about 2 mol %, from about 1.5 mol % to about 2 mol
%, from about 0.1 mol % to about 1.5 mol %, from about 0.5 mol % to
about 1.5 mol %, or from about 1 mol % to about 1.5 mol %.
[1423] In one embodiment, the amount of PEG-lipid in the lipid
composition disclosed herein is about 2 mol %. In one embodiment,
the amount of PEG-lipid in the lipid composition disclosed herein
is about 1.5 mol %.
[1424] In one embodiment, the amount of PEG-lipid in the lipid
composition disclosed herein is at least about 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,
4.7, 4.8, 4.9, or 5 mol %.
[1425] In some aspects, the lipid composition of the pharmaceutical
compositions disclosed herein does not comprise a PEG-lipid.
[1426] (v) Other Ionizable Amino Lipids
[1427] The lipid composition of the pharmaceutical composition
disclosed herein can comprise one or more ionizable amino lipids in
addition to a lipid according to Formula (I), (III), (IV), (V), or
(VI).
[1428] Ionizable lipids can be selected from the non-limiting group
consisting of
3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine
(KL10),
N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanami-
ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane
(KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),
heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate
(DLin-MC3-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),
(13Z,165Z)--N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine (L608),
2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-
-octadeca-9,12-di en-1-yloxy]propan-1-amine (Octyl-CLinDMA),
(2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-
,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA
(2R)), and
(2S)-2-({8-[(3(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(-
9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA
(2S)). In addition to these, an ionizable amino lipid can also be a
lipid including a cyclic amine group.
[1429] Ionizable lipids can also be the compounds disclosed in
International Publication No. WO 2017/075531 A.sub.1, hereby
incorporated by reference in its entirety. For example, the
ionizable amino lipids include, but not limited to:
##STR00167##
and any combination thereof.
[1430] Ionizable lipids can also be the compounds disclosed in
International Publication No. WO 2015/199952 A.sub.1, hereby
incorporated by reference in its entirety. For example, the
ionizable amino lipids include, but not limited to:
##STR00168## ##STR00169##
and any combination thereof.
[1431] Ionizable lipids can further include, but are not limited
to:
##STR00170##
and any combination thereof.
[1432] (vi) Other Lipid Composition Components
[1433] The lipid composition of a pharmaceutical composition
disclosed herein can include one or more components in addition to
those described above. For example, the lipid composition can
include one or more permeability enhancer molecules, carbohydrates,
polymers, surface altering agents (e.g., surfactants), or other
components. For example, a permeability enhancer molecule can be a
molecule described by U.S. Patent Application Publication No.
2005/0222064. Carbohydrates can include simple sugars (e.g.,
glucose) and polysaccharides (e.g., glycogen and derivatives and
analogs thereof).
[1434] A polymer can be included in and/or used to encapsulate or
partially encapsulate a pharmaceutical composition disclosed herein
(e.g., a pharmaceutical composition in lipid nanoparticle form). A
polymer can be biodegradable and/or biocompatible. A polymer can be
selected from, but is not limited to, polyamines, polyethers,
polyamides, polyesters, polycarbamates, polyureas, polycarbonates,
polystyrenes, polyimides, polysulfones, polyurethanes,
polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates,
polyacrylates, polymethacrylates, polyacrylonitriles, and
polyarylates.
[1435] The ratio between the lipid composition and the
polynucleotide range can be from about 10:1 to about 60:1
(wt/wt).
[1436] In some embodiments, the ratio between the lipid composition
and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1,
15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1,
26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1,
37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1,
48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1,
59:1 or 60:1 (wt/wt). In some embodiments, the wt/wt ratio of the
lipid composition to the polynucleotide encoding a therapeutic
agent is about 20:1 or about 15:1.
[1437] In some embodiments, the pharmaceutical composition
disclosed herein can contain more than one polypeptides. For
example, a pharmaceutical composition disclosed herein can contain
two or more polynucleotides (e.g., RNA, e.g., mRNA).
[1438] In one embodiment, the lipid nanoparticles described herein
can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide
weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1,
45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios
such as, but not limited to, 5:1 to about 10:1, from about 5:1 to
about 15:1, from about 5:1 to about 20:1, from about 5:1 to about
25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1,
from about 5:1 to about 40:1, from about 5:1 to about 45:1, from
about 5:1 to about 50:1, from about 5:1 to about 55:1, from about
5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to
about 15:1, from about 10:1 to about 20:1, from about 10:1 to about
25:1, from about 10:1 to about 30:1, from about 10:1 to about 35:1,
from about 10:1 to about 40:1, from about 10:1 to about 45:1, from
about 10:1 to about 50:1, from about 10:1 to about 55:1, from about
10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1
to about 20:1, from about 15:1 to about 25:1, from about 15:1 to
about 30:1, from about 15:1 to about 35:1, from about 15:1 to about
40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1,
from about 15:1 to about 55:1, from about 15:1 to about 60:1 or
from about 15:1 to about 70:1.
[1439] In one embodiment, the lipid nanoparticles described herein
can comprise the polynucleotide in a concentration from
approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1
mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7
mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3
mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9
mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.
[1440] (vii) Nanoparticle Compositions
[1441] In some embodiments, the pharmaceutical compositions
disclosed herein are formulated as lipid nanoparticles (LNP).
Accordingly, the present disclosure also provides nanoparticle
compositions comprising (i) a lipid composition comprising a
delivery agent such as a compound of Formula (I) or (III) as
described herein, and (ii) a polynucleotide encoding a PBGD
polypeptide. In such nanoparticle composition, the lipid
composition disclosed herein can encapsulate the polynucleotide
encoding a PBGD polypeptide.
[1442] Nanoparticle compositions are typically sized on the order
of micrometers or smaller and can include a lipid bilayer.
Nanoparticle compositions encompass lipid nanoparticles (LNPs),
liposomes (e.g., lipid vesicles), and lipoplexes. For example, a
nanoparticle composition can be a liposome having a lipid bilayer
with a diameter of 500 nm or less.
[1443] Nanoparticle compositions include, for example, lipid
nanoparticles (LNPs), liposomes, and lipoplexes. In some
embodiments, nanoparticle compositions are vesicles including one
or more lipid bilayers. In certain embodiments, a nanoparticle
composition includes two or more concentric bilayers separated by
aqueous compartments. Lipid bilayers can be functionalized and/or
crosslinked to one another. Lipid bilayers can include one or more
ligands, proteins, or channels.
[1444] In some embodiments, the nanoparticle compositions of the
present disclosure comprise at least one compound according to
Formula (I), (III), (IV), (V), or (VI). For example, the
nanoparticle composition can include one or more of Compounds
1-147, or one or more of Compounds 1-342. Nanoparticle compositions
can also include a variety of other components. For example, the
nanoparticle composition may include one or more other lipids in
addition to a lipid according to Formula (I), (III), (IV), (V), or
(VI), such as (i) at least one phospholipid, (ii) at least one
structural lipid, (iii) at least one PEG-lipid, or (iv) any
combination thereof. Inclusion of structural lipid can be optional,
for example when lipids according to Formula III are used in the
lipid nanoparticle compositions of the invention.
[1445] In some embodiments, the nanoparticle composition comprises
a compound of Formula (I), (e.g., Compounds 18, 25, 26 or 48). In
some embodiments, the nanoparticle composition comprises a compound
of Formula (I) (e.g., Compounds 18, 25, 26 or 48) and a
phospholipid (e.g., DSPC).
[1446] In some embodiments, the nanoparticle composition comprises
a compound of Formula (III) (e.g., Compound 236). In some
embodiments, the nanoparticle composition comprises a compound of
Formula (III) (e.g., Compound 236) and a phospholipid (e.g., DOPE
or DSPC).
[1447] In some embodiments, the nanoparticle composition comprises
a lipid composition consisting or consisting essentially of
compound of Formula (I) (e.g., Compounds 18, 25, 26 or 48). In some
embodiments, the nanoparticle composition comprises a lipid
composition consisting or consisting essentially of a compound of
Formula (I) (e.g., Compounds 18, 25, 26 or 48) and a phospholipid
(e.g., DSPC).
[1448] In some embodiments, the nanoparticle composition comprises
a lipid composition consisting or consisting essentially of
compound of Formula (III) (e.g., Compound 236). In some
embodiments, the nanoparticle composition comprises a lipid
composition consisting or consisting essentially of a compound of
Formula (III) (e.g., Compound 236) and a phospholipid (e.g., DOPE
or DSPC).
[1449] In one embodiment, a lipid nanoparticle comprises an
ionizable lipid, a structural lipid, a phospholipid, and mRNA. In
some embodiments, the LNP comprises an ionizable lipid, a
PEG-modified lipid, a sterol and a structural lipid. In some
embodiments, the LNP has a molar ratio of about 20-60% ionizable
lipid:about 5-25% structural lipid:about 25-55% sterol; and about
0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises
a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified
lipid, about 38.5% cholesterol and about 10% structural lipid. In
some embodiments, the LNP comprises a molar ratio of about 55%
ionizable lipid, about 2.5% PEG lipid, about 32.5% cholesterol and
about 10% structural lipid. In some embodiments, the ionizable
lipid is an ionizable amino lipid and the structural lipid is a
neutral lipid, and the sterol is a cholesterol. In some
embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 of
ionizable lipid:cholesterol:DSPC: PEG lipid. In some embodiments,
the ionizable lipid is Compound 18 or Compound 236, and the PEG
lipid is Compound 428.
[1450] In some embodiments, the LNP has a molar ratio of
50:38.5:10:1.5 of Compound 18:Cholesterol:Phospholipid:Compound
428. In some embodiments, the LNP has a molar ratio of
50:38.5:10:1.5 of Compound 18:Cholesterol:DSPC:Compound 428.
[1451] In some embodiments, the LNP has a molar ratio of
50:38.5:10:1.5 of Compound 236:Cholesterol:Phospholipid:Compound
428. In some embodiments, the LNP has a molar ratio of
50:38.5:10:1.5 of Compound 236:Cholesterol:DSPC:Compound 428.
[1452] In some embodiments, the LNP has a polydispersity value of
less than 0.4. In some embodiments, the LNP has a net neutral
charge at a neutral pH. In some embodiments, the LNP has a mean
diameter of 50-150 nm. In some embodiments, the LNP has a mean
diameter of 80-100 nm.
[1453] As generally defined herein, the term "lipid" refers to a
small molecule that has hydrophobic or amphiphilic properties.
Lipids may be naturally occurring or synthetic. Examples of classes
of lipids include, but are not limited to, fats, waxes,
sterol-containing metabolites, vitamins, fatty acids,
glycerolipids, glycerophospholipids, sphingolipids, saccharolipids,
and polyketides, and prenol lipids. In some instances, the
amphiphilic properties of some lipids leads them to form liposomes,
vesicles, or membranes in aqueous media.
[1454] In some embodiments, a lipid nanoparticle (LNP) may comprise
an ionizable lipid. As used herein, the term "ionizable lipid" has
its ordinary meaning in the art and may refer to a lipid comprising
one or more charged moieties. In some embodiments, an ionizable
lipid may be positively charged or negatively charged. An ionizable
lipid may be positively charged, in which case it can be referred
to as "cationic lipid." In certain embodiments, an ionizable lipid
molecule may comprise an amine group, and can be referred to as an
ionizable amino lipid. As used herein, a "charged moiety" is a
chemical moiety that carries a formal electronic charge, e.g.,
monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or
-3), etc. The charged moiety may be anionic (i.e., negatively
charged) or cationic (i.e., positively charged). Examples of
positively-charged moieties include amine groups (e.g., primary,
secondary, and/or tertiary amines), ammonium groups, pyridinium
group, guanidine groups, and imidizolium groups. In a particular
embodiment, the charged moieties comprise amine groups. Examples of
negatively-charged groups or precursors thereof, include
carboxylate groups, sulfonate groups, sulfate groups, phosphonate
groups, phosphate groups, hydroxyl groups, and the like. The charge
of the charged moiety may vary, in some cases, with the
environmental conditions, for example, changes in pH may alter the
charge of the moiety, and/or cause the moiety to become charged or
uncharged. In general, the charge density of the molecule may be
selected as desired.
[1455] It should be understood that the terms "charged" or "charged
moiety" does not refer to a "partial negative charge" or "partial
positive charge" on a molecule. The terms "partial negative charge"
and "partial positive charge" are given its ordinary meaning in the
art. A "partial negative charge" may result when a functional group
comprises a bond that becomes polarized such that electron density
is pulled toward one atom of the bond, creating a partial negative
charge on the atom. Those of ordinary skill in the art will, in
general, recognize bonds that can become polarized in this way.
[1456] In some embodiments, the ionizable lipid is an ionizable
amino lipid, sometimes referred to in the art as an "ionizable
cationic lipid". In one embodiment, the ionizable amino lipid may
have a positively charged hydrophilic head and a hydrophobic tail
that are connected via a linker structure.
[1457] In addition to these, an ionizable lipid may also be a lipid
including a cyclic amine group.
[1458] In one embodiment, the ionizable lipid may be selected from,
but not limited to, a ionizable lipid described in International
Publication Nos. WO2013086354 and WO2013116126; the contents of
each of which are herein incorporated by reference in their
entirety.
[1459] In yet another embodiment, the ionizable lipid may be
selected from, but not limited to, formula CLI-CLXXXXII of U.S.
Pat. No. 7,404,969; each of which is herein incorporated by
reference in their entirety.
[1460] In one embodiment, the lipid may be a cleavable lipid such
as those described in International Publication No. WO2012170889,
herein incorporated by reference in its entirety. In one
embodiment, the lipid may be synthesized by methods known in the
art and/or as described in International Publication Nos.
WO2013086354; the contents of each of which are herein incorporated
by reference in their entirety.
[1461] Nanoparticle compositions can be characterized by a variety
of methods. For example, microscopy (e.g., transmission electron
microscopy or scanning electron microscopy) can be used to examine
the morphology and size distribution of a nanoparticle composition.
Dynamic light scattering or potentiometry (e.g., potentiometric
titrations) can be used to measure zeta potentials. Dynamic light
scattering can also be utilized to determine particle sizes.
Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd,
Malvem, Worcestershire, UK) can also be used to measure multiple
characteristics of a nanoparticle composition, such as particle
size, polydispersity index, and zeta potential.
[1462] The size of the nanoparticles can help counter biological
reactions such as, but not limited to, inflammation, or can
increase the biological effect of the polynucleotide.
[1463] As used herein, "size" or "mean size" in the context of
nanoparticle compositions refers to the mean diameter of a
nanoparticle composition.
[1464] In one embodiment, the polynucleotide encoding a PBGD
polypeptide are formulated in lipid nanoparticles having a diameter
from about 10 to about 100 nm such as, but not limited to, about 10
to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm,
about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about
70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20
to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm,
about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about
80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30
to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm,
about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about
90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40
to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm,
about 40 to about 90 nm, about 40 to about 100 nm, about 50 to
about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm,
about 50 to about 90 nm, about 50 to about 100 nm, about 60 to
about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm,
about 60 to about 100 nm, about 70 to about 80 nm, about 70 to
about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm,
about 80 to about 100 nm and/or about 90 to about 100 nm.
[1465] In one embodiment, the nanoparticles have a diameter from
about 10 to 500 nm. In one embodiment, the nanoparticle has a
diameter greater than 100 nm, greater than 150 nm, greater than 200
nm, greater than 250 nm, greater than 300 nm, greater than 350 nm,
greater than 400 nm, greater than 450 nm, greater than 500 nm,
greater than 550 nm, greater than 600 nm, greater than 650 nm,
greater than 700 nm, greater than 750 nm, greater than 800 nm,
greater than 850 nm, greater than 900 nm, greater than 950 nm or
greater than 1000 nm.
[1466] In some embodiments, the largest dimension of a nanoparticle
composition is 1 .mu.m or shorter (e.g., 1 .mu.m, 900 nm, 800 nm,
700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125
nm, 100 nm, 75 nm, 50 nm, or shorter).
[1467] A nanoparticle composition can be relatively homogenous. A
polydispersity index can be used to indicate the homogeneity of a
nanoparticle composition, e.g., the particle size distribution of
the nanoparticle composition. A small (e.g., less than 0.3)
polydispersity index generally indicates a narrow particle size
distribution. A nanoparticle composition can have a polydispersity
index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04,
0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15,
0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In
some embodiments, the polydispersity index of a nanoparticle
composition disclosed herein can be from about 0.10 to about
0.20.
[1468] The zeta potential of a nanoparticle composition can be used
to indicate the electrokinetic potential of the composition. For
example, the zeta potential can describe the surface charge of a
nanoparticle composition. Nanoparticle compositions with relatively
low charges, positive or negative, are generally desirable, as more
highly charged species can interact undesirably with cells,
tissues, and other elements in the body. In some embodiments, the
zeta potential of a nanoparticle composition disclosed herein can
be from about -10 mV to about +20 mV, from about -10 mV to about
+15 mV, from about 10 mV to about +10 mV, from about -10 mV to
about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to
about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to
about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to
about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to
about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to
about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to
about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV
to about +10 mV.
[1469] In some embodiments, the zeta potential of the lipid
nanoparticles can be from about 0 mV to about 100 mV, from about 0
mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV
to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to
about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to
about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to
about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to
about 90 mV, from about 10 mV to about 80 mV, from about 10 mV to
about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to
about 50 mV, from about 10 mV to about 40 mV, from about 10 mV to
about 30 mV, from about 10 mV to about 20 mV, from about 20 mV to
about 100 mV, from about 20 mV to about 90 mV, from about 20 mV to
about 80 mV, from about 20 mV to about 70 mV, from about 20 mV to
about 60 mV, from about 20 mV to about 50 mV, from about 20 mV to
about 40 mV, from about 20 mV to about 30 mV, from about 30 mV to
about 100 mV, from about 30 mV to about 90 mV, from about 30 mV to
about 80 mV, from about 30 mV to about 70 mV, from about 30 mV to
about 60 mV, from about 30 mV to about 50 mV, from about 30 mV to
about 40 mV, from about 40 mV to about 100 mV, from about 40 mV to
about 90 mV, from about 40 mV to about 80 mV, from about 40 mV to
about 70 mV, from about 40 mV to about 60 mV, and from about 40 mV
to about 50 mV. In some embodiments, the zeta potential of the
lipid nanoparticles can be from about 10 mV to about 50 mV, from
about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and
from about 25 mV to about 35 mV. In some embodiments, the zeta
potential of the lipid nanoparticles can be about 10 mV, about 20
mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70
mV, about 80 mV, about 90 mV, and about 100 mV.
[1470] The term "encapsulation efficiency" of a polynucleotide
describes the amount of the polynucleotide that is encapsulated by
or otherwise associated with a nanoparticle composition after
preparation, relative to the initial amount provided. As used
herein, "encapsulation" can refer to complete, substantial, or
partial enclosure, confinement, surrounding, or encasement.
[1471] Encapsulation efficiency is desirably high (e.g., close to
100%). The encapsulation efficiency can be measured, for example,
by comparing the amount of the polynucleotide in a solution
containing the nanoparticle composition before and after breaking
up the nanoparticle composition with one or more organic solvents
or detergents.
[1472] Fluorescence can be used to measure the amount of free
polynucleotide in a solution. For the nanoparticle compositions
described herein, the encapsulation efficiency of a polynucleotide
can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In
some embodiments, the encapsulation efficiency can be at least 80%.
In certain embodiments, the encapsulation efficiency can be at
least 90%.
[1473] The amount of a polynucleotide present in a pharmaceutical
composition disclosed herein can depend on multiple factors such as
the size of the polynucleotide, desired target and/or application,
or other properties of the nanoparticle composition as well as on
the properties of the polynucleotide.
[1474] For example, the amount of an mRNA useful in a nanoparticle
composition can depend on the size (expressed as length, or
molecular mass), sequence, and other characteristics of the mRNA.
The relative amounts of a polynucleotide in a nanoparticle
composition can also vary.
[1475] The relative amounts of the lipid composition and the
polynucleotide present in a lipid nanoparticle composition of the
present disclosure can be optimized according to considerations of
efficacy and tolerability. For compositions including an mRNA as a
polynucleotide, the N:P ratio can serve as a useful metric.
[1476] As the N:P ratio of a nanoparticle composition controls both
expression and tolerability, nanoparticle compositions with low N:P
ratios and strong expression are desirable. N:P ratios vary
according to the ratio of lipids to RNA in a nanoparticle
composition.
[1477] In general, a lower N:P ratio is preferred. The one or more
RNA, lipids, and amounts thereof can be selected to provide an N:P
ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1,
6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1,
26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be
from about 2:1 to about 8:1. In other embodiments, the N:P ratio is
from about 5:1 to about 8:1. In certain embodiments, the N:P ratio
is between 5:1 and 6:1. In one specific aspect, the N:P ratio is
about is about 5.67:1.
[1478] In addition to providing nanoparticle compositions, the
present disclosure also provides methods of producing lipid
nanoparticles comprising encapsulating a polynucleotide. Such
method comprises using any of the pharmaceutical compositions
disclosed herein and producing lipid nanoparticles in accordance
with methods of production of lipid nanoparticles known in the art.
See, e.g., Wang et al. (2015) "Delivery of oligonucleotides with
lipid nanoparticles" Adv. Drug Deliv. Rev. 87:68-80; Silva et al.
(2015) "Delivery Systems for Biopharmaceuticals. Part I:
Nanoparticles and Microparticles" Curr. Pharm. Technol. 16:
940-954; Naseri et al. (2015) "Solid Lipid Nanoparticles and
Nanostructured Lipid Carriers: Structure, Preparation and
Application" Adv. Pharm. Bull. 5:305-13; Silva et al. (2015) "Lipid
nanoparticles for the delivery of biopharmaceuticals" Curr. Pharm.
Biotechnol. 16:291-302, and references cited therein.
23. OTHER DELIVERY AGENTS
[1479] a. Liposomes, Lipoplexes, and Lipid Nanoparticles
[1480] In some embodiments, the compositions or formulations of the
present disclosure comprise a delivery agent, e.g., a liposome, a
lioplexes, a lipid nanoparticle, or any combination thereof. The
polynucleotides described herein (e.g., a polynucleotide comprising
a nucleotide sequence encoding a PBGD polypeptide) can be
formulated using one or more liposomes, lipoplexes, or lipid
nanoparticles. Liposomes, lipoplexes, or lipid nanoparticles can be
used to improve the efficacy of the polynucleotides directed
protein production as these formulations can increase cell
transfection by the polynucleotide; and/or increase the translation
of encoded protein. The liposomes, lipoplexes, or lipid
nanoparticles can also be used to increase the stability of the
polynucleotides.
[1481] Liposomes are artificially-prepared vesicles that can
primarily be composed of a lipid bilayer and can be used as a
delivery vehicle for the administration of pharmaceutical
formulations. Liposomes can be of different sizes. A multilamellar
vesicle (MLV) can be hundreds of nanometers in diameter, and can
contain a series of concentric bilayers separated by narrow aqueous
compartments. A small unicellular vesicle (SUV) can be smaller than
50 nm in diameter, and a large unilamellar vesicle (LUV) can be
between 50 and 500 nm in diameter. Liposome design can include, but
is not limited to, opsonins or ligands to improve the attachment of
liposomes to unhealthy tissue or to activate events such as, but
not limited to, endocytosis. Liposomes can contain a low or a high
pH value in order to improve the delivery of the pharmaceutical
formulations.
[1482] The formation of liposomes can depend on the pharmaceutical
formulation entrapped and the liposomal ingredients, the nature of
the medium in which the lipid vesicles are dispersed, the effective
concentration of the entrapped substance and its potential
toxicity, any additional processes involved during the application
and/or delivery of the vesicles, the optimal size, polydispersity
and the shelf-life of the vesicles for the intended application,
and the batch-to-batch reproducibility and scale up production of
safe and efficient liposomal products, etc.
[1483] As a non-limiting example, liposomes such as synthetic
membrane vesicles can be prepared by the methods, apparatus and
devices described in U.S. Pub. Nos. US20130177638, US20130177637,
US20130177636, US20130177635, US20130177634, US20130177633,
US20130183375, US20130183373, and US20130183372. In some
embodiments, the polynucleotides described herein can be
encapsulated by the liposome and/or it can be contained in an
aqueous core that can then be encapsulated by the liposome as
described in, e.g., Intl. Pub. Nos. WO2012031046, WO2012031043,
WO2012030901, WO2012006378, and WO2013086526; and U.S. Pub. Nos.
US20130189351, US20130195969 and US20130202684. Each of the
references in herein incorporated by reference in its entirety.
[1484] In some embodiments, the polynucleotides described herein
can be formulated in a cationic oil-in-water emulsion where the
emulsion particle comprises an oil core and a cationic lipid that
can interact with the polynucleotide anchoring the molecule to the
emulsion particle. In some embodiments, the polynucleotides
described herein can be formulated in a water-in-oil emulsion
comprising a continuous hydrophobic phase in which the hydrophilic
phase is dispersed. Exemplary emulsions can be made by the methods
described in Intl. Pub. Nos. WO2012006380 and WO201087791, each of
which is herein incorporated by reference in its entirety.
[1485] In some embodiments, the polynucleotides described herein
can be formulated in a lipid-polycation complex. The formation of
the lipid-polycation complex can be accomplished by methods as
described in, e.g., U.S. Pub. No. US20120178702. As a non-limiting
example, the polycation can include a cationic peptide or a
polypeptide such as, but not limited to, polylysine, polyomithine
and/or polyarginine and the cationic peptides described in Intl.
Pub. No. WO2012013326 or U.S. Pub. No. US20130142818. Each of the
references is herein incorporated by reference in its entirety.
[1486] In some embodiments, the polynucleotides described herein
can be formulated in a lipid nanoparticle (LNP) such as those
described in Intl. Pub. Nos. WO2013123523, WO2012170930,
WO2011127255 and WO2008103276; and U.S. Pub. No. US20130171646,
each of which is herein incorporated by reference in its
entirety.
[1487] Lipid nanoparticle formulations typically comprise one or
more lipids. In some embodiments, the lipid is an ionizable lipid
(e.g., an ionizable amino lipid), sometimes referred to in the art
as an "ionizable cationic lipid". In some embodiments, lipid
nanoparticle formulations further comprise other components,
including a phospholipid, a structural lipid, and a molecule
capable of reducing particle aggregation, for example a PEG or
PEG-modified lipid.
[1488] Exemplary ionizable lipids include, but not limited to, any
one of Compounds 1-342 disclosed herein, DLin-MC3-DMA (MC3),
DLin-DMA, DLenDMA, DLin-D-DMA, DLin-K-DMA, DLin-M-C2-DMA,
DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLin-C2K-DMA,
DLin-MP-DMA, DODMA, 98N12-5, C12-200, DLin-C-DAP, DLin-DAC,
DLinDAP, DLinAP, DLin-EG-DMA, DLin-2-DMAP, KL10, KL22, KL25,
Octyl-CLinDMA, Octyl-CLinDMA (2R), Octyl-CLinDMA (2S), and any
combination thereof. Other exemplary ionizable lipids include,
(13Z,16Z)--N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608),
(20Z,23Z)--N,N-dimethylnonacosa-20,23-dien-10-amine,
(17Z,20Z)--N,N-dimemylhexacosa-17,20-dien-9-amine,
(16Z,19Z)--N5N-dimethylpentacosa-16,19-dien-8-amine,
(13Z,16Z)--N,N-dimethyldocosa-13,16-dien-5-amine,
(12Z,15Z)--N,N-dimethylhenicosa-12,15-dien-4-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-6-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-7-amine,
(18Z,21Z)--N,N-dimethylheptacosa-18,21-dien-10-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-5-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-4-amine,
(19Z,22Z)--N,N-dimeihyloctacosa-19,22-dien-9-amine,
(18Z,21Z)--N,N-dimethylheptacosa-18,21-dien-8-amine,
(17Z,20Z)--N,N-dimethylhexacosa-17,20-dien-7-amine,
(16Z,19Z)--N,N-dimethylpentacosa-16,19-dien-6-amine,
(22Z,25Z)--N,N-dimethylhentriaconta-22,25-dien-10-amine,
(21Z,24Z)--N,N-dimethyltriaconta-21,24-dien-9-amine,
(18Z)--N,N-dimetylheptacos-18-en-10-amine,
(17Z)--N,N-dimethylhexacos-17-en-9-amine, (1
9Z,22Z)--N,N-dimethyloctacosa-19,22-dien-7-amine,
N,N-dimethylheptacosan-10-amine,
(20Z,23Z)--N-ethyl-N-methylnonacosa-20,23-dien-10-amine,
1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,
(20Z)--N,N-dimethylheptacos-20-en-10-amine, (15Z)--N,N-dimethyl
eptacos-15-en-10-amine, (14Z)--N,N-dimethylnonacos-14-en-10-amine,
(17Z)--N,N-dimethylnonacos-17-en-10-amine,
(24Z)--N,N-dimethyltritriacont-24-en-10-amine,
(20Z)--N,N-dimethylnonacos-20-en-10-amine,
(22Z)--N,N-dimethylhentriacont-22-en-10-amine,
(16Z)--N,N-dimethylpentacos-16-en-8-amine,
(12Z,15Z)--N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,
1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,
N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]nonadecan-10-amine,
N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,
N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropy-
l]nonadecan-10-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,
N,N-dimethyl-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine,
N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,
1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,
1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,
R--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-
n-2-amine,
S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octy-
loxy)propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrr-
olidine,
(2S)--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-
)-oct-5-en-1-yloxy]propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azet-
idine,
(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-ylo-
xy]propan-2-amine,
(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-
opan-2-amine,
N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-
-amine,
N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-am-
ine;
(2S)--N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(o-
ctyloxy)propan-2-amine,
(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro-
pan-2-amine,
(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylprop-
an-2-amine,
1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2--
amine,
1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)pr-
opan-2-amine,
(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpro-
pan-2-amine,
(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amin-
e,
1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
(2R)--N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1--
yloxy]propan-2-amine,
(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-di-
en-1-yloxy]propan-2-amine,
N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-
methyl}cyclopropyl]octyl}oxy)propan-2-amine,
N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-am-
ine, and
(11E,20Z,23Z)--N,N-dimethylnonacosa-11,20,2-trien-10-amine, and any
combination thereof.
[1489] Phospholipids include, but are not limited to,
glycerophospholipids such as phosphatidylcholines,
phosphatidylethanolamines, phosphatidylserines,
phosphatidylinositols, phosphatidy glycerols, and phosphatidic
acids. Phospholipids also include phosphosphingolipid, such as
sphingomyelin. In some embodiments, the phospholipids are DLPC,
DMPC, DOPC, DPPC, DSPC, DUPC, 18:0 Diether PC, DLnPC, DAPC, DHAPC,
DOPE, 4ME 16:0 PE, DSPE, DLPE, DLnPE, DAPE, DHAPE, DOPG, and any
combination thereof. In some embodiments, the phospholipids are
MPPC, MSPC, PMPC, PSPC, SMPC, SPPC, DHAPE, DOPG, and any
combination thereof. In some embodiments, the amount of
phospholipids (e.g., DSPC) in the lipid composition ranges from
about 1 mol % to about 20 mol %.
[1490] The structural lipids include sterols and lipids containing
sterol moieties. In some embodiments, the structural lipids include
cholesterol, fecosterol, sitosterol, ergosterol, campesterol,
stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid,
alpha-tocopherol, and mixtures thereof. In some embodiments, the
structural lipid is cholesterol. In some embodiments, the amount of
the structural lipids (e.g., cholesterol) in the lipid composition
ranges from about 20 mol % to about 60 mol %.
[1491] The PEG-modified lipids include PEG-modified
phosphatidylethanolamine and phosphatidic acid, PEG-ceramide
conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified
dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such
lipids are also referred to as PEGylated lipids. For example, a PEG
lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG DMPE, PEG-DPPC, or
a PEG-DSPE lipid. In some embodiments, the PEG-lipid are
1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene
glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG),
PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide
(PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or
PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some
embodiments, the PEG moiety has a size of about 1000, 2000, 5000,
10,000, 15,000 or 20,000 daltons. In some embodiments, the amount
of PEG-lipid in the lipid composition ranges from about 0.1 mol %
to about 5 mol %.
[1492] In some embodiments, the LNP formulations described herein
can additionally comprise a permeability enhancer molecule.
Non-limiting permeability enhancer molecules are described in U.S.
Pub. No. US20050222064, herein incorporated by reference in its
entirety.
[1493] The LNP formulations can further contain a phosphate
conjugate. The phosphate conjugate can increase in vivo circulation
times and/or increase the targeted delivery of the nanoparticle.
Phosphate conjugates can be made by the methods described in, e.g.,
Intl. Pub. No. WO2013033438 or U.S. Pub. No. US20130196948. The LNP
formulation can also contain a polymer conjugate (e.g., a water
soluble conjugate) as described in, e.g., U.S. Pub. Nos.
US20130059360, US20130196948, and US20130072709. Each of the
references is herein incorporated by reference in its entirety.
[1494] The LNP formulations can comprise a conjugate to enhance the
delivery of nanoparticles of the present invention in a subject.
Further, the conjugate can inhibit phagocytic clearance of the
nanoparticles in a subject. In some embodiments, the conjugate can
be a "self" peptide designed from the human membrane protein CD47
(e.g., the "self" particles described by Rodriguez et al, Science
2013 339, 971-975, herein incorporated by reference in its
entirety). As shown by Rodriguez et al. the self peptides delayed
macrophage-mediated clearance of nanoparticles which enhanced
delivery of the nanoparticles.
[1495] The LNP formulations can comprise a carbohydrate carrier. As
a non-limiting example, the carbohydrate carrier can include, but
is not limited to, an anhydride-modified phytoglycogen or
glycogen-type material, phytoglycogen octenyl succinate,
phytoglycogen beta-dextrin, anhydride-modified phytoglycogen
beta-dextrin (e.g., Intl. Pub. No. WO2012109121, herein
incorporated by reference in its entirety).
[1496] The LNP formulations can be coated with a surfactant or
polymer to improve the delivery of the particle. In some
embodiments, the LNP can be coated with a hydrophilic coating such
as, but not limited to, PEG coatings and/or coatings that have a
neutral surface charge as described in U.S. Pub. No. US20130183244,
herein incorporated by reference in its entirety.
[1497] The LNP formulations can be engineered to alter the surface
properties of particles so that the lipid nanoparticles can
penetrate the mucosal barrier as described in U.S. Pat. No.
8,241,670 or Intl. Pub. No. WO2013110028, each of which is herein
incorporated by reference in its entirety.
[1498] The LNP engineered to penetrate mucus can comprise a
polymeric material (i.e., a polymeric core) and/or a
polymer-vitamin conjugate and/or a tri-block co-polymer. The
polymeric material can include, but is not limited to, polyamines,
polyethers, polyamides, polyesters, polycarbamates, polyureas,
polycarbonates, poly(styrenes), polyimides, polysulfones,
polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines,
polyisocyanates, polyacrylates, polymethacrylates,
polyacrylonitriles, and polyarylates.
[1499] LNP engineered to penetrate mucus can also include surface
altering agents such as, but not limited to, polynucleotides,
anionic proteins (e.g., bovine serum albumin), surfactants (e.g.,
cationic surfactants such as for example
dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives
(e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin,
polyethylene glycol and poloxamer), mucolytic agents (e.g.,
N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,
acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna,
ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,
gelsolin, thymosin .beta.4 dornase alfa, neltenexine, erdosteine)
and various DNases including rhDNase.
[1500] In some embodiments, the mucus penetrating LNP can be a
hypotonic formulation comprising a mucosal penetration enhancing
coating. The formulation can be hypotonic for the epithelium to
which it is being delivered. Non-limiting examples of hypotonic
formulations can be found in, e.g., Intl. Pub. No. WO2013110028,
herein incorporated by reference in its entirety.
[1501] In some embodiments, the polynucleotide described herein is
formulated as a lipoplex, such as, without limitation, the
ATUPLEX.TM. system, the DACC system, the DBTC system and other
siRNA-lipoplex technology from Silence Therapeutics (London, United
Kingdom), STEMFECT.TM. from STEMGENT.RTM. (Cambridge, Mass.), and
polyethylenimine (PEI) or protamine-based targeted and non-targeted
delivery of nucleic acids (Aleku et al. Cancer Res. 2008
68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012
50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et
al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol.
Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010
80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide et al.
J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther.
4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15;
Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc
Natl Acad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene
Ther. 2008 19:125-132; all of which are incorporated herein by
reference in its entirety).
[1502] In some embodiments, the polynucleotides described herein
are formulated as a solid lipid nanoparticle (SLN), which can be
spherical with an average diameter between 10 to 1000 nm. SLN
possess a solid lipid core matrix that can solubilize lipophilic
molecules and can be stabilized with surfactants and/or
emulsifiers. Exemplary SLN can be those as described in Intl. Pub.
No. WO2013105101, herein incorporated by reference in its
entirety.
[1503] In some embodiments, the polynucleotides described herein
can be formulated for controlled release and/or targeted delivery.
As used herein, "controlled release" refers to a pharmaceutical
composition or compound release profile that conforms to a
particular pattern of release to effect a therapeutic outcome. In
one embodiment, the polynucleotides can be encapsulated into a
delivery agent described herein and/or known in the art for
controlled release and/or targeted delivery. As used herein, the
term "encapsulate" means to enclose, surround or encase. As it
relates to the formulation of the compounds of the invention,
encapsulation can be substantial, complete or partial. The term
"substantially encapsulated" means that at least greater than 50,
60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than
99.999% of the pharmaceutical composition or compound of the
invention can be enclosed, surrounded or encased within the
delivery agent. "Partially encapsulation" means that less than 10,
10, 20, 30, 40 50 or less of the pharmaceutical composition or
compound of the invention can be enclosed, surrounded or encased
within the delivery agent.
[1504] Advantageously, encapsulation can be determined by measuring
the escape or the activity of the pharmaceutical composition or
compound of the invention using fluorescence and/or electron
micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70,
80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99%
of the pharmaceutical composition or compound of the invention are
encapsulated in the delivery agent.
[1505] In some embodiments, the polynucleotide controlled release
formulation can include at least one controlled release coating
(e.g., OPADRY.RTM., EUDRAGIT RL.RTM., EUDRAGIT RS.RTM. and
cellulose derivatives such as ethylcellulose aqueous dispersions
(AQUACOAT.RTM. and SURELEASE.RTM.)). In some embodiments, the
polynucleotide controlled release formulation can comprise a
polymer system as described in U.S. Pub. No. US20130130348, or a
PEG and/or PEG related polymer derivative as described in U.S. Pat.
No. 8,404,222, each of which is incorporated by reference in its
entirety.
[1506] In some embodiments, the polynucleotides described herein
can be encapsulated in a therapeutic nanoparticle, referred to
herein as "therapeutic nanoparticle polynucleotides." Therapeutic
nanoparticles can be formulated by methods described in, e.g.,
Intl. Pub. Nos. WO2010005740, WO2010030763, WO2010005721,
WO2010005723, and WO2012054923; and U.S. Pub. Nos. US20110262491,
US20100104645, US20100087337, US20100068285, US20110274759,
US20100068286, US20120288541, US20120140790, US20130123351 and
US20130230567; and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208
and 8,318,211, each of which is herein incorporated by reference in
its entirety.
[1507] In some embodiments, the therapeutic nanoparticle
polynucleotide can be formulated for sustained release. As used
herein, "sustained release" refers to a pharmaceutical composition
or compound that conforms to a release rate over a specific period
of time. The period of time can include, but is not limited to,
hours, days, weeks, months and years. As a non-limiting example,
the sustained release nanoparticle of the polynucleotides described
herein can be formulated as disclosed in Intl. Pub. No.
WO2010075072 and U.S. Pub. Nos. US20100216804, US20110217377,
US20120201859 and US20130150295, each of which is herein
incorporated by reference in their entirety.
[1508] In some embodiments, the therapeutic nanoparticle
polynucleotide can be formulated to be target specific, such as
those described in Intl. Pub. Nos. WO2008121949, WO2010005726,
WO2010005725, WO2011084521 and WO2011084518; and U.S. Pub. Nos.
US20100069426, US20120004293 and US20100104655, each of which is
herein incorporated by reference in its entirety.
[1509] The LNPs can be prepared using microfluidic mixers or
micromixers. Exemplary microfluidic mixers can include, but are not
limited to, a slit interdigital micromixer including, but not
limited to those manufactured by Microinnova (Allerheiligen bei
Wildon, Austria) and/or a staggered herringbone micromixer (SHM)
(see Zhigaltsev et al., "Bottom-up design and synthesis of limit
size lipid nanoparticle systems with aqueous and triglyceride cores
using millisecond microfluidic mixing," Langmuir 28:3633-40 (2012);
Belliveau et al., "Microfluidic synthesis of highly potent
limit-size lipid nanoparticles for in vivo delivery of siRNA,"
Molecular Therapy-Nucleic Acids. 1:e37 (2012); Chen et al., "Rapid
discovery of potent siRNA-containing lipid nanoparticles enabled by
controlled microfluidic formulation," J. Am. Chem. Soc.
134(16):6948-51 (2012); each of which is herein incorporated by
reference in its entirety). Exemplary micromixers include Slit
Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit
Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or
Impinging-jet (IJMM,) from the Institut fur Mikrotechnik Mainz
GmbH, Mainz Germany. In some embodiments, methods of making LNP
using SHM further comprise mixing at least two input streams
wherein mixing occurs by microstructure-induced chaotic advection
(MICA). According to this method, fluid streams flow through
channels present in a herringbone pattern causing rotational flow
and folding the fluids around each other. This method can also
comprise a surface for fluid mixing wherein the surface changes
orientations during fluid cycling. Methods of generating LNPs using
SHM include those disclosed in U.S. Pub. Nos. US20040262223 and
US20120276209, each of which is incorporated herein by reference in
their entirety.
[1510] In some embodiments, the polynucleotides described herein
can be formulated in lipid nanoparticles using microfluidic
technology (see Whitesides, George M., "The Origins and the Future
of Microfluidics," Nature 442: 368-373 (2006); and Abraham et al.,
"Chaotic Mixer for Microchannels," Science 295: 647-651 (2002);
each of which is herein incorporated by reference in its entirety).
In some embodiments, the polynucleotides can be formulated in lipid
nanoparticles using a micromixer chip such as, but not limited to,
those from Harvard Apparatus (Holliston, Mass.) or Dolomite
Microfluidics (Royston, UK). A micromixer chip can be used for
rapid mixing of two or more fluid streams with a split and
recombine mechanism.
[1511] In some embodiments, the polynucleotides described herein
can be formulated in lipid nanoparticles having a diameter from
about 1 nm to about 100 nm such as, but not limited to, about 1 nm
to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to
about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to
about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to
about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to
about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to
about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to
about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to
about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to
about 80 nm, from about 5 nm to about 90 nm, about 10 to about 20
nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to
about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm,
about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about
30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20
to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm,
about 20 to about 90 nm, about 20 to about 100 nm, about 30 to
about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm,
about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about
90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40
to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm,
about 40 to about 90 nm, about 40 to about 100 nm, about 50 to
about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about
50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70
nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to
about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm,
about 70 to about 100 nm, about 80 to about 90 nm, about 80 to
about 100 nm and/or about 90 to about 100 nm.
[1512] In some embodiments, the lipid nanoparticles can have a
diameter from about 10 to 500 nm. In one embodiment, the lipid
nanoparticle can have a diameter greater than 100 nm, greater than
150 nm, greater than 200 nm, greater than 250 nm, greater than 300
nm, greater than 350 nm, greater than 400 nm, greater than 450 nm,
greater than 500 nm, greater than 550 nm, greater than 600 nm,
greater than 650 nm, greater than 700 nm, greater than 750 nm,
greater than 800 nm, greater than 850 nm, greater than 900 nm,
greater than 950 nm or greater than 1000 nm.
[1513] In some embodiments, the polynucleotides can be delivered
using smaller LNPs. Such particles can comprise a diameter from
below 0.1 .mu.m up to 100 nm such as, but not limited to, less than
0.1 .mu.m, less than 1.0 .mu.m, less than 5 .mu.m, less than 10
.mu.m, less than 15 um, less than 20 um, less than 25 um, less than
30 um, less than 35 um, less than 40 um, less than 50 um, less than
55 um, less than 60 um, less than 65 um, less than 70 um, less than
75 um, less than 80 um, less than 85 um, less than 90 um, less than
95 um, less than 100 um, less than 125 um, less than 150 um, less
than 175 um, less than 200 um, less than 225 um, less than 250 um,
less than 275 um, less than 300 um, less than 325 um, less than 350
um, less than 375 um, less than 400 um, less than 425 um, less than
450 um, less than 475 um, less than 500 um, less than 525 um, less
than 550 um, less than 575 um, less than 600 um, less than 625 um,
less than 650 um, less than 675 um, less than 700 um, less than 725
um, less than 750 um, less than 775 um, less than 800 um, less than
825 um, less than 850 um, less than 875 um, less than 900 um, less
than 925 um, less than 950 um, or less than 975 um.
[1514] The nanoparticles and microparticles described herein can be
geometrically engineered to modulate macrophage and/or the immune
response. The geometrically engineered particles can have varied
shapes, sizes and/or surface charges to incorporate the
polynucleotides described herein for targeted delivery such as, but
not limited to, pulmonary delivery (see, e.g., Intl. Pub. No.
WO2013082111, herein incorporated by reference in its entirety).
Other physical features the geometrically engineering particles can
include, but are not limited to, fenestrations, angled arms,
asymmetry and surface roughness, charge that can alter the
interactions with cells and tissues.
[1515] In some embodiment, the nanoparticles described herein are
stealth nanoparticles or target-specific stealth nanoparticles such
as, but not limited to, those described in U.S. Pub. No.
US20130172406, herein incorporated by reference in its entirety.
The stealth or target-specific stealth nanoparticles can comprise a
polymeric matrix, which can comprise two or more polymers such as,
but not limited to, polyethylenes, polycarbonates, polyanhydrides,
polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides, polyacetals, polyethers, polyesters, poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines,
polyesters, polyanhydrides, polyethers, polyurethanes,
polymethacrylates, polyacrylates, polycyanoacrylates, or
combinations thereof.
b. Lipidoids
[1516] In some embodiments, the compositions or formulations of the
present disclosure comprise a delivery agent, e.g., a lipidoid. The
polynucleotides described herein (e.g., a polynucleotide comprising
a nucleotide sequence encoding a PBGD polypeptide) can be
formulated with lipidoids. Complexes, micelles, liposomes or
particles can be prepared containing these lipidoids and therefore
to achieve an effective delivery of the polynucleotide, as judged
by the production of an encoded protein, following the injection of
a lipidoid formulation via localized and/or systemic routes of
administration. Lipidoid complexes of polynucleotides can be
administered by various means including, but not limited to,
intravenous, intramuscular, or subcutaneous routes.
[1517] The synthesis of lipidoids is described in literature (see
Mahon et al., Bioconjug. Chem. 2010 21:1448-1454; Schroeder et al.,
J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008
26:561-569; Love et al., Proc Natl Acad Sci USA. 2010
107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011
108:12996-3001; all of which are incorporated herein in their
entireties).
[1518] Formulations with the different lipidoids, including, but
not limited to
penta[3-(1-laurylaminopropionyl)]-triethylenetetramine
hydrochloride (TETA-5LAP; also known as 98N12-5, see Murugaiah et
al., Analytical Biochemistry, 401:61 (2010)), C12-200 (including
derivatives and variants), and MD1, can be tested for in vivo
activity. The lipidoid "98N12-5" is disclosed by Akinc et al., Mol
Ther. 2009 17:872-879. The lipidoid "C12-200" is disclosed by Love
et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and
Huang, Molecular Therapy. 2010 669-670. Each of the references is
herein incorporated by reference in its entirety.
[1519] In one embodiment, the polynucleotides described herein can
be formulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids
can be prepared by the methods described in U.S. Pat. No. 8,450,298
(herein incorporated by reference in its entirety).
[1520] The lipidoid formulations can include particles comprising
either 3 or 4 or more components in addition to polynucleotides.
Lipidoids and polynucleotide formulations comprising lipidoids are
described in Intl. Pub. No. WO 2015051214 (herein incorporated by
reference in its entirety.
c. Hyaluronidase
[1521] In some embodiments, the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) and hyaluronidase for injection (e.g.,
intramuscular or subcutaneous injection). Hyaluronidase catalyzes
the hydrolysis of hyaluronan, which is a constituent of the
interstitial barrier. Hyaluronidase lowers the viscosity of
hyaluronan, thereby increases tissue permeability (Frost, Expert
Opin. Drug Deliv. (2007) 4:427-440). Alternatively, the
hyaluronidase can be used to increase the number of cells exposed
to the polynucleotides administered intramuscularly or
subcutaneously.
d. Nanoparticle Mimics
[1522] In some embodiments, the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) is encapsulated within and/or absorbed to a
nanoparticle mimic. A nanoparticle mimic can mimic the delivery
function organisms or particles such as, but not limited to,
pathogens, viruses, bacteria, fungus, parasites, prions and cells.
As a non-limiting example, the polynucleotides described herein can
be encapsulated in a non-viron particle that can mimic the delivery
function of a virus (see e.g., Intl. Pub. No. WO2012006376 and U.S.
Pub. Nos. US20130171241 and US20130195968, each of which is herein
incorporated by reference in its entirety).
e. Nanotubes
[1523] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) attached or otherwise bound to (e.g., through
steric, ionic, covalent and/or other forces) at least one nanotube,
such as, but not limited to, rosette nanotubes, rosette nanotubes
having twin bases with a linker, carbon nanotubes and/or
single-walled carbon nanotubes. Nanotubes and nanotube formulations
comprising a polynucleotide are described in, e.g., Intl. Pub. No.
WO2014152211, herein incorporated by reference in its entirety.
f. Self-Assembled Nanoparticles, or Self-Assembled
Macromolecules
[1524] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in self-assembled nanoparticles, or amphiphilic
macromolecules (AMs) for delivery. AMs comprise biocompatible
amphiphilic polymers that have an alkylated sugar backbone
covalently linked to poly(ethylene glycol). In aqueous solution,
the AMs self-assemble to form micelles. Nucleic acid self-assembled
nanoparticles are described in Intl. Appl. No. PCT/US2014/027077,
and AMs and methods of forming AMs are described in U.S. Pub. No.
US20130217753, each of which is herein incorporated by reference in
its entirety.
g. Inorganic Nanoparticles, Semi-Conductive and Metallic
Nanoparticles
[1525] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in inorganic nanoparticles, or water-dispersible
nanoparticles comprising a semiconductive or metallic material. The
inorganic nanoparticles can include, but are not limited to, clay
substances that are water swellable. The water-dispersible
nanoparticles can be hydrophobic or hydrophilic nanoparticles. As a
non-limiting example, the inorganic, semi-conductive and metallic
nanoparticles are described in, e.g., U.S. Pat. Nos. 5,585,108 and
8,257,745; and U.S. Pub. Nos. US20120228565, US 20120265001 and US
20120283503, each of which is herein incorporated by reference in
their entirety.
h. Surgical Sealants: Gels and Hydrogels
[1526] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in a surgical sealant. Surgical sealants such as
gels and hydrogels are described in Intl. Appl. No.
PCT/US2014/027077, herein incorporated by reference in its
entirety.
i. Suspension Formulations
[1527] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in suspensions. In some embodiments, suspensions
comprise a polynucleotide, water immiscible oil depots, surfactants
and/or co-surfactants and/or co-solvents. Suspensions can be formed
by first preparing an aqueous solution of a polynucleotide and an
oil-based phase comprising one or more surfactants, and then mixing
the two phases (aqueous and oil-based).
[1528] Exemplary oils for suspension formulations can include, but
are not limited to, sesame oil and Miglyol (comprising esters of
saturated coconut and palmkernel oil-derived caprylic and capric
fatty acids and glycerin or propylene glycol), corn oil, soybean
oil, peanut oil, beeswax and/or palm seed oil. Exemplary
surfactants can include, but are not limited to Cremophor,
polysorbate 20, polysorbate 80, polyethylene glycol, transcutol,
Capmul.RTM., labrasol, isopropyl myristate, and/or Span 80. In some
embodiments, suspensions can comprise co-solvents including, but
not limited to ethanol, glycerol and/or propylene glycol.
[1529] In some embodiments, suspensions can provide modulation of
the release of the polynucleotides into the surrounding environment
by diffusion from a water immiscible depot followed by
resolubilization into a surrounding environment (e.g., an aqueous
environment).
[1530] In some embodiments, the polynucleotides can be formulated
such that upon injection, an emulsion forms spontaneously (e.g.,
when delivered to an aqueous phase), which can provide a high
surface area to volume ratio for release of polynucleotides from an
oil phase to an aqueous phase. In some embodiments, the
polynucleotide is formulated in a nanoemulsion, which can comprise
a liquid hydrophobic core surrounded by or coated with a lipid or
surfactant layer. Exemplary nanoemulsions and their preparations
are described in, e.g., U.S. Pat. No. 8,496,945, herein
incorporated by reference in its entirety.
j. Cations and Anions
[1531] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) and a cation or anion, such as Zn2+, Ca2+, Cu2+,
Mg2+ and combinations thereof. Exemplary formulations can include
polymers and a polynucleotide complexed with a metal cation as
described in, e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of
which is herein incorporated by reference in its entirety. In some
embodiments, cationic nanoparticles can contain a combination of
divalent and monovalent cations. The delivery of polynucleotides in
cationic nanoparticles or in one or more depot comprising cationic
nanoparticles can improve polynucleotide bioavailability by acting
as a long-acting depot and/or reducing the rate of degradation by
nucleases.
k. Molded Nanoparticles and Microparticles
[1532] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in molded nanoparticles in various sizes, shapes
and chemistry. For example, the nanoparticles and/or microparticles
can be made using the PRINT.RTM. technology by LIQUIDA
TECHNOLOGIES.RTM. (Morrisville, N.C.) (e.g., International Pub. No.
WO2007024323, herein incorporated by reference in its
entirety).
[1533] In some embodiments, the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) is formulated in microparticles. The
microparticles can contain a core of the polynucleotide and a
cortex of a biocompatible and/or biodegradable polymer, including
but not limited to, poly(u-hydroxy acid), a polyhydroxy butyric
acid, a polycaprolactone, a polyorthoester and a polyanhydride. The
microparticle can have adsorbent surfaces to adsorb
polynucleotides. The microparticles can have a diameter of from at
least 1 micron to at least 100 microns (e.g., at least 1 micron, at
least 10 micron, at least 20 micron, at least 30 micron, at least
50 micron, at least 75 micron, at least 95 micron, and at least 100
micron). In some embodiment, the compositions or formulations of
the present disclosure are microemulsions comprising microparticles
and polynucleotides. Exemplary microparticles, microemulsions and
their preparations are described in, e.g., U.S. Pat. Nos.
8,460,709, 8,309,139 and 8,206,749; U.S. Pub. Nos. US20130129830,
US2013195923 and US20130195898; and Intl. Pub. No. WO2013075068,
each of which is herein incorporated by reference in its
entirety.
L. NanoJackets and NanoLiposomes
[1534] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in NanoJackets and NanoLiposomes by Keystone Nano
(State College, Pa.). NanoJackets are made of materials that are
naturally found in the body including calcium, phosphate and can
also include a small amount of silicates. Nanojackets can have a
size ranging from 5 to 50 nm.
[1535] NanoLiposomes are made of lipids such as, but not limited
to, lipids that naturally occur in the body. NanoLiposomes can have
a size ranging from 60-80 nm. In some embodiments, the
polynucleotides disclosed herein are formulated in a NanoLiposome
such as, but not limited to, Ceramide NanoLiposomes.
m. Cells or Minicells
[1536] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) that is transfected ex vivo into cells, which are
subsequently transplanted into a subject. Cell-based formulations
of the polynucleotide disclosed herein can be used to ensure cell
transfection (e.g., in the cellular carrier), alter the
biodistribution of the polynucleotide (e.g., by targeting the cell
carrier to specific tissues or cell types), and/or increase the
translation of encoded protein.
[1537] Exemplary cells include, but are not limited to, red blood
cells, virosomes, and electroporated cells (see e.g., Godfrin et
al., Expert Opin Biol Ther. 2012 12:127-133; Fang et al., Expert
Opin Biol Ther. 2012 12:385-389; Hu et al., Proc Natl Acad Sci USA
2011 108:10980-10985; Lund et al., Pharm Res. 2010 27:400-420;
Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, Hum Vaccin.
2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all of
which are herein incorporated by reference in its entirety).
[1538] A variety of methods are known in the art and are suitable
for introduction of nucleic acid into a cell, including viral and
non-viral mediated techniques. Examples of typical non-viral
mediated techniques include, but are not limited to,
electroporation, calcium phosphate mediated transfer,
nucleofection, sonoporation, heat shock, magnetofection, liposome
mediated transfer, microinjection, microprojectile mediated
transfer (nanoparticles), cationic polymer mediated transfer
(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the
like) or cell fusion.
[1539] In some embodiments, the polynucleotides described herein
can be delivered in synthetic virus-like particles (VLPs)
synthesized by the methods as described in Intl. Pub Nos.
WO2011085231 and WO2013116656; and U.S. Pub. No. 20110171248, each
of which is herein incorporated by reference in its entirety.
[1540] The technique of sonoporation, or cellular sonication, is
the use of sound (e.g., ultrasonic frequencies) for modifying the
permeability of the cell plasma membrane. Sonoporation methods are
known to deliver nucleic acids in vivo (Yoon and Park, Expert Opin
Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr Pharm
Biotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 2007
14:465-475; U.S. Pub. Nos. US20100196983 and US20100009424; all
herein incorporated by reference in their entirety).
[1541] In some embodiments, the polynucleotides described herein
can be delivered by electroporation. Electroporation techniques are
known to deliver nucleic acids in vivo and clinically (Andre et
al., Curr Gene Ther. 2010 10:267-280; Chiarella et al., Curr Gene
Ther. 2010 10:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all
herein incorporated by reference in their entirety).
Electroporation devices are sold by many companies worldwide
including, but not limited to BTX.RTM. Instruments (Holliston,
Mass.) (e.g., the AgilePulse In Vivo System) and Inovio (Blue Bell,
Pa.) (e.g., Inovio SP-5P intramuscular delivery device or the
CELLECTRA.RTM. 3000 intradermal delivery device).
[1542] In some embodiments, the cells are selected from the group
consisting of mammalian cells, bacterial cells, plant, microbial,
algal and fungal cells. In some embodiments, the cells are
mammalian cells, such as, but not limited to, human, mouse, rat,
goat, horse, rabbit, hamster or cow cells. In a further embodiment,
the cells can be from an established cell line, including, but not
limited to, HeLa, NSO, SP2/0, KEK 293T, Vero, Caco, Caco-2, MDCK,
COS-1, COS-7, K562, Jurkat, CHO-Kl, DG44, CHOK1SV, CHO--S, Huvec,
CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2, IMR-90, MCF-7,
U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO) cells.
[1543] In certain embodiments, the cells are fungal cells, such as,
but not limited to, Chrysosporium cells, Aspergillus cells,
Trichoderma cells, Dictyostelium cells, Candida cells,
Saccharomyces cells, Schizosaccharomyces cells, and Penicillium
cells.
[1544] In certain embodiments, the cells are bacterial cells such
as, but not limited to, E. coli, B. subtilis, or BL21 cells.
Primary and secondary cells to be transfected by the methods of the
invention can be obtained from a variety of tissues and include,
but are not limited to, all cell types that can be maintained in
culture. The primary and secondary cells include, but are not
limited to, fibroblasts, keratinocytes, epithelial cells (e.g.,
mammary epithelial cells, intestinal epithelial cells), endothelial
cells, glial cells, neural cells, formed elements of the blood
(e.g., lymphocytes, bone marrow cells), muscle cells and precursors
of these somatic cell types. Primary cells can also be obtained
from a donor of the same species or from another species (e.g.,
mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat,
horse).
[1545] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein in
bacterial minicells. As a non-limiting example, bacterial minicells
can be those described in Intl. Pub. No. WO2013088250 or U.S. Pub.
No. US20130177499, each of which is herein incorporated by
reference in its entirety.
n. Semi-solid Compositions
[1546] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in a hydrophobic matrix to form a semi-solid or
paste-like composition. As a non-limiting example, the semi-solid
or paste-like composition can be made by the methods described in
Intl. Pub. No. WO201307604, herein incorporated by reference in its
entirety.
o. Exosomes
[1547] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in exosomes, which can be loaded with at least
one polynucleotide and delivered to cells, tissues and/or
organisms. As a non-limiting example, the polynucleotides can be
loaded in the exosomes as described in Intl. Pub. No. WO2013084000,
herein incorporated by reference in its entirety.
p. Silk-Based Delivery
[1548] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) that is formulated for silk-based delivery. The
silk-based delivery system can be formed by contacting a silk
fibroin solution with a polynucleotide described herein. As a
non-limiting example, a sustained release silk-based delivery
system and methods of making such system are described in U.S. Pub.
No. US20130177611, herein incorporated by reference in its
entirety.
q. Amino Acid Lipids
[1549] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) that is formulation with an amino acid lipid.
Amino acid lipids are lipophilic compounds comprising an amino acid
residue and one or more lipophilic tails. Non-limiting examples of
amino acid lipids and methods of making amino acid lipids are
described in U.S. Pat. No. 8,501,824. The amino acid lipid
formulations can deliver a polynucleotide in releasable form that
comprises an amino acid lipid that binds and releases the
polynucleotides. As a non-limiting example, the release of the
polynucleotides described herein can be provided by an acid-labile
linker as described in, e.g., U.S. Pat. Nos. 7,098,032, 6,897,196,
6,426,086, 7,138,382, 5,563,250, and 5,505,931, each of which is
herein incorporated by reference in its entirety.
r. Microvesicles
[1550] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in a microvesicle formulation. Exemplary
microvesicles include those described in U.S. Pub. No.
US20130209544 (herein incorporated by reference in its entirety).
In some embodiments, the microvesicle is an ARRDC1-mediated
microvesicles (ARMMs) as described in Intl. Pub. No. WO2013119602
(herein incorporated by reference in its entirety).
s. Interpolyelectrolyte Complexes
[1551] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in an interpolyelectrolyte complex.
Interpolyelectrolyte complexes are formed when charge-dynamic
polymers are complexed with one or more anionic molecules.
Non-limiting examples of charge-dynamic polymers and
interpolyelectrolyte complexes and methods of making
interpolyelectrolyte complexes are described in U.S. Pat. No.
8,524,368, herein incorporated by reference in its entirety.
t. Crystalline Polymeric Systems
[1552] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in crystalline polymeric systems. Crystalline
polymeric systems are polymers with crystalline moieties and/or
terminal units comprising crystalline moieties. Exemplary polymers
are described in U.S. Pat. No. 8,524,259 (herein incorporated by
reference in its entirety).
u. Polymers, Biodegradable Nanoparticles, and Core-Shell
Nanoparticles
[1553] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) and a natural and/or synthetic polymer. The
polymers include, but not limited to, polyethenes, polyethylene
glycol (PEG), poly(1-lysine)(PLL), PEG grafted to PLL, cationic
lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine
(PEI), cross-linked branched poly(alkylene imines), a polyamine
derivative, a modified poloxamer, elastic biodegradable polymer,
biodegradable copolymer, biodegradable polyester copolymer,
biodegradable polyester copolymer, multiblock copolymers,
poly[.alpha.-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable
cross-linked cationic multi-block copolymers, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates,
polycaprolactones, polyamides, polyacetals, polyethers, polyesters,
poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,
polyurethanes, polyphosphazenes, polyureas, polystyrenes,
polyamines, polylysine, poly(ethylene imine), poly(serine ester),
poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),
amine-containing polymers, dextran polymers, dextran polymer
derivatives or combinations thereof.
[1554] Exemplary polymers include, DYNAMIC POLYCONJUGATE.RTM.
(Arrowhead Research Corp., Pasadena, Calif.) formulations from
MIRUS.RTM. Bio (Madison, Wis.) and Roche Madison (Madison, Wis.),
PHASERX.TM. polymer formulations such as, without limitation,
SMARTT POLYMER TECHNOLOGY.TM. (PHASERX.RTM., Seattle, Wash.),
DMRI/DOPE, poloxamer, VAXFECTIN.RTM. adjuvant from Vical (San
Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals
(Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid)
(PLGA) polymers. RONDEL.TM. (RNAi/Oligonucleotide Nanoparticle
Delivery) polymers (Arrowhead Research Corporation, Pasadena,
Calif.) and pH responsive co-block polymers such as PHASERX.RTM.
(Seattle, Wash.).
[1555] The polymer formulations allow a sustained or delayed
release of the polynucleotide (e.g., following intramuscular or
subcutaneous injection). The altered release profile for the
polynucleotide can result in, for example, translation of an
encoded protein over an extended period of time. The polymer
formulation can also be used to increase the stability of the
polynucleotide. Sustained release formulations can include, but are
not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc),
poloxamer, GELSITE.RTM. (Nanotherapeutics, Inc. Alachua, Fla.),
HYLENEX.RTM. (Halozyme Therapeutics, San Diego Calif.), surgical
sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),
TISSELL.RTM. (Baxter International, Inc. Deerfield, Ill.),
PEG-based sealants, and COSEAL.RTM. (Baxter International, Inc.
Deerfield, Ill.).
[1556] As a non-limiting example modified mRNA can be formulated in
PLGA microspheres by preparing the PLGA microspheres with tunable
release rates (e.g., days and weeks) and encapsulating the modified
mRNA in the PLGA microspheres while maintaining the integrity of
the modified mRNA during the encapsulation process. EVAc are
non-biodegradable, biocompatible polymers that are used extensively
in pre-clinical sustained release implant applications (e.g.,
extended release products Ocusert a pilocarpine ophthalmic insert
for glaucoma or progestasert a sustained release progesterone
intrauterine device; transdermal delivery systems Testoderm,
Duragesic and Selegiline; catheters). Poloxamer F-407 NF is a
hydrophilic, non-ionic surfactant triblock copolymer of
polyoxyethylene-polyoxypropylene-polyoxyethylene having a low
viscosity at temperatures less than 5.degree. C. and forms a solid
gel at temperatures greater than 15.degree. C.
[1557] As a non-limiting example, the polynucleotides described
herein can be formulated with the polymeric compound of PEG grafted
with PLL as described in U.S. Pat. No. 6,177,274. As another
non-limiting example, the polynucleotides described herein can be
formulated with a block copolymer such as a PLGA-PEG block
copolymer (see e.g., U.S. Pub. No. US20120004293 and U.S. Pat. Nos.
8,236,330 and 8,246,968), or a PLGA-PEG-PLGA block copolymer (see
e.g., U.S. Pat. No. 6,004,573). Each of the references is herein
incorporated by reference in its entirety.
[1558] In some embodiments, the polynucleotides described herein
can be formulated with at least one amine-containing polymer such
as, but not limited to polylysine, polyethylene imine,
poly(amidoamine) dendrimers, poly(amine-co-esters) or combinations
thereof. Exemplary polyamine polymers and their use as delivery
agents are described in, e.g., U.S. Pat. Nos. 8,460,696, 8,236,280,
each of which is herein incorporated by reference in its
entirety.
[1559] In some embodiments, the polynucleotides described herein
can be formulated in a biodegradable cationic lipopolymer, a
biodegradable polymer, or a biodegradable copolymer, a
biodegradable polyester copolymer, a biodegradable polyester
polymer, a linear biodegradable copolymer, PAGA, a biodegradable
cross-linked cationic multi-block copolymer or combinations thereof
as described in, e.g., U.S. Pat. Nos. 6,696,038, 6,517,869,
6,267,987, 6,217,912, 6,652,886, 8,057,821, and 8,444,992; U.S.
Pub. Nos. US20030073619, US20040142474, US20100004315, US2012009145
and US20130195920; and Intl Pub. Nos. WO2006063249 and
WO2013086322, each of which is herein incorporated by reference in
its entirety.
[1560] In some embodiments, the polynucleotides described herein
can be formulated in or with at least one cyclodextrin polymer as
described in U.S. Pub. No. US20130184453. In some embodiments, the
polynucleotides described herein can be formulated in or with at
least one crosslinked cation-binding polymers as described in Intl.
Pub. Nos. WO2013106072, WO2013106073 and WO2013106086. In some
embodiments, the polynucleotides described herein can be formulated
in or with at least PEGylated albumin polymer as described in U.S.
Pub. No. US20130231287. Each of the references is herein
incorporated by reference in its entirety.
[1561] In some embodiments, the polynucleotides disclosed herein
can be formulated as a nanoparticle using a combination of
polymers, lipids, and/or other biodegradable agents, such as, but
not limited to, calcium phosphate. Components can be combined in a
core-shell, hybrid, and/or layer-by-layer architecture, to allow
for fine-tuning of the nanoparticle for delivery (Wang et al., Nat
Mater. 2006 5:791-796; Fuller et al., Biomaterials. 2008
29:1526-1532; DeKoker et al., Adv Drug Deliv Rev. 2011 63:748-761;
Endres et al., Biomaterials. 2011 32:7721-7731; Su et al., Mol
Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated by reference
in their entireties). As a non-limiting example, the nanoparticle
can comprise a plurality of polymers such as, but not limited to
hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic
polymers (e.g., PEG) and/or hydrophilic polymers (Intl. Pub. No.
WO20120225129, herein incorporated by reference in its
entirety).
[1562] The use of core-shell nanoparticles has additionally focused
on a high-throughput approach to synthesize cationic cross-linked
nanogel cores and various shells (Siegwart et al., Proc Natl Acad
Sci USA. 2011 108:12996-13001; herein incorporated by reference in
its entirety). The complexation, delivery, and internalization of
the polymeric nanoparticles can be precisely controlled by altering
the chemical composition in both the core and shell components of
the nanoparticle. For example, the core-shell nanoparticles can
efficiently deliver siRNA to mouse hepatocytes after they
covalently attach cholesterol to the nanoparticle.
[1563] In some embodiments, a hollow lipid core comprising a middle
PLGA layer and an outer neutral lipid layer containing PEG can be
used to delivery of the polynucleotides as described herein. In
some embodiments, the lipid nanoparticles can comprise a core of
the polynucleotides disclosed herein and a polymer shell, which is
used to protect the polynucleotides in the core. The polymer shell
can be any of the polymers described herein and are known in the
art. The polymer shell can be used to protect the polynucleotides
in the core.
[1564] Core-shell nanoparticles for use with the polynucleotides
described herein are described in U.S. Pat. No. 8,313,777 or Intl.
Pub. No. WO2013124867, each of which is herein incorporated by
reference in their entirety.
v. Peptides and Proteins
[1565] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) that is formulated with peptides and/or proteins
to increase transfection of cells by the polynucleotide, and/or to
alter the biodistribution of the polynucleotide (e.g., by targeting
specific tissues or cell types), and/or increase the translation of
encoded protein (e.g., Intl. Pub. Nos. WO2012110636 and
WO2013123298. In some embodiments, the peptides can be those
described in U.S. Pub. Nos. US20130129726, US20130137644 and
US20130164219. Each of the references is herein incorporated by
reference in its entirety.
w. Conjugates
[1566] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) that is covalently linked to a carrier or
targeting group, or including two encoding regions that together
produce a fusion protein (e.g., bearing a targeting group and
therapeutic protein or peptide) as a conjugate. The conjugate can
be a peptide that selectively directs the nanoparticle to neurons
in a tissue or organism, or assists in crossing the blood-brain
barrier.
[1567] The conjugates include a naturally occurring substance, such
as a protein (e.g., human serum albumin (HSA), low-density
lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an
carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,
cyclodextrin or hyaluronic acid); or a lipid. The ligand can also
be a recombinant or synthetic molecule, such as a synthetic
polymer, e.g., a synthetic polyamino acid, an oligonucleotide
(e.g., an aptamer). Examples of polyamino acids include polyamino
acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic
acid, styrene-maleic acid anhydride copolymer,
poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic
anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer
(HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),
polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, or polyphosphazine. Example of polyamines include:
polyethylenimine, polylysine (PLL), spermine, spermidine,
polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine, arginine, amidine, protamine, cationic lipid,
cationic porphyrin, quaternary salt of a polyamine, or an alpha
helical peptide.
[1568] In some embodiments, the conjugate can function as a carrier
for the polynucleotide disclosed herein. The conjugate can comprise
a cationic polymer such as, but not limited to, polyamine,
polylysine, polyalkylenimine, and polyethylenimine that can be
grafted to with poly(ethylene glycol). Exemplary conjugates and
their preparations are described in U.S. Pat. No. 6,586,524 and
U.S. Pub. No. US20130211249, each of which herein is incorporated
by reference in its entirety.
[1569] The conjugates can also include targeting groups, e.g., a
cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid
or protein, e.g., an antibody, that binds to a specified cell type
such as a kidney cell. A targeting group can be a thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin
carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent
galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,
vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an
aptamer.
[1570] Targeting groups can be proteins, e.g., glycoproteins, or
peptides, e.g., molecules having a specific affinity for a
co-ligand, or antibodies e.g., an antibody, that binds to a
specified cell type such as a cancer cell, endothelial cell, or
bone cell. Targeting groups can also include hormones and hormone
receptors. They can also include non-peptidic species, such as
lipids, lectins, carbohydrates, vitamins, cofactors, multivalent
lactose, multivalent galactose, N-acetyl-galactosamine,
N-acetyl-glucosamine multivalent mannose, multivalent frucose, or
aptamers. The ligand can be, for example, a lipopolysaccharide, or
an activator of p38 MAP kinase.
[1571] The targeting group can be any ligand that is capable of
targeting a specific receptor. Examples include, without
limitation, folate, GalNAc, galactose, mannose, mannose-6P,
apatamers, integrin receptor ligands, chemokine receptor ligands,
transferrin, biotin, serotonin receptor ligands, PSMA, endothelin,
GCPII, somatostatin, LDL, and HDL ligands. In particular
embodiments, the targeting group is an aptamer. The aptamer can be
unmodified or have any combination of modifications disclosed
herein. As a non-limiting example, the targeting group can be a
glutathione receptor (GR)-binding conjugate for targeted delivery
across the blood-central nervous system barrier as described in,
e.g., U.S. Pub. No. US2013021661012 (herein incorporated by
reference in its entirety).
[1572] In some embodiments, the conjugate can be a synergistic
biomolecule-polymer conjugate, which comprises a long-acting
continuous-release system to provide a greater therapeutic
efficacy. The synergistic biomolecule-polymer conjugate can be
those described in U.S. Pub. No. US20130195799. In some
embodiments, the conjugate can be an aptamer conjugate as described
in Intl. Pat. Pub. No. WO2012040524. In some embodiments, the
conjugate can be an amine containing polymer conjugate as described
in U.S. Pat. No. 8,507,653. Each of the references is herein
incorporated by reference in its entirety. In some embodiments, the
polynucleotides can be conjugated to SMARTT POLYMER TECHNOLOGY.RTM.
(PHASERX.RTM., Inc. Seattle, Wash.).
[1573] In some embodiments, the polynucleotides described herein
are covalently conjugated to a cell penetrating polypeptide, which
can also include a signal sequence or a targeting sequence. The
conjugates can be designed to have increased stability, and/or
increased cell transfection; and/or altered the biodistribution
(e.g., targeted to specific tissues or cell types).
[1574] In some embodiments, the polynucleotides described herein
can be conjugated to an agent to enhance delivery. In some
embodiments, the agent can be a monomer or polymer such as a
targeting monomer or a polymer having targeting blocks as described
in Intl. Pub. No. WO2011062965. In some embodiments, the agent can
be a transport agent covalently coupled to a polynucleotide as
described in, e.g., U.S. Pat. Nos. 6,835,393 and 7,374,778. In some
embodiments, the agent can be a membrane barrier transport
enhancing agent such as those described in U.S. Pat. Nos. 7,737,108
and 8,003,129. Each of the references is herein incorporated by
reference in its entirety.
x. Micro-Organs
[1575] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in a micro-organ that can then express an encoded
polypeptide of interest in a long-lasting therapeutic formulation.
Exemplary micro-organs and formulations are described in Intl. Pub.
No. WO2014152211 (herein incorporated by reference in its
entirety).
y. Pseudovirions
[1576] In some embodiments, the compositions or formulations of the
present disclosure comprise the polynucleotides described herein
(e.g., a polynucleotide comprising a nucleotide sequence encoding a
PBGD polypeptide) in pseudovirions (e.g., pseudovirions developed
by Aura Biosciences, Cambridge, Mass.).
[1577] In some embodiments, the pseudovirion used for delivering
the polynucleotides can be derived from viruses such as, but not
limited to, herpes and papillomaviruses as described in, e.g., U.S.
Pub. Nos. US20130012450, US20130012566, US21030012426 and
US20120207840; and Intl. Pub. No. WO2013009717, each of which is
herein incorporated by reference in its entirety.
[1578] The pseudovirion can be a virus-like particle (VLP) prepared
by the methods described in U.S. Pub. Nos. US20120015899 and
US20130177587, and Intl. Pub. Nos. WO2010047839, WO2013116656,
WO2013106525 and WO2013122262. In one aspect, the VLP can be
bacteriophages MS, Q.beta., R17, fr, GA, Sp, MI, I, MXI, NL95,
AP205, f2, PP7, and the plant viruses Turnip crinkle virus (TCV),
Tomato bushy stunt virus (TBSV), Southern bean mosaic virus (SBMV)
and members of the genus Bromovirus including Broad bean mottle
virus, Brome mosaic virus, Cassia yellow blotch virus, Cowpea
chlorotic mottle virus (CCMV), Melandrium yellow fleck virus, and
Spring beauty latent virus. In another aspect, the VLP can be
derived from the influenza virus as described in U.S. Pub. No.
US20130177587 and U.S. Pat. No. 8,506,967. In one aspect, the VLP
can comprise a B7-1 and/or B7-2 molecule anchored to a lipid
membrane or the exterior of the particle such as described in Intl.
Pub. No. WO2013116656. In one aspect, the VLP can be derived from
norovirus, rotavirus recombinant VP6 protein or double layered
VP2/VP6 such as the VLP as described in Intl. Pub. No.
WO2012049366. Each of the references is herein incorporated by
reference in its entirety.
[1579] In some embodiments, the pseudovirion can be a human
papilloma virus-like particle as described in Intl. Pub. No.
WO2010120266 and U.S. Pub. No. US20120171290. In some embodiments,
the virus-like particle (VLP) can be a self-assembled particle. In
one aspect, the pseudovirions can be virion derived nanoparticles
as described in U.S. Pub. Nos. US20130116408 and US20130115247; and
Intl. Pub. No. WO2013119877. Each of the references is herein
incorporated by reference in their entirety.
[1580] Non-limiting examples of formulations and methods for
formulating the polynucleotides described herein are also provided
in Intl. Pub. No WO2013090648 (incorporated herein by reference in
their entirety).
24. ACCELERATED BLOOD CLEARANCE
[1581] The invention provides compounds, compositions and methods
of use thereof for reducing the effect of ABC on a repeatedly
administered active agent such as a biologically active agent. As
will be readily apparent, reducing or eliminating altogether the
effect of ABC on an administered active agent effectively increases
its half-life and thus its efficacy.
[1582] In some embodiments the term reducing ABC refers to any
reduction in ABC in comparison to a positive reference control ABC
inducing LNP such as an MC3 LNP. ABC inducing LNPs cause a
reduction in circulating levels of an active agent upon a second or
subsequent administration within a given time frame. Thus a
reduction in ABC refers to less clearance of circulating agent upon
a second or subsequent dose of agent, relative to a standard LNP.
The reduction may be, for instance, at least 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, or 100%. In some embodiments the reduction is 10-100%,
10-50%, 20-100%, 20-50%, 30-100%, 30-50%, 40%-100%, 40-80%, 50-90%,
or 50-100%. Alternatively the reduction in ABC may be characterized
as at least a detectable level of circulating agent following a
second or subsequent administration or at least a 2 fold, 3 fold, 4
fold, 5 fold increase in circulating agent relative to circulating
agent following administration of a standard LNP. In some
embodiments the reduction is a 2-100 fold, 2-50 fold, 3-100 fold,
3-50 fold, 3-20 fold, 4-100 fold, 4-50 fold, 4-40 fold, 4-30 fold,
4-25 fold, 4-20 fold, 4-15 fold, 4-10 fold, 4-5 fold, 5-100 fold,
5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold, 5-20 fold, 5-15 fold,
5-10 fold, 6-100 fold, 6-50 fold, 6-40 fold, 6-30 fold, 6-25 fold,
6-20 fold, 6-15 fold, 6-10 fold, 8-100 fold, 8-50 fold, 8-40 fold,
8-30 fold, 8-25 fold, 8-20 fold, 8-15 fold, 8-10 fold, 10-100 fold,
10-50 fold, 10-40 fold, 10-30 fold, 10-25 fold, 10-20 fold, 10-15
fold, 20-100 fold, 20-50 fold, 20-40 fold, 20-30 fold, or 20-25
fold.
[1583] The disclosure provides lipid-comprising compounds and
compositions that are less susceptible to clearance and thus have a
longer half-life in vivo. This is particularly the case where the
compositions are intended for repeated including chronic
administration, and even more particularly where such repeated
administration occurs within days or weeks.
[1584] Significantly, these compositions are less susceptible or
altogether circumvent the observed phenomenon of accelerated blood
clearance (ABC). ABC is a phenomenon in which certain exogenously
administered agents are rapidly cleared from the blood upon second
and subsequent administrations. This phenomenon has been observed,
in part, for a variety of lipid-containing compositions including
but not limited to lipidated agents, liposomes or other lipid-based
delivery vehicles, and lipid-encapsulated agents. Heretofore, the
basis of ABC has been poorly understood and in some cases
attributed to a humoral immune response and accordingly strategies
for limiting its impact in vivo particularly in a clinical setting
have remained elusive.
[1585] This disclosure provides compounds and compositions that are
less susceptible, if at all susceptible, to ABC. In some important
aspects, such compounds and compositions are lipid-comprising
compounds or compositions. The lipid-containing compounds or
compositions of this disclosure, surprisingly, do not experience
ABC upon second and subsequent administration in vivo. This
resistance to ABC renders these compounds and compositions
particularly suitable for repeated use in vivo, including for
repeated use within short periods of time, including days or 1-2
weeks. This enhanced stability and/or half-life is due, in part, to
the inability of these compositions to activate B1a and/or B1b
cells and/or conventional B cells, pDCs and/or platelets.
[1586] This disclosure therefore provides an elucidation of the
mechanism underlying accelerated blood clearance (ABC). It has been
found, in accordance with this disclosure and the inventions
provided herein, that the ABC phenomenon at least as it relates to
lipids and lipid nanoparticles is mediated, at least in part an
innate immune response involving B1a and/or B1b cells, pDC and/or
platelets. B1a cells are normally responsible for secreting natural
antibody, in the form of circulating IgM. This IgM is
poly-reactive, meaning that it is able to bind to a variety of
antigens, albeit with a relatively low affinity for each.
[1587] It has been found in accordance with the invention that some
lipidated agents or lipid-comprising formulations such as lipid
nanoparticles administered in vivo trigger and are subject to ABC.
It has now been found in accordance with the invention that upon
administration of a first dose of the LNP, one or more cells
involved in generating an innate immune response (referred to
herein as sensors) bind such agent, are activated, and then
initiate a cascade of immune factors (referred to herein as
effectors) that promote ABC and toxicity. For instance, B1a and B1b
cells may bind to LNP, become activated (alone or in the presence
of other sensors such as pDC and/or effectors such as IL6) and
secrete natural IgM that binds to the LNP. Pre-existing natural IgM
in the subject may also recognize and bind to the LNP, thereby
triggering complement fixation. After administration of the first
dose, the production of natural IgM begins within 1-2 hours of
administration of the LNP. Typically by about 2-3 weeks the natural
IgM is cleared from the system due to the natural half-life of IgM.
Natural IgG is produced beginning around 96 hours after
administration of the LNP. The agent, when administered in a naive
setting, can exert its biological effects relatively unencumbered
by the natural IgM produced post-activation of the B1a cells or B1b
cells or natural IgG. The natural IgM and natural IgG are
non-specific and thus are distinct from anti-PEG IgM and anti-PEG
IgG.
[1588] Although Applicant is not bound by mechanism, it is proposed
that LNPs trigger ABC and/or toxicity through the following
mechanisms. It is believed that when an LNP is administered to a
subject the LNP is rapidly transported through the blood to the
spleen. The LNPs may encounter immune cells in the blood and/or the
spleen. A rapid innate immune response is triggered in response to
the presence of the LNP within the blood and/or spleen. Applicant
has shown herein that within hours of administration of an LNP
several immune sensors have reacted to the presence of the LNP.
These sensors include but are not limited to immune cells involved
in generating an immune response, such as B cells, pDC, and
platelets. The sensors may be present in the spleen, such as in the
marginal zone of the spleen and/or in the blood. The LNP may
physically interact with one or more sensors, which may interact
with other sensors. In such a case the LNP is directly or
indirectly interacting with the sensors. The sensors may interact
directly with one another in response to recognition of the LNP.
For instance many sensors are located in the spleen and can easily
interact with one another. Alternatively one or more of the sensors
may interact with LNP in the blood and become activated. The
activated sensor may then interact directly with other sensors or
indirectly (e.g., through the stimulation or production of a
messenger such as a cytokine e.g., IL6).
[1589] In some embodiments the LNP may interact directly with and
activate each of the following sensors: pDC, B1a cells, B1b cells,
and platelets. These cells may then interact directly or indirectly
with one another to initiate the production of effectors which
ultimately lead to the ABC and/or toxicity associated with repeated
doses of LNP. For instance, Applicant has shown that LNP
administration leads to pDC activation, platelet aggregation and
activation and B cell activation. In response to LNP platelets also
aggregate and are activated and aggregate with B cells. pDC cells
are activated. LNP has been found to interact with the surface of
platelets and B cells relatively quickly. Blocking the activation
of any one or combination of these sensors in response to LNP is
useful for dampening the immune response that would ordinarily
occur. This dampening of the immune response results in the
avoidance of ABC and/or toxicity.
[1590] The sensors once activated produce effectors. An effector,
as used herein, is an immune molecule produced by an immune cell,
such as a B cell. Effectors include but are not limited to
immunoglobulin such as natural IgM and natural IgG and cytokines
such as IL6. B1a and B1b cells stimulate the production of natural
IgMs within 2-6 hours following administration of an LNP. Natural
IgG can be detected within 96 hours. IL6 levels are increased
within several hours. The natural IgM and IgG circulate in the body
for several days to several weeks. During this time the circulating
effectors can interact with newly administered LNPs, triggering
those LNPs for clearance by the body. For instance, an effector may
recognize and bind to an LNP. The Fc region of the effector may be
recognized by and trigger uptake of the decorated LNP by
macrophage. The macrophage are then transported to the spleen. The
production of effectors by immune sensors is a transient response
that correlates with the timing observed for ABC.
[1591] If the administered dose is the second or subsequent
administered dose, and if such second or subsequent dose is
administered before the previously induced natural IgM and/or IgG
is cleared from the system (e.g., before the 2-3 window time
period), then such second or subsequent dose is targeted by the
circulating natural IgM and/or natural IgG or Fc which trigger
alternative complement pathway activation and is itself rapidly
cleared. When LNP are administered after the effectors have cleared
from the body or are reduced in number, ABC is not observed.
[1592] Thus, it is useful according to aspects of the invention to
inhibit the interaction between LNP and one or more sensors, to
inhibit the activation of one or more sensors by LNP (direct or
indirect), to inhibit the production of one or more effectors,
and/or to inhibit the activity of one or more effectors. In some
embodiments the LNP is designed to limit or block interaction of
the LNP with a sensor. For instance the LNP may have an altered PC
and/or PEG to prevent interactions with sensors. Alternatively or
additionally an agent that inhibits immune responses induced by
LNPs may be used to achieve any one or more of these effects.
[1593] It has also been determined that conventional B cells are
also implicated in ABC. Specifically, upon first administration of
an agent, conventional B cells, referred to herein as CD19(+), bind
to and react against the agent. Unlike B1a and B1b cells though,
conventional B cells are able to mount first an IgM response
(beginning around 96 hours after administration of the LNPs)
followed by an IgG response (beginning around 14 days after
administration of the LNPs) concomitant with a memory response.
Thus conventional B cells react against the administered agent and
contribute to IgM (and eventually IgG) that mediates ABC. The IgM
and IgG are typically anti-PEG IgM and anti-PEG IgG.
[1594] It is contemplated that in some instances, the majority of
the ABC response is mediated through B1a cells and B1a-mediated
immune responses. It is further contemplated that in some
instances, the ABC response is mediated by both IgM and IgG, with
both conventional B cells and B1a cells mediating such effects. In
yet still other instances, the ABC response is mediated by natural
IgM molecules, some of which are capable of binding to natural IgM,
which may be produced by activated B1a cells. The natural IgMs may
bind to one or more components of the LNPs, e.g., binding to a
phospholipid component of the LNPs (such as binding to the PC
moiety of the phospholipid) and/or binding to a PEG-lipid component
of the LNPs (such as binding to PEG-DMG, in particular, binding to
the PEG moiety of PEG-DMG). Since B1a expresses CD36, to which
phosphatidylcholine is a ligand, it is contemplated that the CD36
receptor may mediate the activation of B1a cells and thus
production of natural IgM. In yet still other instances, the ABC
response is mediated primarily by conventional B cells.
[1595] It has been found in accordance with the invention that the
ABC phenomenon can be reduced or abrogated, at least in part,
through the use of compounds and compositions (such as agents,
delivery vehicles, and formulations) that do not activate B1a
cells. Compounds and compositions that do not activate B1a cells
may be referred to herein as B1a inert compounds and compositions.
It has been further found in accordance with the invention that the
ABC phenomenon can be reduced or abrogated, at least in part,
through the use of compounds and compositions that do not activate
conventional B cells. Compounds and compositions that do not
activate conventional B cells may in some embodiments be referred
to herein as CD 19-inert compounds and compositions. Thus, in some
embodiments provided herein, the compounds and compositions do not
activate B1a cells and they do not activate conventional B cells.
Compounds and compositions that do not activate B1a cells and
conventional B cells may in some embodiments be referred to herein
as B1a/CD19-inert compounds and compositions.
[1596] These underlying mechanisms were not heretofore understood,
and the role of B1a and B1b cells and their interplay with
conventional B cells in this phenomenon was also not
appreciated.
[1597] Accordingly, this disclosure provides compounds and
compositions that do not promote ABC. These may be further
characterized as not capable of activating B1a and/or B1b cells,
platelets and/or pDC, and optionally conventional B cells also.
These compounds (e.g., agents, including biologically active agents
such as prophylactic agents, therapeutic agents and diagnostic
agents, delivery vehicles, including liposomes, lipid
nanoparticles, and other lipid-based encapsulating structures,
etc.) and compositions (e.g., formulations, etc.) are particularly
desirable for applications requiring repeated administration, and
in particular repeated administrations that occur within with short
periods of time (e.g., within 1-2 weeks). This is the case, for
example, if the agent is a nucleic acid based therapeutic that is
provided to a subject at regular, closely-spaced intervals. The
findings provided herein may be applied to these and other agents
that are similarly administered and/or that are subject to ABC.
[1598] Of particular interest are lipid-comprising compounds,
lipid-comprising particles, and lipid-comprising compositions as
these are known to be susceptible to ABC. Such lipid-comprising
compounds particles, and compositions have been used extensively as
biologically active agents or as delivery vehicles for such agents.
Thus, the ability to improve their efficacy of such agents, whether
by reducing the effect of ABC on the agent itself or on its
delivery vehicle, is beneficial for a wide variety of active
agents.
[1599] Also provided herein are compositions that do not stimulate
or boost an acute phase response (ARP) associated with repeat dose
administration of one or more biologically active agents.
[1600] The composition, in some instances, may not bind to IgM,
including but not limited to natural IgM.
[1601] The composition, in some instances, may not bind to an acute
phase protein such as but not limited to C-reactive protein.
[1602] The composition, in some instances, may not trigger a CD5(+)
mediated immune response. As used herein, a CD5(+) mediated immune
response is an immune response that is mediated by B1a and/or B1b
cells. Such a response may include an ABC response, an acute phase
response, induction of natural IgM and/or IgG, and the like.
[1603] The composition, in some instances, may not trigger a
CD19(+) mediated immune response. As used herein, a CD19(+)
mediated immune response is an immune response that is mediated by
conventional CD19(+), CD5(-) B cells. Such a response may include
induction of IgM, induction of IgG, induction of memory B cells, an
ABC response, an anti-drug antibody (ADA) response including an
anti-protein response where the protein may be encapsulated within
an LNP, and the like.
[1604] B1a cells are a subset of B cells involved in innate
immunity. These cells are the source of circulating IgM, referred
to as natural antibody or natural serum antibody. Natural IgM
antibodies are characterized as having weak affinity for a number
of antigens, and therefore they are referred to as "poly-specific"
or "poly-reactive", indicating their ability to bind to more than
one antigen. B1a cells are not able to produce IgG. Additionally,
they do not develop into memory cells and thus do not contribute to
an adaptive immune response. However, they are able to secrete IgM
upon activation. The secreted IgM is typically cleared within about
2-3 weeks, at which point the immune system is rendered relatively
naive to the previously administered antigen. If the same antigen
is presented after this time period (e.g., at about 3 weeks after
the initial exposure), the antigen is not rapidly cleared. However,
significantly, if the antigen is presented within that time period
(e.g., within 2 weeks, including within 1 week, or within days),
then the antigen is rapidly cleared. This delay between consecutive
doses has rendered certain lipid-containing therapeutic or
diagnostic agents unsuitable for use.
[1605] In humans, B1a cells are CD19(+), CD20(+), CD27(+), CD43(+),
CD70(-) and CD5(+). In mice, B1a cells are CD19(+), CD5(+), and
CD45 B cell isoform B220(+). It is the expression of CD5 which
typically distinguishes B1a cells from other convention B cells.
B1a cells may express high levels of CD5, and on this basis may be
distinguished from other B-1 cells such as B-1b cells which express
low or undetectable levels of CD5. CD5 is a pan-T cell surface
glycoprotein. B1a cells also express CD36, also known as fatty acid
translocase. CD36 is a member of the class B scavenger receptor
family. CD36 can bind many ligands, including oxidized low density
lipoproteins, native lipoproteins, oxidized phospholipids, and
long-chain fatty acids.
[1606] B1b cells are another subset of B cells involved in innate
immunity. These cells are another source of circulating natural
IgM. Several antigens, including PS, are capable of inducing T cell
independent immunity through B1b activation. CD27 is typically
upregulated on B1b cells in response to antigen activation. Similar
to B1a cells, the Bib cells are typically located in specific body
locations such as the spleen and peritoneal cavity and are in very
low abundance in the blood. The B1b secreted natural IgM is
typically cleared within about 2-3 weeks, at which point the immune
system is rendered relatively naive to the previously administered
antigen. If the same antigen is presented after this time period
(e.g., at about 3 weeks after the initial exposure), the antigen is
not rapidly cleared. However, significantly, if the antigen is
presented within that time period (e.g., within 2 weeks, including
within 1 week, or within days), then the antigen is rapidly
cleared. This delay between consecutive doses has rendered certain
lipid-containing therapeutic or diagnostic agents unsuitable for
use.
[1607] In some embodiments it is desirable to block B1a and/or B1b
cell activation. One strategy for blocking B1a and/or B1b cell
activation involves determining which components of a lipid
nanoparticle promote B cell activation and neutralizing those
components. It has been discovered herein that at least PEG and
phosphatidylcholine (PC) contribute to B1a and B1b cell interaction
with other cells and/or activation. PEG may play a role in
promoting aggregation between B1 cells and platelets, which may
lead to activation. PC (a helper lipid in LNPs) is also involved in
activating the B 1 cells, likely through interaction with the CD36
receptor on the B cell surface. Numerous particles have PEG-lipid
alternatives, PEG-less, and/or PC replacement lipids (e.g. oleic
acid or analogs thereof) have been designed and tested. Applicant
has established that replacement of one or more of these components
within an LNP that otherwise would promote ABC upon repeat
administration, is useful in preventing ABC by reducing the
production of natural IgM and/or B cell activation. Thus, the
invention encompasses LNPs that have reduced ABC as a result of a
design which eliminates the inclusion of B cell triggers.
[1608] Another strategy for blocking B1a and/or B1b cell activation
involves using an agent that inhibits immune responses induced by
LNPs. These types of agents are discussed in more detail below. In
some embodiments these agents block the interaction between B1a/B1b
cells and the LNP or platelets or pDC. For instance the agent may
be an antibody or other binding agent that physically blocks the
interaction. An example of this is an antibody that binds to CD36
or CD6. The agent may also be a compound that prevents or disables
the B1a/B1b cell from signaling once activated or prior to
activation. For instance, it is possible to block one or more
components in the B1a/B1b signaling cascade the results from B cell
interaction with LNP or other immune cells. In other embodiments
the agent may act one or more effectors produced by the B1a/B1b
cells following activation. These effectors include for instance,
natural IgM and cytokines.
[1609] It has been demonstrated according to aspects of the
invention that when activation of pDC cells is blocked, B cell
activation in response to LNP is decreased. Thus, in order to avoid
ABC and/or toxicity, it may be desirable to prevent pDC activation.
Similar to the strategies discussed above, pDC cell activation may
be blocked by agents that interfere with the interaction between
pDC and LNP and/or B cells/platelets. Alternatively agents that act
on the pDC to block its ability to get activated or on its
effectors can be used together with the LNP to avoid ABC.
[1610] Platelets may also play an important role in ABC and
toxicity. Very quickly after a first dose of LNP is administered to
a subject platelets associate with the LNP, aggregate and are
activated. In some embodiments it is desirable to block platelet
aggregation and/or activation. One strategy for blocking platelet
aggregation and/or activation involves determining which components
of a lipid nanoparticle promote platelet aggregation and/or
activation and neutralizing those components. It has been
discovered herein that at least PEG contribute to platelet
aggregation, activation and/or interaction with other cells.
Numerous particles have PEG-lipid alternatives and PEG-less have
been designed and tested. Applicant has established that
replacement of one or more of these components within an LNP that
otherwise would promote ABC upon repeat administration, is useful
in preventing ABC by reducing the production of natural IgM and/or
platelet aggregation. Thus, the invention encompasses LNPs that
have reduced ABC as a result of a design which eliminates the
inclusion of platelet triggers. Alternatively agents that act on
the platelets to block its activity once it is activated or on its
effectors can be used together with the LNP to avoid ABC.
[1611] (i) Measuring ABC Activity and Related Activities
[1612] Various compounds and compositions provided herein,
including LNPs, do not promote ABC activity upon administration in
vivo. These LNPs may be characterized and/or identified through any
of a number of assays, such as but not limited to those described
below, as well as any of the assays disclosed in the Examples
section, include the methods subsection of the Examples.
[1613] In some embodiments the methods involve administering an LNP
without producing an immune response that promotes ABC. An immune
response that promotes ABC involves activation of one or more
sensors, such as B1 cells, pDC, or platelets, and one or more
effectors, such as natural IgM, natural IgG or cytokines such as
IL6. Thus administration of an LNP without producing an immune
response that promotes ABC, at a minimum involves administration of
an LNP without significant activation of one or more sensors and
significant production of one or more effectors. Significant used
in this context refers to an amount that would lead to the
physiological consequence of accelerated blood clearance of all or
part of a second dose with respect to the level of blood clearance
expected for a second dose of an ABC triggering LNP. For instance,
the immune response should be dampened such that the ABC observed
after the second dose is lower than would have been expected for an
ABC triggering LNP.
[1614] (ii) B1a or B1b Activation Assay
[1615] Certain compositions provided in this disclosure do not
activate B cells, such as B1a or B1b cells (CD19+CD5+) and/or
conventional B cells (CD19+CD5-). Activation of B1a cells, B1b
cells, or conventional B cells may be determined in a number of
ways, some of which are provided below. B cell population may be
provided as fractionated B cell populations or unfractionated
populations of splenocytes or peripheral blood mononuclear cells
(PBMC). If the latter, the cell population may be incubated with
the LNP of choice for a period of time, and then harvested for
further analysis. Alternatively, the supernatant may be harvested
and analyzed.
[1616] (iii) Upregulation of Activation Marker Cell Surface
Expression
[1617] Activation of B1a cells, B1b cells, or conventional B cells
may be demonstrated as increased expression of B cell activation
markers including late activation markers such as CD86. In an
exemplary non-limiting assay, unfractionated B cells are provided
as a splenocyte population or as a PBMC population, incubated with
an LNP of choice for a particular period of time, and then stained
for a standard B cell marker such as CD19 and for an activation
marker such as CD86, and analyzed using for example flow cytometry.
A suitable negative control involves incubating the same population
with medium, and then performing the same staining and
visualization steps. An increase in CD86 expression in the test
population compared to the negative control indicates B cell
activation.
[1618] (iv) Pro-Inflammatory Cytokine Release
[1619] B cell activation may also be assessed by cytokine release
assay. For example, activation may be assessed through the
production and/or secretion of cytokines such as IL-6 and/or
TNF-alpha upon exposure with LNPs of interest.
[1620] Such assays may be performed using routine cytokine
secretion assays well known in the art. An increase in cytokine
secretion is indicative of B cell activation.
[1621] (v) LNP Binding/Association to and/or Uptake by B Cells
[1622] LNP association or binding to B cells may also be used to
assess an LNP of interest and to further characterize such LNP.
Association/binding and/or uptake/internalization may be assessed
using a detectably labeled, such as fluorescently labeled, LNP and
tracking the location of such LNP in or on B cells following
various periods of incubation.
[1623] The invention further contemplates that the compositions
provided herein may be capable of evading recognition or detection
and optionally binding by downstream mediators of ABC such as
circulating IgM and/or acute phase response mediators such as acute
phase proteins (e.g., C-reactive protein (CRP).
[1624] (vi) Methods of Use for Reducing ABC
[1625] Also provided herein are methods for delivering LNPs, which
may encapsulate an agent such as a therapeutic agent, to a subject
without promoting ABC.
[1626] In some embodiments, the method comprises administering any
of the LNPs described herein, which do not promote ABC, for
example, do not induce production of natural IgM binding to the
LNPs, do not activate B1a and/or B1b cells. As used herein, an LNP
that "does not promote ABC" refers to an LNP that induces no immune
responses that would lead to substantial ABC or a substantially low
level of immune responses that is not sufficient to lead to
substantial ABC. An LNP that does not induce the production of
natural IgMs binding to the LNP refers to LNPs that induce either
no natural IgM binding to the LNPs or a substantially low level of
the natural IgM molecules, which is insufficient to lead to
substantial ABC. An LNP that does not activate B1a and/or Bib cells
refer to LNPs that induce no response of B1a and/or B1b cells to
produce natural IgM binding to the LNPs or a substantially low
level of B1a and/or B1b responses, which is insufficient to lead to
substantial ABC.
[1627] In some embodiments the terms do not activate and do not
induce production are a relative reduction to a reference value or
condition. In some embodiments the reference value or condition is
the amount of activation or induction of production of a molecule
such as IgM by a standard LNP such as an MC3 LNP. In some
embodiments the relative reduction is a reduction of at least 30%,
for example at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%. In other embodiments the terms do not activate cells such as
B cells and do not induce production of a protein such as IgM may
refer to an undetectable amount of the active cells or the specific
protein.
[1628] (vii) Platelet Effects and Toxicity
[1629] The invention is further premised in part on the elucidation
of the mechanism underlying dose-limiting toxicity associated with
LNP administration. Such toxicity may involve coagulopathy,
disseminated intravascular coagulation (DIC, also referred to as
consumptive coagulopathy), whether acute or chronic, and/or
vascular thrombosis. In some instances, the dose-limiting toxicity
associated with LNPs is acute phase response (APR) or complement
activation-related psudoallergy (CARPA).
[1630] As used herein, coagulopathy refers to increased coagulation
(blood clotting) in vivo. The findings reported in this disclosure
are consistent with such increased coagulation and significantly
provide insight on the underlying mechanism. Coagulation is a
process that involves a number of different factors and cell types,
and heretofore the relationship between and interaction of LNPs and
platelets has not been understood in this regard. This disclosure
provides evidence of such interaction and also provides compounds
and compositions that are modified to have reduced platelet effect,
including reduced platelet association, reduced platelet
aggregation, and/or reduced platelet aggregation. The ability to
modulate, including preferably down-modulate, such platelet effects
can reduce the incidence and/or severity of coagulopathy post-LNP
administration. This in turn will reduce toxicity relating to such
LNP, thereby allowing higher doses of LNPs and importantly their
cargo to be administered to patients in need thereof.
[1631] CARPA is a class of acute immune toxicity manifested in
hypersensitivity reactions (HSRs), which may be triggered by
nanomedicines and biologicals. Unlike allergic reactions, CARPA
typically does not involve IgE but arises as a consequence of
activation of the complement system, which is part of the innate
immune system that enhances the body's abilities to clear
pathogens. One or more of the following pathways, the classical
complement pathway (CP), the alternative pathway (AP), and the
lectin pathway (LP), may be involved in CARPA. Szebeni, Molecular
Immunology, 61:163-173 (2014).
[1632] The classical pathway is triggered by activation of the
C1-complex, which contains. C1q, C1r, C1s, or C1qr2s2. Activation
of the C1-complex occurs when C1q binds to IgM or IgG complexed
with antigens, or when C1q binds directly to the surface of the
pathogen. Such binding leads to conformational changes in the C1q
molecule, which leads to the activation of C1r, which in turn,
cleave C1s. The Clr2s2 component now splits C4 and then C2,
producing C4a, C4b, C2a, and C2b. C4b and C2b bind to form the
classical pathway C3-convertase (C4b2b complex), which promotes
cleavage of C3 into C3a and C3b. C3b then binds the C3 convertase
to from the C5 convertase (C4b2b3b complex). The alternative
pathway is continuously activated as a result of spontaneous C3
hydrolysis. Factor P (properdin) is a positive regulator of the
alternative pathway. Oligomerization of properdin stabilizes the C3
convertase, which can then cleave much more C3. The C3 molecules
can bind to surfaces and recruit more B, D, and P activity, leading
to amplification of the complement activation.
[1633] Acute phase response (APR) is a complex systemic innate
immune responses for preventing infection and clearing potential
pathogens. Numerous proteins are involved in APR and C-reactive
protein is a well-characterized one.
[1634] It has been found, in accordance with the invention, that
certain LNP are able to associate physically with platelets almost
immediately after administration in vivo, while other LNP do not
associate with platelets at all or only at background levels.
Significantly, those LNPs that associate with platelets also
apparently stabilize the platelet aggregates that are formed
thereafter. Physical contact of the platelets with certain LNPs
correlates with the ability of such platelets to remain aggregated
or to form aggregates continuously for an extended period of time
after administration. Such aggregates comprise activated platelets
and also innate immune cells such as macrophages and B cells.
25. METHODS OF USE
[1635] The polynucleotides, pharmaceutical compositions and
formulations described herein are used in the preparation,
manufacture and therapeutic use to treat and/or prevent
PBGD-related diseases, disorders or conditions. In some
embodiments, the polynucleotides, compositions and formulations of
the invention are used to treat AIP. AIP patients can suffer from
acute attacks and can be treated during or after an acute attack
(e.g., severe pain). Patients having reoccurring attacks, e.g.,
several attacks per year, can be treated "prophylactically", i.e.,
to reduce the risk of and/or prevent recurring attacks.
[1636] In some embodiments, the polynucleotides, pharmaceutical
compositions and formulations of the invention are used in methods
for reducing the levels of porphobilinogen in a subject in need
thereof. For instance, one aspect of the invention provides a
method of alleviating the signs or symptoms of AIP in a subject
comprising the administration of a composition or formulation
comprising a polynucleotide encoding PBGD to that subject (e.g, an
mRNA encoding a PBGD polypeptide).
[1637] In some embodiments, the polynucleotides, pharmaceutical
compositions and formulations of the invention are used to reduce
the level of a metabolite associated with AIP (e.g., the substrate
or product, i.e., hydroxymethylbilane or porphobilinogen), the
method comprising administering to the subject an effective amount
of a polynucleotide encoding a PBGD polypeptide.
[1638] In some embodiments, the administration of an effective
amount of a polynucleotide, pharmaceutical composition or
formulation of the invention reduces the levels of a biomarker of
AIP, e.g., porphobilinogen (PBG), aminolevulinate acid (ALA),
porphyrin, alanine transaminase (ALT), aspartate transaminase
(AST), bilirubin, or any combination thereof. In some embodiments,
the administration of the polynucleotide, pharmaceutical
composition or formulation of the invention results in reduction in
the level of one or more biomarkers of AIP, e.g., porphobilinogen,
within a short period of time (e.g., within about 6 hours, within
about 8 hours, within about 12 hours, within about 16 hours, within
about 20 hours, or within about 24 hours) after administration of
the polynucleotide, pharmaceutical composition or formulation of
the invention.
[1639] Replacement therapy is a potential treatment for AIP. Thus,
in certain aspects of the invention, the polynucleotides, e.g.,
mRNA, disclosed herein comprise one or more sequences encoding a
PBGD polypeptide that is suitable for use in gene replacement
therapy for AIP. In some embodiments, the present disclosure treats
a lack of PBGD or PBGD activity, or decreased or abnomal PBGD
activity in a subject by providing a polynucleotide, e.g., mRNA,
that encodes a PBGD polypeptide to the subject. In some
embodiments, the polynucleotide is sequence-optimized. In some
embodiments, the polynucleotide (e.g., an mRNA) comprises a nucleic
acid sequence (e.g., an ORF) encoding a PBGD polypeptide, wherein
the nucleic acid is sequence-optimized, e.g., by modifying its G/C,
uridine, or thymidine content, and/or the polynucleotide comprises
at least one chemically modified nucleoside. In some embodiments,
the polynucleotide comprises a miRNA binding site, e.g., a miRNA
binding site that binds miRNA-142 and/or a miRNA binding site that
binds miRNA-126.
[1640] In some embodiments, the administration of a composition or
formulation comprising polynucleotide, pharmaceutical composition
or formulation of the invention to a subject results in a decrease
in porphobilinogen in cells to a level at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, or to 100% lower than the level
observed prior to the administration of the composition or
formulation.
[1641] In some embodiments, the administration of the
polynucleotide, pharmaceutical composition or formulation of the
invention results in expression of PBGD protein in cells of the
subject. In some embodiments, administering the polynucleotide,
pharmaceutical composition or formulation of the invention results
in an increase of PBGD expression and/or enzymatic activity in the
subject. For example, in some embodiments, the polynucleotides of
the present invention are used in methods of administering a
composition or formulation comprising an mRNA encoding a PBGD
polypeptide to a subject, wherein the method results in an increase
of PBGD expression and/or enzymatic activity in at least some cells
of a subject.
[1642] In some embodiments, the administration of a composition or
formulation comprising an mRNA encoding a PBGD polypeptide to a
subject results in an increase of PBGD expression and/or enzymatic
activity in cells subject to a level at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or to 100% or more of the expression
and/or activity level expected in a normal subject, e.g., a human
not suffering from AIP.
[1643] In some embodiments, the administration of the
polynucleotide, pharmaceutical composition or formulation of the
invention results in expression of PBGD protein in at least some of
the cells of a subject that persists for a period of time
sufficient to allow significant porphobilinogen metabolism to
occur.
[1644] In some embodiments, the expression and/or enzymatic
activity of the encoded polypeptide is increased. In some
embodiments, the polynucleotide increases PBGD expression and/or
enzymatic activity levels in cells when introduced into those
cells, e.g., by at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or to 100% with respect to the PBGD expression and/or
enzymatic activity level in the cells before the polypeptide is
introduced in the cells.
[1645] In some embodiments, the method or use comprises
administering a polynucleotide, e.g., mRNA, comprising a nucleotide
sequence having sequence similarity to a polynucleotide selected
from the group of SEQ ID NOs: 9 to 33 and 89 to 117 (See TABLE 2)
or a polynucleotide selected from the group of SEQ ID NOs: 118-148,
e.g., 133, 141, 144, or 145 (See TABLE 5), wherein the
polynucleotide encodes an PBGD polypeptide.
[1646] Other aspects of the present disclosure relate to
transplantation of cells containing polynucleotides to a mammalian
subject. Administration of cells to mammalian subjects is known to
those of ordinary skill in the art, and includes, but is not
limited to, local implantation (e.g., topical or subcutaneous
administration), organ delivery or systemic injection (e.g.,
intravenous injection or inhalation), and the formulation of cells
in pharmaceutically acceptable carriers.
[1647] The present disclosure also provides methods to increase
hepatic PBGD activity (for example, expressed as pmol
uroporphyrinogen/mg protein/hour) in a subject in need thereof,
e.g., a subject with AIP, comprising administering to the subject a
therapeutically effective amount of a composition or formulation
comprising mRNA encoding a PBGD polypeptide disclosed herein, e.g.,
a human PBGD isoform, a mutant thereof (such as gain of function SM
double mutant), or a fusion protein comprising a human PBGD isoform
or a mutant thereof (for example a fusion protein comprising a PBGD
moiety and a human apoliprotein A.sub.1 moeity).
[1648] In some aspects, the hepatic PBGD activity measured after
administration to a subject in need thereof, e.g., a subject with
AIP, is at least the normal PBGD activity level observed in healthy
human subjects. In some aspects, such normal PBGD activity level
observed in healthy human subjects is 5.+-.0.2 units (pmol
uroporphyrinogen/mg protein/hour units). In some aspects, the
hepatic PBGD activity measured after administration is at higher
than the PBGD activity level observed in AIP patients, e.g.,
untreated AIP patients. In some aspects, the hepatic PBGD activity
level observed in AIP patients is 2.+-.0.2 units (pmol
uroporphyrinogen/mg protein/hour units). In some aspects, the
increase in hepatic PBGD activity (for example, expressed as pmol
uroporphyrinogen/mg protein/hour) in a subject in need thereof,
e.g., a subject with AIP, after administering to the subject a
therapeutically effective amount of a composition or formulation
comprising mRNA encoding a PBGD polypeptide disclosed herein is at
least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.6, 0.7, 0.8, 0.9, 1.0,
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,
2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,
5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,
6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,
7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,
8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 pmol
uroporphyrinogen/mg protein/hour units (i) above the normal PBGD
activity level observed in healthy human subjects or (ii) above the
PBGD activity level observed in AIP patients, e.g., untreated AIP
patients. In some aspects, the increase in hepatic PBGD activity
above the PBGD activity level observed in AIP patients (e.g., about
2 pmol uroporphyrinogen/mg protein/hour units) after administering
to the subject a composition or formulation comprising an mRNA
encoding a PBGD polypeptide disclosed herein (e.g., after a single
dose administration) is maintained for at least 1 day, 2 days, 3
days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10
days.
[1649] When an AIP attack occurs there is an accumulation of the
porphyrin precursors ALA (aminolevulinate) and PBG
(Porphobilinogen). ALA and PBG can be measured in urine using
methods known in the art. The present disclosure also provides a
method to decrease ALA levels in a subject in need thereof, e.g.,
untreated AIP patients, comprising administering to the subject a
therapeutically effective amount of a composition or formulation
comprising mRNA encoding a PBGD polypeptide disclosed herein. In
some aspects, the ALA levels are urinary ALA excretion levels. Also
provided is a method to decrease PBG levels in a subject in need
thereof, e.g., untreated AIP patients, comprising administering to
the subject a therapeutically effection amount of a composition or
formulation comprising mRNA encoding a PBGD polypeptide disclosed
herein. In some aspects, the PBG levels are urinary PBG excretion
levels.
[1650] In some aspects, the present disclosure provides a method to
protect a subject suffering from AIP against an increase in heme
precursors (e.g., ALA and/or PBG) in an AIP attack comprising
administering to the subject a therapeutically effective amount of
a composition or formulation comprising mRNA encoding a PBGD
polypeptide disclosed herein. Also provided is a method to reduce
the accumulation of heme precursors (e.g., ALA and/or PBG) in a
subject suffering from AIP comprising administering to the subject
a therapeutically effective amount of a composition or formulation
comprising mRNA encoding a PBGD polypeptide disclosed herein.
[1651] The present disclosure also provides a method to treat,
prevent, or ameliorate pain in an AIP patient comprising
administering to the subject a therapeutically effective amount of
a composition or formulation comprising mRNA encoding a PBGD
polypeptide disclosed herein. In some aspects, the administration
of a therapeutically effective amount of a composition or
formulation comprising mRNA encoding a PBGD polypeptide disclosed
herein to subject suffering from AIP results in a reduction in
pain. In some aspects, the reduction in pain is complete (pain
annulment). In some aspects, the pain is severe pain.
[1652] Also provided is a method to treat, prevent, or ameliorate
neuropathy in an AIP patient comprising administering to the
subject a therapeutically effective amount of a composition or
formulation comprising mRNA encoding a PBGD polypeptide disclosed
herein. In some aspects, the administration of a therapeutically
effective amount of a composition or formulation comprising mRNA
encoding a PBGD polypeptide disclosed herein to subject suffering
from AIP results in a reduction in neuropathy. In some aspects, the
reduction in neuropathy is complete (neuropathy annulment). In some
aspects the neuropathy is peripheral neuropathy.
[1653] Also provided is a method to increase survival an AIP
patient comprising administering to the subject a therapeutically
effective amount of a composition or formulation comprising mRNA
encoding a PBGD polypeptide disclosed herein.
[1654] In some aspects, the dose of mRNA encoding a PBGD
polypeptide disclosed herein is at least about 0.1 nmol/kg, at
least about 0.2 nmol/kg, at least about 0.3 nmol/kg, at least about
0.4 nmol/kg, at least about 0.5 nmol/kg, at least about 0.6
nmol/kg, at least about 0.7 nmol/kg, at least about 0.8 nmol/kg, at
least about 0.9 nmol/kg, at least about 1 nmol/kg, at least about
1.1 nmol/kg, at least about 1.2 nmol/kg, at least about 1.3
nmol/kg, at least about 1.4 nmol/kg, at least about 1.5 nmol/kg, at
least about 1.6 nmol/kg, at least about 1.7 nmol/kg, at least about
1.8 nmol/kg, at least about 1.9 nmol/kg, at least about 2 nmol/kg,
at least about 2.5 nmol/kg, at least about 3 nmol/kg, at least
about 3.5 nmol/kg, at least about 4 nmol/kg, at least about 4.5
nmol/kg, or at least about 5 nmol/kg. In some aspects, the dose of
mRNA encoding a PBGD polypeptide disclosed herein is at least about
0.05 mg/kg, at least about 0.1 mg/kg, at least about 0.15 mg/kg, at
least about 0.2 mg/kg, at least about 0.25 mg/kg, at least about
0.3 mg/kg, at least about 0.35 mg/kg, at least about 0.4 mg/kg, at
least about 0.45 mg/kg, at least about 0.5 mg/kg, at least about
0.55 mg/kg, at least about 0.6 mg/kg, at least about 0.7 mg/kg, at
least about 0.75 mg/kg, at least about 0.8 mg/kg, at least about
0.85 mg/kg, at least about 0.9 mg/kg, at least about 0.95 mg/kg, or
at least about 1 mg/kg.
[1655] In some aspects of the method disclosed herein, the AIP
patient is an aymptomatic patient.
[1656] In some embodiments, the polynucleotides (e.g., mRNA),
pharmaceutical compositions and formulations used in the methods of
the invention comprise a uracil-modified sequence encoding a PBGD
polypeptide disclosed herein and a miRNA binding site disclosed
herein, e.g., a miRNA binding site that binds to miR-142 and/or a
miRNA binding site that binds to miR-126. In some embodiments, the
uracil-modified sequence encoding a PBGD polypeptide comprises at
least one chemically modified nucleobase, e.g., 5-methoxyuracil. In
some embodiments, at least 95% of a type of nucleobase (e.g.,
uracil) in a uracil-modified sequence encoding a PBGD polypeptide
of the invention are modified nucleobases. In some embodiments, at
least 95% of uricil in a uracil-modified sequence encoding a PBGD
polypeptide is 5-methoxyuridine. In some embodiments, the
polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein is
formulated with a delivery agent comprising, e.g., a compound
having the Formula (I), e.g., any of Compounds 1-232, e.g.,
Compound 18; a compound having the Formula (III), (IV), (V), or
(VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a
compound having the Formula (VIII), e.g., any of Compounds 419-428,
e.g., Compound 428, or any combination thereof. In some
embodiments, the delivery agent comprises Compound 18, DSPC,
Cholesterol, and Compound 428, e.g., with a mole ratio of about
50:10:38.5:1.5.
[1657] The skilled artisan will appreciate that the therapeutic
effectiveness of a drug or a treatment of the instant invention can
be characterized or determined by measuring the level of expression
of an encoded protein (e.g., enzyme) in a sample or in samples
taken from a subject (e.g., from a preclinical test subject
(rodent, primate, etc.) or from a clinical subject (human).
Likewise, the therapeutic effectiveness of a drug or a treatment of
the instant invention can be characterized or determined by
measuring the level of activity of an encoded protein (e.g.,
enzyme) in a sample or in samples taken from a subject (e.g., from
a preclinical test subject (rodent, primate, etc.) or from a
clinical subject (human). Furthermore, the therapeutic
effectiveness of a drug or a treatment of the instant invention can
be characterized or determined by measuring the level of an
appropriate biomarker in sample(s) taken from a subject. Levels of
protein and/or biomarkers can be determined post-administration
with a single dose of an mRNA therapeutic of the invention or can
be determined and/or monitored at several time points following
administration with a single dose or can be determined and/or
monitored throughout a course of treatment, e.g., a multi-dose
treatment.
[1658] AIP is characterized by acute episodes and asymptomatic
periods. Accordingly, AIP patients can be asymptomatic carriers or
suffer from recurrent acute attack. AIP patients commonly show high
aminolevulinic acid (ALA) and porphobilinogen (PBG) blood and
urinary levels and their concentrations further increase during
acute attacks. Unless otherwise specified, the methods of treating
AIP patients or human subjects disclosed herein include treatment
of both asymptomatic "high excreter" (ASHE) patients, who carry a
genetic mutation of acute intermittent porphyria (AIP) and have
elevated levels of biomarkers, e.g., ALA and PBG, and recurrent
acute attack patients.
PBGD Protein Expression Levels
[1659] Certain aspects of the invention feature measurement,
determination and/or monitoring of the expression level or levels
of porphobilinogen deaminase (PBGD) protein in a subject, for
example, in an animal (e.g., rodents, primates, and the like) or in
a human subject. Animals include normal, healthy or wildtype
animals, as well as animal models for use in understanding acute
intermittent porphyria (AIP) and treatments thereof. Exemplary
animal models include rodent models, for example, PBGD deficient
mice also referred to as AIP mice. AIP mice are compound
heterozygotes of two different disruptions of the PBGD gene: Ti
strain [C57BL/6-pbgdtml(neo)Uam and T2 strain
(C57BL/6-pbgdtm2(neo)Uam] (Lindberg R L, et al. "Porphobilinogen
deaminase deficiency in mice causes a neuropathy resembling that of
human hepatic porphyria." Nat Genet. 1996; 12:195-199). Acute
porphyria attacks are triggered by phenobarbital challenges in AIP
mice. AIP mice subjected to phenobarbital challenge biochemically
mimic AIP, e.g., induction of the porphyrin precursors, and develop
physical signs and symptoms that mimic those in human subjects with
AIP.
[1660] PBGD protein expression levels can be measured or determined
by any art-recognized method for determining protein levels in
biological samples, e.g., needle liver biopsy. The term "level" or
"level of a protein" as used herein, preferably means the weight,
mass or concentration of the protein within a sample or a subject.
It will be understood by the skilled artisan that in certain
embodiments the sample may be subjected, e.g., to any of the
following: purification, precipitation, separation, e.g.
centrifugation and/or HPLC, and subsequently subjected to
determining the level of the protein, e.g., using mass and/or
spectrometric analysis. In exemplary embodiments, enzyme-linked
immunosorbent assay (ELISA) can be used to determine protein
expression levels. In other exemplary embodiments, protein
purification, separation and LC-MS can be used as a means for
determining the level of a protein according to the invention. In
some embodiments, an mRNA therapy of the invention (e.g., a single
intravenous dose) results in increased PBGD protein expression
levels in the liver tissue of the subject (e.g., 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,
30-fold, 40-fold, 50-fold increase and/or increased to at least
50%, at least 60%, at least 70%, at least 75%, 80%, at least 85%,
at least 90%, at least 95%, or at least 100% of normal levels) for
at least 6 hours, at least 12 hours, at least 24 hours, at least 36
hours, at least 48 hours, at least 60 hours, at least 72 hours, at
least 84 hours, at least 96 hours, at least 108 hours, at least 122
hours after administration of a single dose of the mRNA
therapy.
PBGD Protein Activity
[1661] In AIP patients, PBGD enzymatic activity is reduced, e.g.,
to about 50% of normal. Further aspects of the invention feature
measurement, determination and/or monitoring of the activity
level(s) (i.e., enzymatic activity level(s)) of PBGD protein in a
subject, for example, in an animal (e.g., rodent, primate, and the
like) or in a human subject. Activity levels can be measured or
determined by any art-recognized method for determining enzymatic
activity levels in biological samples. The term "activity level" or
"enzymatic activity level" as used herein, preferably means the
activity of the enzyme per volume, mass or weight of sample or
total protein within a sample. In exemplary embodiments, the
"activity level" or "enzymatic activity level" is described in
terms of units per milliliter of fluid (e.g., bodily fluid, e.g.,
serum, plasma, urine and the like) or is described in terms of
units per weight of tissue or per weight of protein (e.g., total
protein) within a sample. Units ("U") of enzyme activity can be
described in terms of weight or mass of substrate hydrolyzed per
unit time. Exemplary embodiments of the invention feature PBGD
activity described in terms of U/ml plasma or U/mg protein
(tissue), where units ("U") are described in terms of nmol
substrate hydrolyzed per hour (or nmol/hr). An exemplary enzymatic
assay features uroporphyrin production (URO). PBGD activity in
tissue (e.g., liver) can be quantitated, e.g., as pmol of
uroporphyrin produced per mg of protein per hour.
[1662] In exemplary embodiments, an mRNA therapy of the invention
features a pharmaceutical composition comprising a dose of mRNA
effective to result in at least 5 U/mg, at least 10 U/mg, at least
20 U/mg, at least 30 U/mg, at least 40 U/mg, at least 50 U/mg, at
least 60 U/mg, at least 70 U/mg, at least 80 U/mg, at least 90
U/mg, at least 100 U/mg, or at least 150 U/mg of PBGD activity in
tissue (e.g., liver) between 6 and 12 hours, or between 12 and 24,
between 24 and 48, or between 48 and 72 hours post administration
(e.g., at 48 or at 72 hours post administration).
[1663] In exemplary embodiments, an mRNA therapy of the invention
features a pharmaceutical composition comprising a single
intravenous dose of mRNA that results in the above-described levels
of activity. In another embodiment, an mRNA therapy of the
invention features a pharmaceutical composition which can be
administered in multiple single unit intravenous doses of mRNA that
maintain the above-described levels of activity.
PBGD Biomarkers
[1664] Further aspects of the invention feature determining the
level (or levels) of a biomarker, e.g., urinary aminolevulinate
acid (ALA) excretion, urinary porphobilinogen (PBG) excretion,
urinary porphyrin excretion, serum transaminases (e.g., alanine
transaminase (ALT) or aspartate transaminase (AST)) and/or serum
bilirubin, determined in a sample as compared to a level (e.g., a
reference level) of the same or another biomarker in another
sample, e.g., from the same patient, from another patient, from a
control and/or from the same or different time points, and/or a
physiologic level, and/or an elevated level, and/or a
supraphysiologic level, and/or a level of a control. The skilled
artisan will be familiar with physiologic levels of biomarkers, for
example, levels in normal or wildtype animals, normal or healthy
subjects, and the like, in particular, the level or levels
characteristic of subjects who are healthy and/or normal
functioning. As used herein, the phrase "elevated level" means
amounts greater than normally found in a normal or wildtype
preclinical animal or in a normal or healthy subject, e.g. a human
subject. As used herein, the term "supraphysiologic" means amounts
greater than normally found in a normal or wildtype preclinical
animal or in a normal or healthy subject, e.g. a human subject,
optionally producing a significantly enhanced physiologic response.
As used herein, the term "comparing" or "compared to" preferably
means the mathematical comparison of the two or more values, e.g.,
of the levels of the biomarker(s). It will thus be readily apparent
to the skilled artisan whether one of the values is higher, lower
or identical to another value or group of values if at least two of
such values are compared with each other. Comparing or comparison
to can be in the context, for example, of comparing to a control
value, e.g., as compared to a reference serum ALT level, a
reference serum AST level and/or a reference serum bilirubin level
in said subject prior to administration (e.g., in a person
suffering from AIP) or in a normal or healthy subject. Comparing or
comparison to can also be in the context, for example, of comparing
to a control value, e.g., as compared to a reference urinary ALA,
PBG, and/or porphyrin excretion level in said subject prior to
administration (e.g., in a person suffering from AIP) or in a
normal or healthy subject.
[1665] As used herein, a "control" is preferably a sample from a
subject wherein the AIP status of said subject is known. In one
embodiment, a control is a sample of a healthy patient. In another
embodiment, the control is a sample from at least one subject
having a known AIP status, for example, a severe, mild, or healthy
AIP status, e.g. a control patient. In another embodiment, the
control is a sample from a subject not being treated for AIP. In a
still further embodiment, the control is a sample from a single
subject or a pool of samples from different subjects and/or samples
taken from the subject(s) at different time points.
[1666] The term "level" or "level of a biomarker" as used herein,
preferably means the mass, weight or concentration of a biomarker
of the invention within a sample or a subject. Biomarkers of the
invention include, for example, aminolevulinate acid (ALA) (e.g.,
normal urinary excretion levels <about 3.9 mmol ALA/mol
creatinine), porphobilinogen (PBG) (e.g., normal urinary excretion
levels <about 1.6 or 1.5 mmol PBG/mol creatinine or 0-4 mg/L),
porphyrin (e.g., normal urinary excretion levels <145 .mu.g/1),
transaminases (e.g., alanine transaminase (ALT) (e.g., normal serum
levels <about 10-35 U/L) or aspartate transaminase (AST) (e.g.,
normal serum levels <about 15-41 U/L)) and/or bilirubin (e.g.,
normal serum levels <about 1.2 mg/dl). It will be understood by
the skilled artisan that in certain embodiments the sample may be
subjected to, e.g., one or more of the following: substance
purification, precipitation, separation, e.g. centrifugation and/or
HPLC and subsequently subjected to determining the level of the
biomarker, e.g. using mass spectrometric analysis. In exemplary
embodiments, LC-MS can be used as a means for determining the level
of a biomarker according to the invention.
[1667] Certain embodiments an mRNA of the invention can be used
express a PBGD polypeptide at a level sufficient to reduce urinary
excretion of: (i) aminolevulinate acid (ALA) to less than about 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or
1 mmol ALA/mol creatinine for at least 24 hours, at least 48 hours,
at least 72 hours, at least 96 hours, or at least 120 hours
post-administration, and/or (ii) porphobilinogen (PBG) to less than
about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mmol
PBG/mol creatinine for at least 24 hours, at least 48 hours, at
least 72 hours, at least 96 hours, or at least 120 hours
post-administration.
[1668] The term "determining the level" of a biomarker as used
herein can mean methods which include quantifying an amount of at
least one substance in a sample from a subject, for example, in a
bodily fluid from the subject (e.g., serum, plasma, urine, blood,
lymph, fecal, etc.) or in a tissue of the subject (e.g., liver,
heart, spleen kidney, etc.).
[1669] The term "reference level" as used herein can refer to
levels (e.g., of a biomarker) in a subject prior to administration
of an mRNA therapy of the invention (e.g., in a person suffering
from AIP) or in a normal or healthy subject.
[1670] As used herein, the term "normal subject" or "healthy
subject" refers to a subject not suffering from symptoms associated
with AIP. Moreover, a subject will be considered to be normal (or
healthy) if it has no mutation of the functional portions or
domains of the porphobilinogen deaminase (PBGD) gene (also referred
to as hydroxymethylbilane synthase (HMBS) and uroporphyrinogen I
synthase) and/or no mutation of the PBGD gene resulting in a
reduction of or deficiency of the enzyme PBGD (also known as
uroporphyrinogen I synthase or hydroxymethylbilane synthase) or the
activity thereof, resulting in symptoms associated with AIP. Said
mutations will be detected if a sample from the subject is
subjected to a genetic testing for such PBGD mutations. In
exemplary embodiments of the present invention, a sample from a
healthy subject is used as a control sample, or the known or
standardized value for the level of biomarker from samples of
healthy or normal subjects is used as a control.
[1671] In some embodiments, comparing the level of the biomarker in
a sample from a subject in need of treatment for AIP and/or
prevention of an acute porphyria attack, or in a subject being
treated for AIP to a control level of the biomarker comprises
comparing the level of the biomarker in the sample from the subject
(in need of treatment or being treated for AIP) to a baseline or
reference level, wherein if a level of the biomarker in the sample
from the subject (in need of treatment or being treated for AIP) is
elevated, increased or higher compared to the baseline or reference
level, this is indicative that the subject is suffering from AIP
and/or is in need of treatment; and/or wherein if a level of the
biomarker in the sample from the subject (in need of treatment or
being treated for AIP) is decreased or lower compared to the
baseline level this is indicative that the subject is not suffering
from, is successfully being treated for AIP, or is not in need of
treatment for AIP. The stronger the reduction (e.g., at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least
6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at
least 20-fold, at least-30 fold, at least 40-fold, at least 50-fold
reduction and/or at least 10%, at least 20%, at least 30% at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, at least 95%, at least 98%, at least 99%, or at least
100% reduction) of the level of a biomarker, e.g., aminolevulinate
acid (ALA), porphobilinogen (PBG), porphyrin, transaminases (e.g.,
alanine transaminase (ALT) or aspartate transaminase (AST)) and/or
bilirubin, within a certain time period, e.g., within 6 hours,
within 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72
hours, and/or for a certain duration of time, e.g., 48 hours, 72
hours, 96 hours, 120 hours, 144 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7
months, 8 months, 9 months, 10 months, 11 months, 12 months, 18
months, 24 months, etc. the more successful is a therapy, such as
for example an mRNA therapy of the invention (e.g., a single dose
or a multiple regimen).
[1672] A reduction of at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least 95%, at
least 98%, at least 99%, at least 100% or more of the level of
biomarker, in particular, in bodily fluid (e.g., plasma, serum,
urine, e.g., urinary sediment) or in tissue(s) in a subject (e.g.,
liver, heart, spleen, kidney, brain or lung), for example a ALA,
PBG, porphyrin, a transaminases (e.g., ALT or AST), and/or serum
bilirubin, within 1, 2, 3, 4, 5, 6 or more days following
administration is indicative of a dose suitable for successful
treatment AIP, wherein reduction as used herein, preferably means
that the level of biomarker determined at the end of a specified
time period (e.g., post-administration, for example, of a single
intravenous dose) is compared to the level of the same biomarker
determined at the beginning of said time period (e.g.,
pre-administration of said dose). Exemplary time periods include
12, 24, 48, 72, 96, 120 or 144 hours post administration, in
particular 24, 48, 72 or 96 hours post administration.
[1673] A sustained reduction in substrate levels (e.g., biomarkers
such as ALA, PBG, porphyrin, a transaminases (e.g., ALT or AST),
and/or serum bilirubin) is particularly indicative of mRNA
therapeutic dosing and/or administration regimens successful for
treatment of AIP. Such sustained reduction can be referred to
herein as "duration" of effect. In exemplary embodiments, a
reduction of at least about 40%, at least about 50%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, or at
least about 95% at least about 96%, at least about 97%, at least
about 98%, at least about 99%, at least about 100% or more of the
level of biomarker, in particular, in a bodily fluid (e.g., plasma,
serum, urine, e.g., urinary sediment) or in tissue(s) in a subject
(e.g., liver, heart, spleen, kidney, brain or lung), for example
ALA, PBG, porphyrin, a transaminases (e.g., ALT or AST), and/or
serum bilirubin, within 1, 2, 3, 4, 5, 6, 7, 8 or more days
following administration is indicative of a successful therapeutic
approach. In exemplary embodiments, sustained reduction in
substrate (e.g., biomarker) levels in one or more samples (e.g.,
fluids and/or tissues) is preferred. For example, mRNA therapies
resulting in sustained reduction in ALA, PBG, porphyrin, a
transaminases (e.g., ALT or AST), and/or serum bilirubin (as
defined herein), optionally in combination with sustained reduction
of said biomarker in at least one tissue, preferably two, three,
four, five or more tissues, is indicative of successful
treatment.
[1674] In some embodiments, a single dose of an mRNA therapy of the
invention is about 0.2 to about 0.8 mpk. about 0.3 to about 0.7
mpk, about 0.4 to about 0.8 mpk, or about 0.5 mpk. In another
embodiment, a single dose of an mRNA therapy of the invention is
less than 1.5 mpk, less than 1.25 mpk, less than 1 mpk, or less
than 0.75 mpk. 26. Compositions and Formulations for Use
[1675] Certain aspects of the invention are directed to
compositions or formulations comprising any of the polynucleotides
disclosed above.
[1676] In some embodiments, the composition or formulation
comprises:
[1677] (i) a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising
a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a
PBGD polypeptide (e.g., the wild-type sequence, functional
fragment, or variant thereof), wherein the polynucleotide comprises
at least one chemically modified nucleobase, e.g., 5-methoxyuracil
(e.g., wherein at least about 25%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, at
least about 99%, or 100% of the uracils are 5-methoxyuracils), and
wherein the polynucleotide further comprises a miRNA binding site,
e.g., a miRNA binding site that binds to miR-142 (e.g., a
miR-142-3p or miR-142-5p binding site) and/or a miRNA binding site
that binds to miR-126 (e.g., a miR-126-3p or miR-126-5p binding
site); and (ii) a delivery agent comprising, e.g., a compound
having the Formula (I), e.g., any of Compounds 1-232, e.g.,
Compound 18; a compound having the Formula (III), (IV), (V), or
(VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a
compound having the Formula (VIII), e.g., any of Compounds 419-428,
e.g., Compound 428, or any combination thereof.
[1678] In some embodiments, the uracil or thymine content of the
ORF relative to the theoretical minimum uracil or thymine content
of a nucleotide sequence encoding the PBGD polypeptide (% U.sub.TM
or % T.sub.TM), is between about 100% and about 150%.
[1679] In some embodiments, the polynucleotides, compositions or
formulations above are used to treat and/or prevent a PBGD-related
diseases, disorders or conditions, e.g., AIP.
27. FORMS OF ADMINISTRATION
[1680] The polynucleotides, pharmaceutical compositions and
formulations of the invention described above can be administered
by any route that results in a therapeutically effective outcome.
These include, but are not limited to enteral (into the intestine),
gastroenteral, epidural (into the dura matter), oral (by way of the
mouth), transdermal, peridural, intracerebral (into the cerebrum),
intracerebroventricular (into the cerebral ventricles),
epicutaneous (application onto the skin), intradermal, (into the
skin itself), subcutaneous (under the skin), nasal administration
(through the nose), intravenous (into a vein), intravenous bolus,
intravenous drip, intraarterial (into an artery), intramuscular
(into a muscle), intracardiac (into the heart), intraosseous
infusion (into the bone marrow), intrathecal (into the spinal
canal), intraperitoneal, (infusion or injection into the
peritoneum), intravesical infusion, intravitreal, (through the
eye), intracavemous injection (into a pathologic cavity)
intracavitary (into the base of the penis), intravaginal
administration, intrauterine, extra-amniotic administration,
transdermal (diffusion through the intact skin for systemic
distribution), transmucosal (diffusion through a mucous membrane),
transvaginal, insufflation (snorting), sublingual, sublabial,
enema, eye drops (onto the conjunctiva), in ear drops, auricular
(in or by way of the ear), buccal (directed toward the cheek),
conjunctival, cutaneous, dental (to a tooth or teeth),
electro-osmosis, endocervical, endosinusial, endotracheal,
extracorporeal, hemodialysis, infiltration, interstitial,
intra-abdominal, intra-amniotic, intra-articular, intrabiliary,
intrabronchial, intrabursal, intracartilaginous (within a
cartilage), intracaudal (within the cauda equine), intracistemal
(within the cistema magna cerebellomedularis), intracomeal (within
the cornea), dental intracomal, intracoronary (within the coronary
arteries), intracorporus cavernosum (within the dilatable spaces of
the corporus cavemosa of the penis), intradiscal (within a disc),
intraductal (within a duct of a gland), intraduodenal (within the
duodenum), intradural (within or beneath the dura), intraepidermal
(to the epidermis), intraesophageal (to the esophagus),
intragastric (within the stomach), intragingival (within the
gingivae), intraileal (within the distal portion of the small
intestine), intralesional (within or introduced directly to a
localized lesion), intraluminal (within a lumen of a tube),
intralymphatic (within the lymph), intramedullary (within the
marrow cavity of a bone), intrameningeal (within the meninges),
intraocular (within the eye), intraovarian (within the ovary),
intrapericardial (within the pericardium), intrapleural (within the
pleura), intraprostatic (within the prostate gland), intrapulmonary
(within the lungs or its bronchi), intrasinal (within the nasal or
periorbital sinuses), intraspinal (within the vertebral column),
intrasynovial (within the synovial cavity of a joint),
intratendinous (within a tendon), intratesticular (within the
testicle), intrathecal (within the cerebrospinal fluid at any level
of the cerebrospinal axis), intrathoracic (within the thorax),
intratubular (within the tubules of an organ), intratympanic
(within the aurus media), intravascular (within a vessel or
vessels), intraventricular (within a ventricle), iontophoresis (by
means of electric current where ions of soluble salts migrate into
the tissues of the body), irrigation (to bathe or flush open wounds
or body cavities), laryngeal (directly upon the larynx),
nasogastric (through the nose and into the stomach), occlusive
dressing technique (topical route administration that is then
covered by a dressing that occludes the area), ophthalmic (to the
external eye), oropharyngeal (directly to the mouth and pharynx),
parenteral, percutaneous, periarticular, peridural, perineural,
periodontal, rectal, respiratory (within the respiratory tract by
inhaling orally or nasally for local or systemic effect),
retrobulbar (behind the pons or behind the eyeball),
intramyocardial (entering the myocardium), soft tissue,
subarachnoid, subconjunctival, submucosal, topical, transplacental
(through or across the placenta), transtracheal (through the wall
of the trachea), transtympanic (across or through the tympanic
cavity), ureteral (to the ureter), urethral (to the urethra),
vaginal, caudal block, diagnostic, nerve block, biliary perfusion,
cardiac perfusion, photopheresis or spinal. In specific
embodiments, compositions can be administered in a way that allows
them cross the blood-brain barrier, vascular barrier, or other
epithelial barrier. In some embodiments, a formulation for a route
of administration can include at least one inactive ingredient.
[1681] The polynucleotides of the present invention (e.g., a
polynucleotide comprising a nucleotide sequence encoding a PBGD
polypeptide or a functional fragment or variant thereof) can be
delivered to a cell naked. As used herein in, "naked" refers to
delivering polynucleotides free from agents that promote
transfection. The naked polynucleotides can be delivered to the
cell using routes of administration known in the art and described
herein.
[1682] The polynucleotides of the present invention (e.g., a
polynucleotide comprising a nucleotide sequence encoding a PBGD
polypeptide or a functional fragment or variant thereof) can be
formulated, using the methods described herein. The formulations
can contain polynucleotides that can be modified and/or unmodified.
The formulations can further include, but are not limited to, cell
penetration agents, a pharmaceutically acceptable carrier, a
delivery agent, a bioerodible or biocompatible polymer, a solvent,
and a sustained-release delivery depot. The formulated
polynucleotides can be delivered to the cell using routes of
administration known in the art and described herein.
[1683] A pharmaceutical composition for parenteral administration
can comprise at least one inactive ingredient. Any or none of the
inactive ingredients used can have been approved by the US Food and
Drug Administration (FDA). A non-exhaustive list of inactive
ingredients for use in pharmaceutical compositions for parenteral
administration includes hydrochloric acid, mannitol, nitrogen,
sodium acetate, sodium chloride and sodium hydroxide.
[1684] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions can be formulated according to
the known art using suitable dispersing agents, wetting agents,
and/or suspending agents. Sterile injectable preparations can be
sterile injectable solutions, suspensions, and/or emulsions in
nontoxic parenterally acceptable diluents and/or solvents, for
example, as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution, U.S.P., and isotonic sodium chloride solution. Sterile,
fixed oils are conventionally employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. Fatty acids such as
oleic acid can be used in the preparation of injectables. The
sterile formulation can also comprise adjuvants such as local
anesthetics, preservatives and buffering agents.
[1685] Injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, and/or by
incorporating sterilizing agents in the form of sterile solid
compositions that can be dissolved or dispersed in sterile water or
other sterile injectable medium prior to use.
[1686] Injectable formulations can be for direct injection into a
region of a tissue, organ and/or subject. As a non-limiting
example, a tissue, organ and/or subject can be directly injected a
formulation by intramyocardial injection into the ischemic region.
(See, e.g., Zangi et al. Nature Biotechnology 2013; the contents of
which are herein incorporated by reference in its entirety).
[1687] In order to prolong the effect of an active ingredient, it
is often desirable to slow the absorption of the active ingredient
from subcutaneous or intramuscular injection. This can be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the drug then depends upon its rate of dissolution
which, in turn, can depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered
drug form is accomplished by dissolving or suspending the drug in
an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the drug in biodegradable polymers such
as polylactide-polyglycolide. Depending upon the ratio of drug to
polymer and the nature of the particular polymer employed, the rate
of drug release can be controlled. Examples of other biodegradable
polymers include poly(orthoesters) and poly(anhydrides). Depot
injectable formulations are prepared by entrapping the drug in
liposomes or microemulsions that are compatible with body
tissues.
28. KITS AND DEVICES
[1688] a. Kits
[1689] The invention provides a variety of kits for conveniently
and/or effectively using the claimed nucleotides of the present
invention. Typically kits will comprise sufficient amounts and/or
numbers of components to allow a user to perform multiple
treatments of a subject(s) and/or to perform multiple
experiments.
[1690] In one aspect, the present invention provides kits
comprising the molecules (polynucleotides) of the invention.
[1691] Said kits can be for protein production, comprising a first
polynucleotide comprising a translatable region. The kit can
further comprise packaging and instructions and/or a delivery agent
to form a formulation composition. The delivery agent can comprise
a saline, a buffered solution, a lipidoid or any delivery agent
disclosed herein.
[1692] In some embodiments, the buffer solution can include sodium
chloride, calcium chloride, phosphate and/or EDTA. In another
embodiment, the buffer solution can include, but is not limited to,
saline, saline with 2 mM calcium, 5% sucrose, 5% sucrose with 2 mM
calcium, 5% Mannitol, 5% Mannitol with 2 mM calcium, Ringer's
lactate, sodium chloride, sodium chloride with 2 mM calcium and
mannose (See, e.g., U.S. Pub. No. 20120258046; herein incorporated
by reference in its entirety). In a further embodiment, the buffer
solutions can be precipitated or it can be lyophilized. The amount
of each component can be varied to enable consistent, reproducible
higher concentration saline or simple buffer formulations. The
components can also be varied in order to increase the stability of
modified RNA in the buffer solution over a period of time and/or
under a variety of conditions. In one aspect, the present invention
provides kits for protein production, comprising: a polynucleotide
comprising a translatable region, provided in an amount effective
to produce a desired amount of a protein encoded by the
translatable region when introduced into a target cell; a second
polynucleotide comprising an inhibitory nucleic acid, provided in
an amount effective to substantially inhibit the innate immune
response of the cell; and packaging and instructions.
[1693] In one aspect, the present invention provides kits for
protein production, comprising a polynucleotide comprising a
translatable region, wherein the polynucleotide exhibits reduced
degradation by a cellular nuclease, and packaging and
instructions.
[1694] In one aspect, the present invention provides kits for
protein production, comprising a polynucleotide comprising a
translatable region, wherein the polynucleotide exhibits reduced
degradation by a cellular nuclease, and a mammalian cell suitable
for translation of the translatable region of the first nucleic
acid.
b. Devices
[1695] The present invention provides for devices that can
incorporate polynucleotides that encode polypeptides of interest.
These devices contain in a stable formulation the reagents to
synthesize a polynucleotide in a formulation available to be
immediately delivered to a subject in need thereof, such as a human
patient
[1696] Devices for administration can be employed to deliver the
polynucleotides of the present invention according to single,
multi- or split-dosing regimens taught herein. Such devices are
taught in, for example, International Application Publ. No.
WO2013151666, the contents of which are incorporated herein by
reference in their entirety.
[1697] Method and devices known in the art for multi-administration
to cells, organs and tissues are contemplated for use in
conjunction with the methods and compositions disclosed herein as
embodiments of the present invention. These include, for example,
those methods and devices having multiple needles, hybrid devices
employing for example lumens or catheters as well as devices
utilizing heat, electric current or radiation driven
mechanisms.
[1698] According to the present invention, these
multi-administration devices can be utilized to deliver the single,
multi- or split doses contemplated herein. Such devices are taught
for example in, International Application Publ. No. WO2013151666,
the contents of which are incorporated herein by reference in their
entirety.
[1699] In some embodiments, the polynucleotide is administered
subcutaneously or intramuscularly via at least 3 needles to three
different, optionally adjacent, sites simultaneously, or within a
60 minutes period (e.g., administration to 4, 5, 6, 7, 8, 9, or 10
sites simultaneously or within a 60 minute period).
c. Methods and Devices Utilizing Catheters and/or Lumens
[1700] Methods and devices using catheters and lumens can be
employed to administer the polynucleotides of the present invention
on a single, multi- or split dosing schedule. Such methods and
devices are described in International Application Publication No.
WO2013151666, the contents of which are incorporated herein by
reference in their entirety.
d. Methods and Devices Utilizing Electrical Current
[1701] Methods and devices utilizing electric current can be
employed to deliver the polynucleotides of the present invention
according to the single, multi- or split dosing regimens taught
herein. Such methods and devices are described in International
Application Publication No. WO2013151666, the contents of which are
incorporated herein by reference in their entirety.
29. DEFINITIONS
[1702] In order that the present disclosure can be more readily
understood, certain terms are first defined. As used in this
application, except as otherwise expressly provided herein, each of
the following terms shall have the meaning set forth below.
Additional definitions are set forth throughout the
application.
[1703] The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process.
[1704] In this specification and the appended claims, the singular
forms "a", "an" and "the" include plural referents unless the
context clearly dictates otherwise. The terms "a" (or "an"), as
well as the terms "one or more," and "at least one" can be used
interchangeably herein. In certain aspects, the term "a" or "an"
means "single." In other aspects, the term "a" or "an" includes
"two or more" or "multiple."
[1705] Furthermore, "and/or" where used herein is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. Thus, the term "and/or" as
used in a phrase such as "A and/or B" herein is intended to include
"A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the
term "and/or" as used in a phrase such as "A, B, and/or C" is
intended to encompass each of the following aspects: A, B, and C;
A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A
(alone); B (alone); and C (alone).
[1706] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[1707] Wherever aspects are described herein with the language
"comprising," otherwise analogous aspects described in terms of
"consisting of" and/or "consisting essentially of" are also
provided.
[1708] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Where a range of
values is recited, it is to be understood that each intervening
integer value, and each fraction thereof, between the recited upper
and lower limits of that range is also specifically disclosed,
along with each subrange between such values. The upper and lower
limits of any range can independently be included in or excluded
from the range, and each range where either, neither or both limits
are included is also encompassed within the invention. Where a
value is explicitly recited, it is to be understood that values
which are about the same quantity or amount as the recited value
are also within the scope of the invention. Where a combination is
disclosed, each subcombination of the elements of that combination
is also specifically disclosed and is within the scope of the
invention. Conversely, where different elements or groups of
elements are individually disclosed, combinations thereof are also
disclosed. Where any element of an invention is disclosed as having
a plurality of alternatives, examples of that invention in which
each alternative is excluded singly or in any combination with the
other alternatives are also hereby disclosed; more than one element
of an invention can have such exclusions, and all combinations of
elements having such exclusions are hereby disclosed.
[1709] Nucleotides are referred to by their commonly accepted
single-letter codes. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation. Nucleobases are
referred to herein by their commonly known one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Accordingly, A represents adenine, C represents cytosine, G
represents guanine, T represents thymine, U represents uracil.
[1710] Amino acids are referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Unless
otherwise indicated, amino acid sequences are written left to right
in amino to carboxy orientation.
[1711] About: The term "about" as used in connection with a
numerical value throughout the specification and the claims denotes
an interval of accuracy, familiar and acceptable to a person
skilled in the art. Such interval of accuracy is +10%.
[1712] Where ranges are given, endpoints are included. Furthermore,
unless otherwise indicated or otherwise evident from the context
and understanding of one of ordinary skill in the art, values that
are expressed as ranges can assume any specific value or subrange
within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless
the context clearly dictates otherwise.
[1713] Administered in combination: As used herein, the term
"administered in combination" or "combined administration" means
that two or more agents are administered to a subject at the same
time or within an interval such that there can be an overlap of an
effect of each agent on the patient. In some embodiments, they are
administered within about 60, 30, 15, 10, 5, or 1 minute of one
another. In some embodiments, the administrations of the agents are
spaced sufficiently closely together such that a combinatorial
(e.g., a synergistic) effect is achieved.
[1714] Amino acid substitution: The term "amino acid substitution"
refers to replacing an amino acid residue present in a parent or
reference sequence (e.g., a wild type PBGD sequence) with another
amino acid residue. An amino acid can be substituted in a parent or
reference sequence (e.g., a wild type PBGD polypeptide sequence),
for example, via chemical peptide synthesis or through recombinant
methods known in the art. Accordingly, a reference to a
"substitution at position X" refers to the substitution of an amino
acid present at position X with an alternative amino acid residue.
In some aspects, substitution patterns can be described according
to the schema AnY, wherein A is the single letter code
corresponding to the amino acid naturally or originally present at
position n, and Y is the substituting amino acid residue. In other
aspects, substitution patterns can be described according to the
schema An(YZ), wherein A is the single letter code corresponding to
the amino acid residue substituting the amino acid naturally or
originally present at position X, and Y and Z are alternative
substituting amino acid residue, i.e., and Y and Z are alternative
substituting amino acid residues.
[1715] In the context of the present disclosure, substitutions
(even when they referred to as amino acid substitution) are
conducted at the nucleic acid level, i.e., substituting an amino
acid residue with an alternative amino acid residue is conducted by
substituting the codon encoding the first amino acid with a codon
encoding the second amino acid.
[1716] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans at any stage of development. In some embodiments,
"animal" refers to non-human animals at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, or a pig). In some embodiments, animals include,
but are not limited to, mammals, birds, reptiles, amphibians, fish,
and worms. In some embodiments, the animal is a transgenic animal,
genetically-engineered animal, or a clone.
[1717] Approximately: As used herein, the term "approximately," as
applied to one or more values of interest, refers to a value that
is similar to a stated reference value. In certain embodiments, the
term "approximately" refers to a range of values that fall within
25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater
than or less than) of the stated reference value unless otherwise
stated or otherwise evident from the context (except where such
number would exceed 100% of a possible value).
[1718] Associated with: As used herein with respect to a disease,
the term "associated with" means that the symptom, measurement,
characteristic, or status in question is linked to the diagnosis,
development, presence, or progression of that disease. As
association can, but need not, be causatively linked to the
disease. For example, signs, symptoms, sequelae, or any effects
causing a decrease in the quality of life of a patient of AIP are
considered associated with AIP and in some embodiments of the
present invention can be treated, ameliorated, or prevented by
administering the polynucleotides of the present invention to a
subject in need thereof.
[1719] When used with respect to two or more moieties, the terms
"associated with," "conjugated," "linked," "attached," and
"tethered," when used with respect to two or more moieties, means
that the moieties are physically associated or connected with one
another, either directly or via one or more additional moieties
that serves as a linking agent, to form a structure that is
sufficiently stable so that the moieties remain physically
associated under the conditions in which the structure is used,
e.g., physiological conditions. An "association" need not be
strictly through direct covalent chemical bonding. It can also
suggest ionic or hydrogen bonding or a hybridization based
connectivity sufficiently stable such that the "associated"
entities remain physically associated.
[1720] Bifunctional: As used herein, the term "bifunctional" refers
to any substance, molecule or moiety that is capable of or
maintains at least two functions. The functions can affect the same
outcome or a different outcome. The structure that produces the
function can be the same or different. For example, bifunctional
modified RNAs of the present invention can encode a PBGD peptide (a
first function) while those nucleosides that comprise the encoding
RNA are, in and of themselves, capable of extending the half-life
of the RNA (second function). In this example, delivery of the
bifunctional modified RNA to a subject suffereing from a protein
defficiency would produce not only a peptide or protein molecule
that can ameliorate or treat a disease or conditions, but would
also maintain a population modified RNA present in the subject for
a prolonged period of time. In other aspects, a bifunction modified
mRNA can be a chimeric or quimeric molecule comprising, for
example, an RNA encoding a PBGD peptide (a first function) and a
second protein either fused to first protein or co-expressed with
the first protein, for example, and ApoA1 protein.
[1721] Biocompatible: As used herein, the term "biocompatible"
means compatible with living cells, tissues, organs or systems
posing little to no risk of injury, toxicity or rejection by the
immune system.
[1722] Biodegradable: As used herein, the term "biodegradable"
means capable of being broken down into innocuous products by the
action of living things.
[1723] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any substance
that has activity in a biological system and/or organism. For
instance, a substance that, when administered to an organism, has a
biological effect on that organism, is considered to be
biologically active. In particular embodiments, a polynucleotide of
the present invention can be considered biologically active if even
a portion of the polynucleotide is biologically active or mimics an
activity considered biologically relevant.
[1724] Chimera: As used herein, "chimera" is an entity having two
or more incongruous or heterogeneous parts or regions. For example,
a chimeric molecule can comprise a first part comprising a PBGD
polypeptide, and a second part (e.g., genetically fused to the
first part) comprising a second therapeutic protein (e.g., a
protein with a distinct enzymatic activity, an antigen binding
moiety, or a moiety capable of extending the plasma half life of
PBGD, for example, an Fc region of an antibody).
[1725] Sequence Optimization: The term "sequence optimization"
refers to a process or series of processes by which nucleobases in
a reference nucleic acid sequence are replaced with alternative
nucleobases, resulting in a nucleic acid sequence with improved
properties, e.g., improved protein expression or decreased
immunogenicity.
[1726] In general, the goal in sequence optimization is to produce
a synonymous nucleotide sequence than encodes the same polypeptide
sequence encoded by the reference nucleotide sequence. Thus, there
are no amino acid substitutions (as a result of codon optimization)
in the polypeptide encoded by the codon optimized nucleotide
sequence with respect to the polypeptide encoded by the reference
nucleotide sequence.
[1727] Codon substitution: The terms "codon substitution" or "codon
replacement" in the context of sequence optimization refer to
replacing a codon present in a reference nucleic acid sequence with
another codon. A codon can be substituted in a reference nucleic
acid sequence, for example, via chemical peptide synthesis or
through recombinant methods known in the art. Accordingly,
references to a "substitution" or "replacement" at a certain
location in a nucleic acid sequence (e.g., an mRNA) or within a
certain region or subsequence of a nucleic acid sequence (e.g., an
mRNA) refer to the substitution of a codon at such location or
region with an alternative codon.
[1728] As used herein, the terms "coding region" and "region
encoding" and grammatical variants thereof, refer to an Open
Reading Frame (ORF) in a polynucleotide that upon expression yields
a polypeptide or protein.
[1729] Compound: As used herein, the term "compound," is meant to
include all stereoisomers and isotopes of the structure depicted.
As used herein, the term "stereoisomer" means any geometric isomer
(e.g., cis- and trans-isomer), enantiomer, or diastereomer of a
compound. The present disclosure encompasses any and all
stereoisomers of the compounds described herein, including
stereomerically pure forms (e.g., geometrically pure,
enantiomerically pure, or diastereomerically pure) and enantiomeric
and stereoisomeric mixtures, e.g., racemates. Enantiomeric and
stereomeric mixtures of compounds and means of resolving them into
their component enantiomers or stereoisomers are well-known.
"Isotopes" refers to atoms having the same atomic number but
different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium. Further, a compound, salt, or complex of the
present disclosure can be prepared in combination with solvent or
water molecules to form solvates and hydrates by routine
methods.
[1730] Contacting: As used herein, the term "contacting" means
establishing a physical connection between two or more entities.
For example, contacting a mammalian cell with a nanoparticle
composition means that the mammalian cell and a nanoparticle are
made to share a physical connection. Methods of contacting cells
with external entities both in vivo and ex vivo are well known in
the biological arts. For example, contacting a nanoparticle
composition and a mammalian cell disposed within a mammal can be
performed by varied routes of administration (e.g., intravenous,
intramuscular, intradermal, and subcutaneous) and can involve
varied amounts of nanoparticle compositions. Moreover, more than
one mammalian cell can be contacted by a nanoparticle
composition.
[1731] Conservative amino acid substitution: A "conservative amino
acid substitution" is one in which the amino acid residue in a
protein sequence is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art, including basic side
chains (e.g., lysine, arginine, or histidine), acidic side chains
(e.g., aspartic acid or glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
or cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, or tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or
histidine). Thus, if an amino acid in a polypeptide is replaced
with another amino acid from the same side chain family, the amino
acid substitution is considered to be conservative. In another
aspect, a string of amino acids can be conservatively replaced with
a structurally similar string that differs in order and/or
composition of side chain family members.
[1732] Non-conservative amino acid substitution: Non-conservative
amino acid substitutions include those in which (i) a residue
having an electropositive side chain (e.g., Arg, His or Lys) is
substituted for, or by, an electronegative residue (e.g., Glu or
Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted
for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or
Val), (iii) a cysteine or proline is substituted for, or by, any
other residue, or (iv) a residue having a bulky hydrophobic or
aromatic side chain (e.g., Val, His, Ile or Trp) is substituted
for, or by, one having a smaller side chain (e.g., Ala or Ser) or
no side chain (e.g., Gly).
[1733] Other amino acid substitutions can be readily identified by
workers of ordinary skill. For example, for the amino acid alanine,
a substitution can be taken from any one of D-alanine, glycine,
beta-alanine, L-cysteine and D-cysteine. For lysine, a replacement
can be any one of D-lysine, arginine, D-arginine, homo-arginine,
methionine, D-methionine, omithine, or D-omithine. Generally,
substitutions in functionally important regions that can be
expected to induce changes in the properties of isolated
polypeptides are those in which (i) a polar residue, e.g., serine
or threonine, is substituted for (or by) a hydrophobic residue,
e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a
cysteine residue is substituted for (or by) any other residue;
(iii) a residue having an electropositive side chain, e.g., lysine,
arginine or histidine, is substituted for (or by) a residue having
an electronegative side chain, e.g., glutamic acid or aspartic
acid; or (iv) a residue having a bulky side chain, e.g.,
phenylalanine, is substituted for (or by) one not having such a
side chain, e.g., glycine. The likelihood that one of the foregoing
non-conservative substitutions can alter functional properties of
the protein is also correlated to the position of the substitution
with respect to functionally important regions of the protein: some
non-conservative substitutions can accordingly have little or no
effect on biological properties.
[1734] Conserved: As used herein, the term "conserved" refers to
nucleotides or amino acid residues of a polynucleotide sequence or
polypeptide sequence, respectively, that are those that occur
unaltered in the same position of two or more sequences being
compared. Nucleotides or amino acids that are relatively conserved
are those that are conserved amongst more related sequences than
nucleotides or amino acids appearing elsewhere in the
sequences.
[1735] In some embodiments, two or more sequences are said to be
"completely conserved" if they are 100% identical to one another.
In some embodiments, two or more sequences are said to be "highly
conserved" if they are at least 70% identical, at least 80%
identical, at least 90% identical, or at least 95% identical to one
another. In some embodiments, two or more sequences are said to be
"highly conserved" if they are about 70% identical, about 80%
identical, about 90% identical, about 95%, about 98%, or about 99%
identical to one another. In some embodiments, two or more
sequences are said to be "conserved" if they are at least 30%
identical, at least 40% identical, at least 50% identical, at least
60% identical, at least 70% identical, at least 80% identical, at
least 90% identical, or at least 95% identical to one another. In
some embodiments, two or more sequences are said to be "conserved"
if they are about 30% identical, about 40% identical, about 50%
identical, about 60% identical, about 70% identical, about 80%
identical, about 90% identical, about 95% identical, about 98%
identical, or about 99% identical to one another. Conservation of
sequence can apply to the entire length of an polynucleotide or
polypeptide or can apply to a portion, region or feature
thereof.
[1736] Controlled Release: As used herein, the term "controlled
release" refers to a pharmaceutical composition or compound release
profile that conforms to a particular pattern of release to effect
a therapeutic outcome.
[1737] Cyclic or Cyclized: As used herein, the term "cyclic" refers
to the presence of a continuous loop. Cyclic molecules need not be
circular, only joined to form an unbroken chain of subunits. Cyclic
molecules such as the engineered RNA or mRNA of the present
invention can be single units or multimers or comprise one or more
components of a complex or higher order structure.
[1738] Cytotoxic: As used herein, "cytotoxic" refers to killing or
causing injurious, toxic, or deadly effect on a cell (e.g., a
mammalian cell (e.g., a human cell)), bacterium, virus, fungus,
protozoan, parasite, prion, or a combination thereof.
[1739] Delivering: As used herein, the term "delivering" means
providing an entity to a destination. For example, delivering a
polynucleotide to a subject can involve administering a
nanoparticle composition including the polynucleotide to the
subject (e.g., by an intravenous, intramuscular, intradermal, or
subcutaneous route). Administration of a nanoparticle composition
to a mammal or mammalian cell can involve contacting one or more
cells with the nanoparticle composition.
[1740] Delivery Agent: As used herein, "delivery agent" refers to
any substance that facilitates, at least in part, the in vivo, in
vitro, or ex vivo delivery of a polynucleotide to targeted
cells.
[1741] Destabilized: As used herein, the term "destable,"
"destabilize," or "destabilizing region" means a region or molecule
that is less stable than a starting, wild-type or native form of
the same region or molecule.
[1742] Diastereomer: As used herein, the term "diastereomer," means
stereoisomers that are not mirror images of one another and are
non-superimposable on one another.
[1743] Digest: As used herein, the term "digest" means to break
apart into smaller pieces or components. When referring to
polypeptides or proteins, digestion results in the production of
peptides.
[1744] Distal: As used herein, the term "distal" means situated
away from the center or away from a point or region of
interest.
[1745] Domain: As used herein, when referring to polypeptides, the
term "domain" refers to a motif of a polypeptide having one or more
identifiable structural or functional characteristics or properties
(e.g., binding capacity, serving as a site for protein-protein
interactions).
[1746] Dosing regimen: As used herein, a "dosing regimen" or a
"dosing regimen" is a schedule of administration or physician
determined regimen of treatment, prophylaxis, or palliative
care.
[1747] Effective Amount: As used herein, the term "effective
amount" of an agent is that amount sufficient to effect beneficial
or desired results, for example, clinical results, and, as such, an
"effective amount" depends upon the context in which it is being
applied. For example, in the context of administering an agent that
treats a protein deficiency (e.g., a PBGD deficiency), an effective
amount of an agent is, for example, an amount of mRNA expressing
sufficient PBGD to ameliorate, reduce, eliminate, or prevent the
signs and symptoms associated with the PBGD deficiency, as compared
to the severity of the symptom observed without administration of
the agent. The term "effective amount" can be used interchangeably
with "effective dose," "therapeutically effective amount," or
"therapeutically effective dose."
[1748] Enantiomer: As used herein, the term "enantiomer" means each
individual optically active form of a compound of the invention,
having an optical purity or enantiomeric excess (as determined by
methods standard in the art) of at least 80% (i.e., at least 90% of
one enantiomer and at most 10% of the other enantiomer), at least
90%, or at least 98%.
[1749] Encapsulate: As used herein, the term "encapsulate" means to
enclose, surround or encase.
[1750] Encapsulation Efficiency: As used herein, "encapsulation
efficiency" refers to the amount of a polynucleotide that becomes
part of a nanoparticle composition, relative to the initial total
amount of polynucleotide used in the preparation of a nanoparticle
composition. For example, if 97 mg of polynucleotide are
encapsulated in a nanoparticle composition out of a total 100 mg of
polynucleotide initially provided to the composition, the
encapsulation efficiency can be given as 97%. As used herein,
"encapsulation" can refer to complete, substantial, or partial
enclosure, confinement, surrounding, or encasement.
[1751] Encoded protein cleavage signal: As used herein, "encoded
protein cleavage signal" refers to the nucleotide sequence that
encodes a protein cleavage signal.
[1752] Engineered: As used herein, embodiments of the invention are
"engineered" when they are designed to have a feature or property,
whether structural or chemical, that varies from a starting point,
wild type or native molecule.
[1753] Enhanced Delivery: As used herein, the term "enhanced
delivery" means delivery of more (e.g., at least 1.5 fold more, at
least 2-fold more, at least 3-fold more, at least 4-fold more, at
least 5-fold more, at least 6-fold more, at least 7-fold more, at
least 8-fold more, at least 9-fold more, at least 10-fold more) of
a polynucleotide by a nanoparticle to a target tissue of interest
(e.g., mammalian liver) compared to the level of delivery of a
polynucleotide by a control nanoparticle to a target tissue of
interest (e.g., MC3, KC2, or DLinDMA). The level of delivery of a
nanoparticle to a particular tissue can be measured by comparing
the amount of protein produced in a tissue to the weight of said
tissue, comparing the amount of polynucleotide in a tissue to the
weight of said tissue, comparing the amount of protein produced in
a tissue to the amount of total protein in said tissue, or
comparing the amount of polynucleotide in a tissue to the amount of
total polynucleotide in said tissue. It will be understood that the
enhanced delivery of a nanoparticle to a target tissue need not be
determined in a subject being treated, it can be determined in a
surrogate such as an animal model (e.g., a rat model).
[1754] Exosome: As used herein, "exosome" is a vesicle secreted by
mammalian cells or a complex involved in RNA degradation.
[1755] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an mRNA template from a DNA sequence (e.g., by
transcription); (2) processing of an mRNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end processing); (3)
translation of an mRNA into a polypeptide or protein; and (4)
post-translational modification of a polypeptide or protein.
[1756] Ex Vivo: As used herein, the term "ex vivo" refers to events
that occur outside of an organism (e.g., animal, plant, or microbe
or cell or tissue thereof). Ex vivo events can take place in an
environment minimally altered from a natural (e.g., in vivo)
environment.
[1757] Feature: As used herein, a "feature" refers to a
characteristic, a property, or a distinctive element. When
referring to polypeptides, "features" are defined as distinct amino
acid sequence-based components of a molecule. Features of the
polypeptides encoded by the polynucleotides of the present
invention include surface manifestations, local conformational
shape, folds, loops, half-loops, domains, half-domains, sites,
termini or any combination thereof.
[1758] Formulation: As used herein, a "formulation" includes at
least a polynucleotide and one or more of a carrier, an excipient,
and a delivery agent.
[1759] Fragment: A "fragment," as used herein, refers to a portion.
For example, fragments of proteins can comprise polypeptides
obtained by digesting full-length protein isolated from cultured
cells. In some embodiments, a fragment is a subsequences of a full
length protein (e.g., PBGD) wherein N-terminal, and/or C-terminal,
and/or internal subsequences have been deleted. In some preferred
aspects of the present invention, the fragments of a protein of the
present invention are functional fragments.
[1760] Functional: As used herein, a "functional" biological
molecule is a biological molecule in a form in which it exhibits a
property and/or activity by which it is characterized. Thus, a
functional fragment of a polynucleotide of the present invention is
a polynucleotide capable of expressing a functional PBGD fragment.
As used herein, a functional fragment of PBGD refers to a fragment
of wild type PBGD (i.e., a fragment of any of its naturally
occurring isoforms), or a mutant or variant thereof, wherein the
fragment retains a least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85%, at least about 90%, or at least about 95% of the biological
activity of the corresponding full length protein.
[1761] Helper Lipid: As used herein, the term "helper lipid" refers
to a compound or molecule that includes a lipidic moiety (for
insertion into a lipid layer, e.g., lipid bilayer) and a polar
moiety (for interaction with physiologic solution at the surface of
the lipid layer). Typically the helper lipid is a phospholipid. A
function of the helper lipid is to "complement" the amino lipid and
increase the fusogenicity of the bilayer and/or to help facilitate
endosomal escape, e.g., of nucleic acid delivered to cells. Helper
lipids are also believed to be a key structural component to the
surface of the LNP.
[1762] Homology: As used herein, the term "homology" refers to the
overall relatedness between polymeric molecules, e.g. between
nucleic acid molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Generally, the term
"homology" implies an evolutionary relationship between two
molecules. Thus, two molecules that are homologous will have a
common evolutionary ancestor. In the context of the present
invention, the term homology encompasses both to identity and
similarity.
[1763] In some embodiments, polymeric molecules are considered to
be "homologous" to one another if at least 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the
monomers in the molecule are identical (exactly the same monomer)
or are similar (conservative substitutions). The term "homologous"
necessarily refers to a comparison between at least two sequences
(polynucleotide or polypeptide sequences).
[1764] Identity: As used herein, the term "identity" refers to the
overall monomer conservation between polymeric molecules, e.g.,
between polynucleotide molecules (e.g. DNA molecules and/or RNA
molecules) and/or between polypeptide molecules. Calculation of the
percent identity of two polynucleotide sequences, for example, can
be performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second nucleic acid sequences for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a sequence aligned
for comparison purposes is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, or 100% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which needs
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. When comparing DNA and RNA, thymine (T) and uracil (U)
can be considered equivalent.
[1765] Suitable software programs are available from various
sources, and for alignment of both protein and nucleotide
sequences. One suitable program to determine percent sequence
identity is bl2seq, part of the BLAST suite of program available
from the U.S. government's National Center for Biotechnology
Information BLAST web site (blast.ncbi.nlm.nih.gov). Bl2seq
performs a comparison between two sequences using either the BLASTN
or BLASTP algorithm. BLASTN is used to compare nucleic acid
sequences, while BLASTP is used to compare amino acid sequences.
Other suitable programs are, e.g., Needle, Stretcher, Water, or
Matcher, part of the EMBOSS suite of bioinformatics programs and
also available from the European Bioinformatics Institute (EBI) at
www.ebi.ac.uk/Tools/psa.
[1766] Sequence alignments can be conducted using methods known in
the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal
Omega), MUSCLE, etc.
[1767] Different regions within a single polynucleotide or
polypeptide target sequence that aligns with a polynucleotide or
polypeptide reference sequence can each have their own percent
sequence identity. It is noted that the percent sequence identity
value is rounded to the nearest tenth. For example, 80.11, 80.12,
80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16,
80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted
that the length value will always be an integer.
[1768] In certain aspects, the percentage identity "% ID" of a
first amino acid sequence (or nucleic acid sequence) to a second
amino acid sequence (or nucleic acid sequence) is calculated as %
ID=100.times.(Y/Z), where Y is the number of amino acid residues
(or nucleobases) scored as identical matches in the alignment of
the first and second sequences (as aligned by visual inspection or
a particular sequence alignment program) and Z is the total number
of residues in the second sequence. If the length of a first
sequence is longer than the second sequence, the percent identity
of the first sequence to the second sequence will be higher than
the percent identity of the second sequence to the first
sequence.
[1769] One skilled in the art will appreciate that the generation
of a sequence alignment for the calculation of a percent sequence
identity is not limited to binary sequence-sequence comparisons
exclusively driven by primary sequence data. It will also be
appreciated that sequence alignments can be generated by
integrating sequence data with data from heterogeneous sources such
as structural data (e.g., crystallographic protein structures),
functional data (e.g., location of mutations), or phylogenetic
data. A suitable program that integrates heterogeneous data to
generate a multiple sequence alignment is T-Coffee, available at
www.tcoffee.org, and alternatively available, e.g., from the EBI.
It will also be appreciated that the final alignment used to
calculate percent sequence identity can be curated either
automatically or manually.
[1770] Immune response: The term "immune response" refers to the
action of, for example, lymphocytes, antigen presenting cells,
phagocytic cells, granulocytes, and soluble macromolecules produced
by the above cells or the liver (including antibodies, cytokines,
and complement) that results in selective damage to, destruction
of, or elimination from the human body of invading pathogens, cells
or tissues infected with pathogens, cancerous cells, or, in cases
of autoimmunity or pathological inflammation, normal human cells or
tissues. In some cases, the administration of a nanoparticle
comprising a lipid component and an encapsulated therapeutic agent
can trigger an immune response, which can be caused by (i) the
encapsulated therapeutic agent (e.g., an mRNA), (ii) the expression
product of such encapsulated therapeutic agent (e.g., a polypeptide
encoded by the mRNA), (iii) the lipid component of the
nanoparticle, or (iv) a combination thereof.
[1771] Inflammatory response: "Inflammatory response" refers to
immune responses involving specific and non-specific defense
systems. A specific defense system reaction is a specific immune
system reaction to an antigen. Examples of specific defense system
reactions include antibody responses. A non-specific defense system
reaction is an inflammatory response mediated by leukocytes
generally incapable of immunological memory, e.g., macrophages,
eosinophils and neutrophils. In some aspects, an immune response
includes the secretion of inflammatory cytokines, resulting in
elevated inflammatory cytokine levels.
[1772] Inflammatory cytokines: The term "inflammatory cytokine"
refers to cytokines that are elevated in an inflammatory response.
Examples of inflammatory cytokines include interleukin-6 (IL-6),
CXCL1 (chemokine (C--X--C motif) ligand 1; also known as GROc,
interferon-.gamma. (IFN.gamma.), tumor necrosis factor .alpha.
(TNF.alpha.), interferon .gamma.-induced protein 10 (IP-10), or
granulocyte-colony stimulating factor (G-CSF). The term
inflammatory cytokines includes also other cytokines associated
with inflammatory responses known in the art, e.g., interleukin-1
(IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12),
interleukin-13 (11-13), interferon .alpha. (IFN-.alpha.), etc.
[1773] In Vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, in a Petri dish, etc.,
rather than within an organism (e.g., animal, plant, or
microbe).
[1774] In Vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant, or microbe or
cell or tissue thereof).
[1775] Insertional and deletional variants: "Insertional variants"
when referring to polypeptides are those with one or more amino
acids inserted immediately adjacent to an amino acid at a
particular position in a native or starting sequence. "Immediately
adjacent" to an amino acid means connected to either the
alpha-carboxy or alpha-amino functional group of the amino acid.
"Deletional variants" when referring to polypeptides are those with
one or more amino acids in the native or starting amino acid
sequence removed. Ordinarily, deletional variants will have one or
more amino acids deleted in a particular region of the
molecule.
[1776] Intact: As used herein, in the context of a polypeptide, the
term "intact" means retaining an amino acid corresponding to the
wild type protein, e.g., not mutating or substituting the wild type
amino acid. Conversely, in the context of a nucleic acid, the term
"intact" means retaining a nucleobase corresponding to the wild
type nucleic acid, e.g., not mutating or substituting the wild type
nucleobase.
[1777] Ionizable amino lipid: The term "ionizable amino lipid"
includes those lipids having one, two, three, or more fatty acid or
fatty alkyl chains and a pH-titratable amino head group (e.g., an
alkylamino or dialkylamino head group). An ionizable amino lipid is
typically protonated (i.e., positively charged) at a pH below the
pKa of the amino head group and is substantially not charged at a
pH above the pKa. Such ionizable amino lipids include, but are not
limited to DLin-MC3-DMA (MC3) and
(13Z,165Z)--N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine
(L608).
[1778] Isolated: As used herein, the term "isolated" refers to a
substance or entity that has been separated from at least some of
the components with which it was associated (whether in nature or
in an experimental setting). Isolated substances (e.g.,
polynucleotides or polypeptides) can have varying levels of purity
in reference to the substances from which they have been isolated.
Isolated substances and/or entities can be separated from at least
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, or more of the other components
with which they were initially associated. In some embodiments,
isolated substances are more than about 80%, about 85%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99%, or more than about 99% pure. As
used herein, a substance is "pure" if it is substantially free of
other components.
[1779] Substantially isolated: By "substantially isolated" is meant
that the compound is substantially separated from the environment
in which it was formed or detected. Partial separation can include,
for example, a composition enriched in the compound of the present
disclosure. Substantial separation can include compositions
containing at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, at
least about 97%, or at least about 99% by weight of the compound of
the present disclosure, or salt thereof.
[1780] A polynucleotide, vector, polypeptide, cell, or any
composition disclosed herein which is "isolated" is a
polynucleotide, vector, polypeptide, cell, or composition which is
in a form not found in nature. Isolated polynucleotides, vectors,
polypeptides, or compositions include those which have been
purified to a degree that they are no longer in a form in which
they are found in nature. In some aspects, a polynucleotide,
vector, polypeptide, or composition which is isolated is
substantially pure.
[1781] Isomer: As used herein, the term "isomer" means any
tautomer, stereoisomer, enantiomer, or diastereomer of any compound
of the invention. It is recognized that the compounds of the
invention can have one or more chiral centers and/or double bonds
and, therefore, exist as stereoisomers, such as double-bond isomers
(i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers
(i.e., (+) or (-)) or cis/trans isomers). According to the
invention, the chemical structures depicted herein, and therefore
the compounds of the invention, encompass all of the corresponding
stereoisomers, that is, both the stereomerically pure form (e.g.,
geometrically pure, enantiomerically pure, or diastereomerically
pure) and enantiomeric and stereoisomeric mixtures, e.g.,
racemates. Enantiomeric and stereoisomeric mixtures of compounds of
the invention can typically be resolved into their component
enantiomers or stereoisomers by well-known methods, such as
chiral-phase gas chromatography, chiral-phase high performance
liquid chromatography, crystallizing the compound as a chiral salt
complex, or crystallizing the compound in a chiral solvent.
Enantiomers and stereoisomers can also be obtained from
stereomerically or enantiomerically pure intermediates, reagents,
and catalysts by well-known asymmetric synthetic methods.
[1782] Linker: As used herein, a "linker" refers to a group of
atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or
groups such as, but not limited to, carbon, amino, alkylamino,
oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The
linker can be attached to a modified nucleoside or nucleotide on
the nucleobase or sugar moiety at a first end, and to a payload,
e.g., a detectable or therapeutic agent, at a second end. The
linker can be of sufficient length as to not interfere with
incorporation into a nucleic acid sequence. The linker can be used
for any useful purpose, such as to form polynucleotide multimers
(e.g., through linkage of two or more chimeric polynucleotides
molecules or IVT polynucleotides) or polynucleotides conjugates, as
well as to administer a payload, as described herein. Examples of
chemical groups that can be incorporated into the linker include,
but are not limited to, alkyl, alkenyl, alkynyl, amido, amino,
ether, thioether, ester, alkylene, heteroalkylene, aryl, or
heterocyclyl, each of which can be optionally substituted, as
described herein. Examples of linkers include, but are not limited
to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or
propylene glycol monomeric units, e.g., diethylene glycol,
dipropylene glycol, triethylene glycol, tripropylene glycol,
tetraethylene glycol, or tetraethylene glycol), and dextran
polymers and derivatives thereof. Other examples include, but are
not limited to, cleavable moieties within the linker, such as, for
example, a disulfide bond (--S--S--) or an azo bond (--N.dbd.N--),
which can be cleaved using a reducing agent or photolysis.
Non-limiting examples of a selectively cleavable bond include an
amido bond can be cleaved for example by the use of
tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,
and/or photolysis, as well as an ester bond can be cleaved for
example by acidic or basic hydrolysis.
[1783] Methods ofAdministration: As used herein, "methods of
administration" can include intravenous, intramuscular,
intradermal, subcutaneous, or other methods of delivering a
composition to a subject. A method of administration can be
selected to target delivery (e.g., to specifically deliver) to a
specific region or system of a body.
[1784] Modified: As used herein "modified" refers to a changed
state or structure of a molecule of the invention. Molecules can be
modified in many ways including chemically, structurally, and
functionally. In some embodiments, the mRNA molecules of the
present invention are modified by the introduction of non-natural
nucleosides and/or nucleotides, e.g., as it relates to the natural
ribonucleotides A, U, G, and C. Noncanonical nucleotides such as
the cap structures are not considered "modified" although they
differ from the chemical structure of the A, C, G, U
ribonucleotides.
[1785] Mucus: As used herein, "mucus" refers to the natural
substance that is viscous and comprises mucin glycoproteins.
[1786] Nanoparticle Composition: As used herein, a "nanoparticle
composition" is a composition comprising one or more lipids.
Nanoparticle compositions are typically sized on the order of
micrometers or smaller and can include a lipid bilayer.
Nanoparticle compositions encompass lipid nanoparticles (LNPs),
liposomes (e.g., lipid vesicles), and lipoplexes. For example, a
nanoparticle composition can be a liposome having a lipid bilayer
with a diameter of 500 nm or less.
[1787] Naturally occurring: As used herein, "naturally occurring"
means existing in nature without artificial aid.
[1788] Non-human vertebrate: As used herein, a "non-human
vertebrate" includes all vertebrates except Homo sapiens, including
wild and domesticated species. Examples of non-human vertebrates
include, but are not limited to, mammals, such as alpaca, banteng,
bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea
pig, horse, llama, mule, pig, rabbit, reindeer, sheep water
buffalo, and yak.
[1789] Nucleic acid sequence: The terms "nucleic acid sequence,"
"nucleotide sequence," or "polynucleotide sequence" are used
interchangeably and refer to a contiguous nucleic acid sequence.
The sequence can be either single stranded or double stranded DNA
or RNA, e.g., an mRNA.
[1790] The term "nucleic acid," in its broadest sense, includes any
compound and/or substance that comprises a polymer of nucleotides.
These polymers are often referred to as polynucleotides. Exemplary
nucleic acids or polynucleotides of the invention include, but are
not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids
(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),
peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including
LNA having a .beta.-D-ribo configuration, .alpha.-LNA having an
a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having
a 2'-amino functionalization, and 2'-amino-.alpha.-LNA having a
2'-amino functionalization), ethylene nucleic acids (ENA),
cyclohexenyl nucleic acids (CeNA) or hybrids or combinations
thereof.
[1791] The phrase "nucleotide sequence encoding" refers to the
nucleic acid (e.g., an mRNA or DNA molecule) coding sequence which
encodes a polypeptide. The coding sequence can further include
initiation and termination signals operably linked to regulatory
elements including a promoter and polyadenylation signal capable of
directing expression in the cells of an individual or mammal to
which the nucleic acid is administered. The coding sequence can
further include sequences that encode signal peptides.
[1792] Off-target: As used herein, "off target" refers to any
unintended effect on any one or more target, gene, or cellular
transcript.
[1793] Open reading frame: As used herein, "open reading frame" or
"ORF" refers to a sequence which does not contain a stop codon in a
given reading frame.
[1794] Operably linked: As used herein, the phrase "operably
linked" refers to a functional connection between two or more
molecules, constructs, transcripts, entities, moieties or the
like.
[1795] Optionally substituted: Herein a phrase of the form
"optionally substituted X" (e.g., optionally substituted alkyl) is
intended to be equivalent to "X, wherein X is optionally
substituted" (e.g., "alkyl, wherein said alkyl is optionally
substituted"). It is not intended to mean that the feature "X"
(e.g., alkyl) per se is optional.
[1796] Part: As used herein, a "part" or "region" of a
polynucleotide is defined as any portion of the polynucleotide that
is less than the entire length of the polynucleotide.
[1797] Patient: As used herein, "patient" refers to a subject who
can seek or be in need of treatment, requires treatment, is
receiving treatment, will receive treatment, or a subject who is
under care by a trained professional for a particular disease or
condition. For example, AIP patients can suffer from acute attacks
and can be treated during or after an acute attack (e.g., severe
pain). Patients having reoccurring attacks can be treated
"prophylactically", i.e., to reduce the risk of or prevent
recurring attacks.
[1798] PBGD Associated Disease: As use herein the terms
"PBGD-associated disease" or "PBGD-associated disorder" refer to
diseases or disorders, respectively, which result from aberrant
PBGD activity (e.g., decreased activity or increased activity). As
a non-limiting example, acute intermittent porphyria is a PBGD
associated disease. Numerous clinical variants of acute
intermittent porphyria are know in the art. See, e.g., www.omim.
org/entry/609806.
[1799] The terms "PBGD enzymatic activity," "PBGD activity," and
"porphobilinogen deaminase activity" are used interchangeably in
the present disclosure and refer to PBGD's ability to catalyze the
stepwise enzymatic condensation of 4 porphobilinogen units into
hydroxymethylbilane. Accordingly, a fragment or variant retaining
or having PBGD enzymatic activity or PBGD activity refers to a
fragment or variant that has measurable enzymatic catalyzing the
enzymatic condensation of porphobilinogen units into
hydroxymethylbilane.
[1800] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds,
materials, compositions, and/or dosage forms that are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[1801] Pharmaceutically acceptable excipients: The phrase
"pharmaceutically acceptable excipient," as used herein, refers any
ingredient other than the compounds described herein (for example,
a vehicle capable of suspending or dissolving the active compound)
and having the properties of being substantially nontoxic and
non-inflammatory in a patient. Excipients can include, for example:
antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes (colors), emollients, emulsifiers, fillers
(diluents), film formers or coatings, flavors, fragrances, glidants
(flow enhancers), lubricants, preservatives, printing inks,
sorbents, suspensing or dispersing agents, sweeteners, and waters
of hydration. Exemplary excipients include, but are not limited to:
butylated hydroxytoluene (BHT), calcium carbonate, calcium
phosphate (dibasic), calcium stearate, croscarmellose, crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,
ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, lactose, magnesium stearate, maltitol, mannitol,
methionine, methylcellulose, methyl paraben, microcrystalline
cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch glycolate, sorbitol, starch (corn), stearic acid,
sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
and xylitol.
[1802] Pharmaceutically acceptable salts: The present disclosure
also includes pharmaceutically acceptable salts of the compounds
described herein. As used herein, "pharmaceutically acceptable
salts" refers to derivatives of the disclosed compounds wherein the
parent compound is modified by converting an existing acid or base
moiety to its salt form (e.g., by reacting the free base group with
a suitable organic acid). Examples of pharmaceutically acceptable
salts include, but are not limited to, mineral or organic acid
salts of basic residues such as amines; alkali or organic salts of
acidic residues such as carboxylic acids; and the like.
Representative acid addition salts include acetate, acetic acid,
adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene
sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,
glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,
hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like, as well as
nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine, and the like. The pharmaceutically
acceptable salts of the present disclosure include the conventional
non-toxic salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. The pharmaceutically
acceptable salts of the present disclosure can be synthesized from
the parent compound that contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts can be
prepared by reacting the free acid or base forms of these compounds
with a stoichiometric amount of the appropriate base or acid in
water or in an organic solvent, or in a mixture of the two;
generally, nonaqueous media like ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile are used. Lists of suitable salts are
found in Remington's Pharmaceutical Sciences, 17.sup.th ed., Mack
Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical
Salts: Properties, Selection, and Use, P. H. Stahl and C. G.
Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of
Pharmaceutical Science, 66, 1-19 (1977), each of which is
incorporated herein by reference in its entirety.
[1803] Pharmaceutically acceptable solvate: The term
"pharmaceutically acceptable solvate," as used herein, means a
compound of the invention wherein molecules of a suitable solvent
are incorporated in the crystal lattice. A suitable solvent is
physiologically tolerable at the dosage administered. For example,
solvates can be prepared by crystallization, recrystallization, or
precipitation from a solution that includes organic solvents,
water, or a mixture thereof. Examples of suitable solvents are
ethanol, water (for example, mono-, di-, and tri-hydrates),
N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),
N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC),
1,3-dimethyl-2-imidazolidinone (DMEU),
1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU),
acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water
is the solvent, the solvate is referred to as a "hydrate."
[1804] Pharmacokinetic: As used herein, "pharmacokinetic" refers to
any one or more properties of a molecule or compound as it relates
to the determination of the fate of substances administered to a
living organism. Pharmacokinetics is divided into several areas
including the extent and rate of absorption, distribution,
metabolism and excretion. This is commonly referred to as ADME
where: (A) Absorption is the process of a substance entering the
blood circulation; (D) Distribution is the dispersion or
dissemination of substances throughout the fluids and tissues of
the body; (M) Metabolism (or Biotransformation) is the irreversible
transformation of parent compounds into daughter metabolites; and
(E) Excretion (or Elimination) refers to the elimination of the
substances from the body. In rare cases, some drugs irreversibly
accumulate in body tissue.
[1805] Physicochemical: As used herein, "physicochemical" means of
or relating to a physical and/or chemical property.
[1806] Polynucleotide: The term "polynucleotide" as used herein
refers to polymers of nucleotides of any length, including
ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures
thereof. This term refers to the primary structure of the molecule.
Thus, the term includes triple-, double- and single-stranded
deoxyribonucleic acid ("DNA"), as well as triple-, double- and
single-stranded ribonucleic acid ("RNA"). It also includes
modified, for example by alkylation, and/or by capping, and
unmodified forms of the polynucleotide. More particularly, the term
"polynucleotide" includes polydeoxyribonucleotides (containing
2-deoxy-D-ribose), polyribonucleotides (containing D-ribose),
including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or
unspliced, any other type of polynucleotide which is an N- or
C-glycoside of a purine or pyrimidine base, and other polymers
containing normucleotidic backbones, for example, polyamide (e.g.,
peptide nucleic acids "PNAs") and polymorpholino polymers, and
other synthetic sequence-specific nucleic acid polymers providing
that the polymers contain nucleobases in a configuration which
allows for base pairing and base stacking, such as is found in DNA
and RNA. In particular aspects, the polynucleotide comprises an
mRNA. In other aspect, the mRNA is a synthetic mRNA. In some
aspects, the synthetic mRNA comprises at least one unnatural
nucleobase. In some aspects, all nucleobases of a certain class
have been replaced with unnatural nucleobases (e.g., all uridines
in a polynucleotide disclosed herein can be replaced with an
unnatural nucleobase, e.g., 5-methoxyuridine). In some aspects, the
polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises
only natural nucleobases, i.e., A (adenosine), G (guanosine), C
(cytidine), and T (thymidine) in the case of a synthetic DNA, or A,
C, G, and U (uridine) in the case of a synthetic RNA.
[1807] The skilled artisan will appreciate that the T bases in the
codon maps disclosed herein are present in DNA, whereas the T bases
would be replaced by U bases in corresponding RNAs. For example, a
codon-nucleotide sequence disclosed herein in DNA form, e.g., a
vector or an in-vitro translation (IVT) template, would have its T
bases transcribed as U based in its corresponding transcribed mRNA.
In this respect, both codon-optimized DNA sequences (comprising T)
and their corresponding mRNA sequences (comprising U) are
considered codon-optimized nucleotide sequence of the present
invention. A skilled artisan would also understand that equivalent
codon-maps can be generated by replaced one or more bases with
non-natural bases. Thus, e.g., a TTC codon (DNA map) would
correspond to a UUC codon (RNA map), which in turn would correspond
to a WPC codon (RNA map in which U has been replaced with
pseudouridine).
[1808] Standard A-T and G-C base pairs form under conditions which
allow the formation of hydrogen bonds between the N3-H and C4-oxy
of thymidine and the N1 and C6-NH2, respectively, of adenosine and
between the C2-oxy, N3 and C4-NH2, of cytidine and the C2-NH2,
N'--H and C6-oxy, respectively, of guanosine. Thus, for example,
guanosine (2-amino-6-oxy-9-O-D-ribofuranosyl-purine) can be
modified to form isoguanosine
(2-oxy-6-amino-9-O-D-ribofuranosyl-purine). Such modification
results in a nucleoside base which will no longer effectively form
a standard base pair with cytosine. However, modification of
cytosine (1-3-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form
isocytosine (1-(3-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-)
results in a modified nucleotide which will not effectively base
pair with guanosine but will form a base pair with isoguanosine
(U.S. Pat. No. 5,681,702 to Collins et al.). Isocytosine is
available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can
be prepared by the method described by Switzer et al. (1993)
Biochemistry 32:10489-10496 and references cited therein;
2'-deoxy-5-methyl-isocytidine can be prepared by the method of Tor
et al., 1993, J. Am. Chem. Soc. 115:4461-4467 and references cited
therein; and isoguanine nucleotides can be prepared using the
method described by Switzer et al., 1993, supra, and Mantsch et
al., 1993, Biochem. 14:5593-5601, or by the method described in
U.S. Pat. No. 5,780,610 to Collins et al. Other nonnatural base
pairs can be synthesized by the method described in Piccirilli et
al., 1990, Nature 343:33-37, for the synthesis of
2,6-diaminopyrimidine and its complement
(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such
modified nucleotide units which form unique base pairs are known,
such as those described in Leach et al. (1992) J. Am. Chem. Soc.
114:3675-3683 and Switzer et al., supra.
[1809] Polypeptide: The terms "polypeptide," "peptide," and
"protein" are used interchangeably herein to refer to polymers of
amino acids of any length. The polymer can comprise modified amino
acids. The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids such as
homocysteine, omithine, p-acetylphenylalanine, D-amino acids, and
creatine), as well as other modifications known in the art.
[1810] The term, as used herein, refers to proteins, polypeptides,
and peptides of any size, structure, or function. Polypeptides
include encoded polynucleotide products, naturally occurring
polypeptides, synthetic polypeptides, homologs, orthologs,
paralogs, fragments and other equivalents, variants, and analogs of
the foregoing. A polypeptide can be a monomer or can be a
multi-molecular complex such as a dimer, trimer or tetramer. They
can also comprise single chain or multichain polypeptides. Most
commonly disulfide linkages are found in multichain polypeptides.
The term polypeptide can also apply to amino acid polymers in which
one or more amino acid residues are an artificial chemical analogue
of a corresponding naturally occurring amino acid. In some
embodiments, a "peptide" can be less than or equal to 50 amino
acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50
amino acids long.
[1811] Polypeptide variant: As used herein, the term "polypeptide
variant" refers to molecules that differ in their amino acid
sequence from a native or reference sequence. The amino acid
sequence variants can possess substitutions, deletions, and/or
insertions at certain positions within the amino acid sequence, as
compared to a native or reference sequence. Ordinarily, variants
will possess at least about 50% identity, at least about 60%
identity, at least about 70% identity, at least about 80% identity,
at least about 90% identity, at least about 95% identity, at least
about 99% identity to a native or reference sequence. In some
embodiments, they will be at least about 80%, or at least about 90%
identical to a native or reference sequence.
[1812] Polypeptide per unit drug (PUD): As used herein, a PUD or
product per unit drug, is defined as a subdivided portion of total
daily dose, usually 1 mg, pg, kg, etc., of a product (such as a
polypeptide) as measured in body fluid or tissue, usually defined
in concentration such as pmol/mL, mmol/mL, etc. divided by the
measure in the body fluid.
[1813] Preventing: As used herein, the term "preventing" refers to
partially or completely delaying onset of an infection, disease,
disorder and/or condition; partially or completely delaying onset
of one or more signs, symptoms, features, or clinical
manifestations of a particular infection, disease, disorder, and/or
condition; partially or completely delaying onset of one or more
signs, symptoms, features, or manifestations of a particular
infection, disease, disorder, and/or condition; partially or
completely delaying progression from an infection, a particular
disease, disorder and/or condition; and/or decreasing the risk of
developing pathology associated with the infection, the disease,
disorder, and/or condition.
[1814] Proliferate: As used herein, the term "proliferate" means to
grow, expand or increase or cause to grow, expand or increase
rapidly. "Proliferative" means having the ability to proliferate.
"Anti-proliferative" means having properties counter to or
inapposite to proliferative properties.
[1815] Prophylactic: As used herein, "prophylactic" refers to a
therapeutic or course of action used to prevent the spread of
disease.
[1816] Prophylaxis: As used herein, a "prophylaxis" refers to a
measure taken to maintain health and prevent the spread of disease.
An "immune prophylaxis" refers to a measure to produce active or
passive immunity to prevent the spread of disease.
[1817] Protein cleavage site: As used herein, "protein cleavage
site" refers to a site where controlled cleavage of the amino acid
chain can be accomplished by chemical, enzymatic or photochemical
means.
[1818] Protein cleavage signal: As used herein "protein cleavage
signal" refers to at least one amino acid that flags or marks a
polypeptide for cleavage.
[1819] Protein of interest: As used herein, the terms "proteins of
interest" or "desired proteins" include those provided herein and
fragments, mutants, variants, and alterations thereof.
[1820] Proximal: As used herein, the term "proximal" means situated
nearer to the center or to a point or region of interest.
[1821] Pseudouridine: As used herein, pseudouridine (w) refers to
the C-glycoside isomer of the nucleoside uridine. A "pseudouridine
analog" is any modification, variant, isoform or derivative of
pseudouridine. For example, pseudouridine analogs include but are
not limited to 1-carboxymethyl-pseudouridine,
1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine,
1-taurinomethyl-4-thio-pseudouridine, 1-methylpseudouridine
(m.sup.1.psi.), 1-methyl-4-thio-pseudouridine
(m.sup.1s.sup.4.psi.), 4-thio-1-methyl-pseudouridine,
3-methyl-pseudouridine (m.sup.3.psi.),
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,
4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,
1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp.sup.3
.psi.), and 2'-O-methyl-pseudouridine (.psi.n).
[1822] Purified: As used herein, "purify," "purified,"
"purification" means to make substantially pure or clear from
unwanted components, material defilement, admixture or
imperfection.
[1823] Reference Nucleic Acid Sequence: The term "reference nucleic
acid sequence" or "reference nucleic acid" or "reference nucleotide
sequence" or "reference sequence" refers to a starting nucleic acid
sequence (e.g., a RNA, e.g., an mRNA sequence) that can be sequence
optimized. In some embodiments, the reference nucleic acid sequence
is a wild type nucleic acid sequence, a fragment or a variant
thereof. In some embodiments, the reference nucleic acid sequence
is a previously sequence optimized nucleic acid sequence.
[1824] Salts: In some aspects, the pharmaceutical composition for
delivery disclosed herein and comprises salts of some of their
lipid constituents. The term "salt" includes any anionic and
cationic complex. Non-limiting examples of anions include inorganic
and organic anions, e.g., fluoride, chloride, bromide, iodide,
oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen
phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate,
nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate,
sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate,
benzoate, citrate, tartrate, lactate, acrylate, polyacrylate,
fumarate, maleate, itaconate, glycolate, gluconate, malate,
mandelate, tiglate, ascorbate, salicylate, polymethacrylate,
perchlorate, chlorate, chlorite, hypochlorite, bromate,
hypobromite, iodate, an alkylsulfonate, an arylsulfonate, arsenate,
arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,
hydroxide, peroxide, permanganate, and mixtures thereof.
[1825] Sample: As used herein, the term "sample" or "biological
sample" refers to a subset of its tissues, cells or component parts
(e.g., body fluids, including but not limited to blood, mucus,
lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva,
amniotic fluid, amniotic cord blood, urine, vaginal fluid and
semen). A sample further can include a homogenate, lysate or
extract prepared from a whole organism or a subset of its tissues,
cells or component parts, or a fraction or portion thereof,
including but not limited to, for example, plasma, serum, spinal
fluid, lymph fluid, the external sections of the skin, respiratory,
intestinal, and genitourinary tracts, tears, saliva, milk, blood
cells, tumors, or organs. A sample further refers to a medium, such
as a nutrient broth or gel, which can contain cellular components,
such as proteins or nucleic acid molecule.
[1826] Signal Sequence: As used herein, the phrases "signal
sequence," "signal peptide," and "transit peptide" are used
interchangeably and refer to a sequence that can direct the
transport or localization of a protein to a certain organelle, cell
compartment, or extracellular export. The term encompasses both the
signal sequence polypeptide and the nucleic acid sequence encoding
the signal sequence. Thus, references to a signal sequence in the
context of a nucleic acid refer in fact to the nucleic acid
sequence encoding the signal sequence polypeptide.
[1827] Signal transduction pathway: A "signal transduction pathway"
refers to the biochemical relationship between a variety of signal
transduction molecules that play a role in the transmission of a
signal from one portion of a cell to another portion of a cell. As
used herein, the phrase "cell surface receptor" includes, for
example, molecules and complexes of molecules capable of receiving
a signal and the transmission of such a signal across the plasma
membrane of a cell.
[1828] Similarity: As used herein, the term "similarity" refers to
the overall relatedness between polymeric molecules, e.g. between
polynucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of percent
similarity of polymeric molecules to one another can be performed
in the same manner as a calculation of percent identity, except
that calculation of percent similarity takes into account
conservative substitutions as is understood in the art.
[1829] Single unit dose: As used herein, a "single unit dose" is a
dose of any therapeutic administered in one dose/at one time/single
route/single point of contact, i.e., single administration
event.
[1830] Split dose: As used herein, a "split dose" is the division
of single unit dose or total daily dose into two or more doses.
[1831] Specific delivery: As used herein, the term "specific
delivery," "specifically deliver," or "specifically delivering"
means delivery of more (e.g., at least 1.5 fold more, at least
2-fold more, at least 3-fold more, at least 4-fold more, at least
5-fold more, at least 6-fold more, at least 7-fold more, at least
8-fold more, at least 9-fold more, at least 10-fold more) of a
polynucleotide by a nanoparticle to a target tissue of interest
(e.g., mammalian liver) compared to an off-target tissue (e.g.,
mammalian spleen). The level of delivery of a nanoparticle to a
particular tissue can be measured by comparing the amount of
protein produced in a tissue to the weight of said tissue,
comparing the amount of polynucleotide in a tissue to the weight of
said tissue, comparing the amount of protein produced in a tissue
to the amount of total protein in said tissue, or comparing the
amount of polynucleotide in a tissue to the amount of total
polynucleotide in said tissue. For example, for renovascular
targeting, a polynucleotide is specifically provided to a mammalian
kidney as compared to the liver and spleen if 1.5, 2-fold, 3-fold,
5-fold, 10-fold, 15 fold, or 20 fold more polynucleotide per 1 g of
tissue is delivered to a kidney compared to that delivered to the
liver or spleen following systemic administration of the
polynucleotide. It will be understood that the ability of a
nanoparticle to specifically deliver to a target tissue need not be
determined in a subject being treated, it can be determined in a
surrogate such as an animal model (e.g., a rat model).
[1832] Stable: As used herein "stable" refers to a compound that is
sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and in some cases capable of
formulation into an efficacious therapeutic agent.
[1833] Stabilized: As used herein, the term "stabilize,"
"stabilized," "stabilized region" means to make or become
stable.
[1834] Stereoisomer: As used herein, the term "stereoisomer" refers
to all possible different isomeric as well as conformational forms
that a compound can possess (e.g., a compound of any formula
described herein), in particular all possible stereochemically and
conformationally isomeric forms, all diastereomers, enantiomers
and/or conformers of the basic molecular structure. Some compounds
of the present invention can exist in different tautomeric forms,
all of the latter being included within the scope of the present
invention.
[1835] Subject: By "subject" or "individual" or "animal" or
"patient" or "mammal," is meant any subject, particularly a
mammalian subject, for whom diagnosis, prognosis, or therapy is
desired. Mammalian subjects include, but are not limited to,
humans, domestic animals, farm animals, zoo animals, sport animals,
pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice,
horses, cattle, cows; primates such as apes, monkeys, orangutans,
and chimpanzees; canids such as dogs and wolves; felids such as
cats, lions, and tigers; equids such as horses, donkeys, and
zebras; bears, food animals such as cows, pigs, and sheep;
ungulates such as deer and giraffes; rodents such as mice, rats,
hamsters and guinea pigs; and so on. In certain embodiments, the
mammal is a human subject. In other embodiments, a subject is a
human patient. In a particular embodiment, a subject is a human
patient in need of treatment.
[1836] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical characteristics rarely, if
ever, go to completion and/or proceed to completeness or achieve or
avoid an absolute result. The term "substantially" is therefore
used herein to capture the potential lack of completeness inherent
in many biological and chemical characteristics.
[1837] Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
[1838] Substantially simultaneous: As used herein and as it relates
to plurality of doses, the term means within 2 seconds.
[1839] Suffering from: An individual who is "suffering from" a
disease, disorder, and/or condition has been diagnosed with or
displays one or more signs or symptoms of the disease, disorder,
and/or condition.
[1840] Susceptible to: An individual who is "susceptible to" a
disease, disorder, and/or condition has not been diagnosed with
and/or cannot exhibit signs or symptoms of the disease, disorder,
and/or condition but harbors a propensity to develop a disease or
its signs or symptoms. In some embodiments, an individual who is
susceptible to a disease, disorder, and/or condition (for example,
AIP) can be characterized by one or more of the following: (1) a
genetic mutation associated with development of the disease,
disorder, and/or condition; (2) a genetic polymorphism associated
with development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
and/or nucleic acid associated with the disease, disorder, and/or
condition; (4) habits and/or lifestyles associated with development
of the disease, disorder, and/or condition; (5) a family history of
the disease, disorder, and/or condition; and (6) exposure to and/or
infection with a microbe associated with development of the
disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[1841] Sustained release: As used herein, the term "sustained
release" refers to a pharmaceutical composition or compound release
profile that conforms to a release rate over a specific period of
time.
[1842] Synthetic: The term "synthetic" means produced, prepared,
and/or manufactured by the hand of man. Synthesis of
polynucleotides or other molecules of the present invention can be
chemical or enzymatic.
[1843] Targeted Cells: As used herein, "targeted cells" refers to
any one or more cells of interest. The cells can be found in vitro,
in vivo, in situ or in the tissue or organ of an organism. The
organism can be an animal, for example a mammal, a human, a subject
or a patient.
[1844] Target tissue: As used herein "target tissue" refers to any
one or more tissue types of interest in which the delivery of a
polynucleotide would result in a desired biological and/or
pharmacological effect. Examples of target tissues of interest
include specific tissues, organs, and systems or groups thereof. In
particular applications, a target tissue can be a liver, a kidney,
a lung, a spleen, or vascular endothelium in vessels (e.g.,
intra-coronary or intra-femoral). An "off-target tissue" refers to
any one or more tissue types in which the expression of the encoded
protein does not result in a desired biological and/or
pharmacological effect.
[1845] The presence of a therapeutic agent in an off-target issue
can be the result of: (i) leakage of a polynucleotide from the
administration site to peripheral tissue or distant off-target
tissue via diffusion or through the bloodstream (e.g., a
polynucleotide intended to express a polypeptide in a certain
tissue would reach the off-target tissue and the polypeptide would
be expressed in the off-target tissue); or (ii) leakage of an
polypeptide after administration of a polynucleotide encoding such
polypeptide to peripheral tissue or distant off-target tissue via
diffusion or through the bloodstream (e.g., a polynucleotide would
expressed a polypeptide in the target tissue, and the polypeptide
would diffuse to peripheral tissue).
[1846] Targeting sequence: As used herein, the phrase "targeting
sequence" refers to a sequence that can direct the transport or
localization of a protein or polypeptide.
[1847] Terminus: As used herein the terms "termini" or "terminus,"
when referring to polypeptides, refers to an extremity of a peptide
or polypeptide. Such extremity is not limited only to the first or
final site of the peptide or polypeptide but can include additional
amino acids in the terminal regions. The polypeptide based
molecules of the invention can be characterized as having both an
N-terminus (terminated by an amino acid with a free amino group
(NH.sub.2)) and a C-terminus (terminated by an amino acid with a
free carboxyl group (COOH)). Proteins of the invention are in some
cases made up of multiple polypeptide chains brought together by
disulfide bonds or by non-covalent forces (multimers, oligomers).
These sorts of proteins will have multiple N- and C-termini.
Alternatively, the termini of the polypeptides can be modified such
that they begin or end, as the case can be, with a non-polypeptide
based moiety such as an organic conjugate.
[1848] Therapeutic Agent: The term "therapeutic agent" refers to an
agent that, when administered to a subject, has a therapeutic,
diagnostic, and/or prophylactic effect and/or elicits a desired
biological and/or pharmacological effect. For example, in some
embodiments, an mRNA encoding a PBGD polypeptide can be a
therapeutic agent.
[1849] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of an agent to
be delivered (e.g., nucleic acid, drug, therapeutic agent,
diagnostic agent, prophylactic agent, etc.) that is sufficient,
when administered to a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
signs or symptoms of, diagnose, prevent, and/or delay the onset of
the infection, disease, disorder, and/or condition.
[1850] Therapeutically effective outcome: As used herein, the term
"therapeutically effective outcome" means an outcome that is
sufficient in a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
signs or symptoms of, diagnose, prevent, and/or delay the onset of
the infection, disease, disorder, and/or condition.
[1851] Total daily dose: As used herein, a "total daily dose" is an
amount given or prescribed in 24 hr. period. The total daily dose
can be administered as a single unit dose or a split dose.
[1852] Transcription factor: As used herein, the term
"transcription factor" refers to a DNA-binding protein that
regulates transcription of DNA into RNA, for example, by activation
or repression of transcription. Some transcription factors effect
regulation of transcription alone, while others act in concert with
other proteins. Some transcription factor can both activate and
repress transcription under certain conditions. In general,
transcription factors bind a specific target sequence or sequences
highly similar to a specific consensus sequence in a regulatory
region of a target gene. Transcription factors can regulate
transcription of a target gene alone or in a complex with other
molecules.
[1853] Transcription: As used herein, the term "transcription"
refers to methods to produce mRNA (e.g., an mRNA sequence or
template) from DNA (e.g., a DNA template or sequence).
[1854] Transfection: As used herein, "transfection" refers to the
introduction of a polynucleotide (e.g., exogenous nucleic acids)
into a cell wherein a polypeptide encoded by the polynucleotide is
expressed (e.g., mRNA) or the polypeptide modulates a cellular
function (e.g., siRNA, miRNA). As used herein, "expression" of a
nucleic acid sequence refers to translation of a polynucleotide
(e.g., an mRNA) into a polypeptide or protein and/or
post-translational modification of a polypeptide or protein.
Methods of transfection include, but are not limited to, chemical
methods, physical treatments and cationic lipids or mixtures.
[1855] Treating, treatment, therapy: As used herein, the term
"treating" or "treatment" or "therapy" refers to partially or
completely alleviating, ameliorating, improving, relieving,
delaying onset of, inhibiting progression of, reducing severity of,
and/or reducing incidence of one or more signs, symptoms or
features of a disease, e.g., acute intermittent porphyria. For
example, "treating" acute intermittent porphyria can refer to
diminishing signs or symptoms associate with the disease, prolong
the lifespan (increase the survival rate) of patients, reducing the
severity of the disease, preventing or delaying the onset of the
disease, etc. Treatment can be administered to a subject who does
not exhibit signs of a disease, disorder, and/or condition and/or
to a subject who exhibits only early signs of a disease, disorder,
and/or condition for the purpose of decreasing the risk of
developing pathology associated with the disease, disorder, and/or
condition.
[1856] Unmodified: As used herein, "unmodified" refers to any
substance, compound or molecule prior to being changed in some way.
Unmodified can, but does not always, refer to the wild type or
native form of a biomolecule. Molecules can undergo a series of
modifications whereby each modified molecule can serve as the
"unmodified" starting molecule for a subsequent modification.
[1857] Uracil: Uracil is one of the four nucleobases in the nucleic
acid of RNA, and it is represented by the letter U. Uracil can be
attached to a ribose ring, or more specifically, a ribofuranose via
a .beta.-N.sub.1-glycosidic bond to yield the nucleoside uridine.
The nucleoside uridine is also commonly abbreviated according to
the one letter code of its nucleobase, i.e., U. Thus, in the
context of the present disclosure, when a monomer in a
polynucleotide sequence is U, such U is designated interchangeably
as a "uracil" or a "uridine."
[1858] Uridine Content: The terms "uridine content" or "uracil
content" are interchangeable and refer to the amount of uracil or
uridine present in a certain nucleic acid sequence. Uridine content
or uracil content can be expressed as an absolute value (total
number of uridine or uracil in the sequence) or relative (uridine
or uracil percentage respect to the total number of nucleobases in
the nucleic acid sequence).
[1859] Uridine-Modified Sequence: The terms "uridine-modified
sequence" refers to a sequence optimized nucleic acid (e.g., a
synthetic mRNA sequence) with a different overall or local uridine
content (higher or lower uridine content) or with different uridine
patterns (e.g., gradient distribution or clustering) with respect
to the uridine content and/or uridine patterns of a candidate
nucleic acid sequence. In the content of the present disclosure,
the terms "uridine-modified sequence" and "uracil-modified
sequence" are considered equivalent and interchangeable.
[1860] A "high uridine codon" is defined as a codon comprising two
or three uridines, a "low uridine codon" is defined as a codon
comprising one uridine, and a "no uridine codon" is a codon without
any uridines. In some embodiments, a uridine-modified sequence
comprises substitutions of high uridine codons with low uridine
codons, substitutions of high uridine codons with no uridine
codons, substitutions of low uridine codons with high uridine
codons, substitutions of low uridine codons with no uridine codons,
substitution of no uridine codons with low uridine codons,
substitutions of no uridine codons with high uridine codons, and
combinations thereof. In some embodiments, a high uridine codon can
be replaced with another high uridine codon. In some embodiments, a
low uridine codon can be replaced with another low uridine codon.
In some embodiments, a no uridine codon can be replaced with
another no uridine codon. A uridine-modified sequence can be
uridine enriched or uridine rarefied.
[1861] Uridine Enriched: As used herein, the terms "uridine
enriched" and grammatical variants refer to the increase in uridine
content (expressed in absolute value or as a percentage value) in
an sequence optimized nucleic acid (e.g., a synthetic mRNA
sequence) with respect to the uridine content of the corresponding
candidate nucleic acid sequence. Uridine enrichment can be
implemented by substituting codons in the candidate nucleic acid
sequence with synonymous codons containing less uridine
nucleobases. Uridine enrichment can be global (i.e., relative to
the entire length of a candidate nucleic acid sequence) or local
(i.e., relative to a subsequence or region of a candidate nucleic
acid sequence).
[1862] Uridine Rarefied: As used herein, the terms "uridine
rarefied" and grammatical variants refer to a decrease in uridine
content (expressed in absolute value or as a percentage value) in
an sequence optimized nucleic acid (e.g., a synthetic mRNA
sequence) with respect to the uridine content of the corresponding
candidate nucleic acid sequence. Uridine rarefication can be
implemented by substituting codons in the candidate nucleic acid
sequence with synonymous codons containing less uridine
nucleobases. Uridine rarefication can be global (i.e., relative to
the entire length of a candidate nucleic acid sequence) or local
(i.e., relative to a subsequence or region of a candidate nucleic
acid sequence).
[1863] Variant: The term variant as used in present disclosure
refers to both natural variants (e.g, polymorphisms, isoforms, etc)
and artificial variants in which at least one amino acid residue in
a native or starting sequence (e.g., a wild type sequence) has been
removed and a different amino acid inserted in its place at the
same position. These variants can de described as "substitutional
variants." The substitutions can be single, where only one amino
acid in the molecule has been substituted, or they can be multiple,
where two or more amino acids have been substituted in the same
molecule. If amino acids are inserted or deleted, the resulting
variant would be an "insertional variant" or a "deletional variant"
respectively.
30. EMBODIMENTS
[1864] Throughout this section, the term embodiment is abbreviated
as `E` followed by an ordinal. For example, E1 is equivalent to
Embodiment 1.
[1865] E1. A polynucleotide comprising an open reading frame (ORF)
encoding a porphobilinogen deaminase (PBGD) polypeptide, wherein
the uracil or thymine content of the ORF relative to the
theoretical minimum uracil or thymine content of a nucleotide
sequence encoding the PBGD polypeptide (% U.sub.TM or % T.sub.TM),
is between about 100% and about 150%.
[1866] E2. The polynucleotide of E1, wherein the % U.sub.TM or %
T.sub.TM is between about 105% and about 145%, between about 105%
and about 140%, between about 110% and about 140%, between about
110% and about 145%, between about 115% and about 135%, between
about 105% and about 135%, between about 110% and about 135%,
between about 115% and about 145%, or between about 115% and about
140%.
[1867] E3. The polynucleotide of E2, wherein the % U.sub.TL or %
T.sub.TM is between (i) 110%, 111%, 112%, 113%, 114%, 115%, 116%,
117%, or 118% and (ii) 132%, 133%, 134%, 135%, 136%, 137%, 138%,
139%, or 140%.
[1868] E4. The polynucleotide of any one of E1 to E3, wherein the
uracil or thymine content of the ORF relative to the uracil or
thymine content of the corresponding wild-type ORF (% U.sub.WT or %
T.sub.WT) is less than 100%.
[1869] E5. The polynucleotide of E4, wherein the % U.sub.WT or %
T.sub.WT is less than about 95%, less than about 90%, less than
about 85%, less than 80%, less than 79%, less than 78%, less than
77%, less than 76%, less than 75%, less than 74%, or less than
73%.
[1870] E6. The polynucleotide of E4, wherein the % U.sub.WT or %
T.sub.WT is between 65% and 73%.
[1871] E7. The polynucleotide of any one of E1 to E6, wherein the
uracil or thymine content in the ORF relative to the total
nucleotide content in the ORF (% U.sub.TL or % T.sub.TL) is less
than about 50%, less than about 40%, less than about 30%, or less
than about 19%.
[1872] E8. The polynucleotide of E7, wherein the % U.sub.TL or %
T.sub.TL is less than about 19%.
[1873] E9. The polynucleotide of any one of E1 to E8, wherein the %
U.sub.TL or % T.sub.TL is between about 13% and about 15%.
[1874] E10. The polynucleotide of any one of E1 to E9, wherein the
guanine content of the ORF with respect to the theoretical maximum
guanine content of a nucleotide sequence encoding the PBGD
polypeptide (% G.sub.TMX) is at least 69%, at least 70%, at least
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or about 100%.
[1875] E11. The polynucleotide of E10, wherein the % G.sub.TMX is
between about 70% and about 80%, between about 71% and about 79%,
between about 71% and about 78%, or between about 71% and about
77%.
[1876] E12. The polynucleotide of any one of E1 to E11, wherein the
cytosine content of the ORF relative to the theoretical maximum
cytosine content of a nucleotide sequence encoding the PBGD
polypeptide (% C.sub.TMX) is at least 59%, at least 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
about 100%.
[1877] E13. The polynucleotide of E12, wherein the % C.sub.TMX is
between about 60% and about 80%, between about 62% and about 80%,
between about 63% and about 79%, or between about 68% and about
76%.
[1878] E14. The polynucleotide of any one of E1 to E13, wherein the
guanine and cytosine content (G/C) of the ORF relative to the
theoretical maximum G/C content in a nucleotide sequence encoding
the PBGD polypeptide (% G/C.sub.TMX) is at least about 81%, at
least about 85%, at least about 90%, at least about 95%, or about
100%.
[1879] E15. The polynucleotide of any one of E1 to E13, wherein the
% G/C.sub.TMX is between about 80% and about 100%, between about
85% and about 99%, between about 90% and about 97%, or between
about 91% and about 96%.
[1880] E16. The polynucleotide of any one of E1 to E15, wherein the
G/C content in the ORF relative to the G/C content in the
corresponding wild-type ORF (% G/C.sub.WT) is at least 102%, at
least 103%, at least 104%, at least 105%, at least 106%, at least
107%, at least 110%, at least 115%, or at least 120%.
[1881] E17. The polynucleotide of any one of E1 to E15, wherein the
average G/C content in the 3.sup.rd codon position in the ORF is at
least 20%, at least 21%, at least 22%, at least 23%, at least 24%,
at least 25%, at least 26%, at least 27%, at least 28%, at least
29%, or at least 30% higher than the average G/C content in the
3.sup.rd codon position in the corresponding wild-type ORF.
[1882] E18. The polynucleotide of any one of E1 to E17, wherein the
ORF further comprises at least one low-frequency codon.
[1883] E19. The polynucleotide of any one of E1 to E18,
[1884] (i) wherein the ORF is at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or 100% identical to PBGD-CO3 or PBGD-CO25,
[1885] (ii) wherein the ORF is at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% identical to PBGD-CO1, PBGD-CO2, PBGD-CO7, PBGD-CO11,
PBGD-CO13, PBGD-CO14, PBGD-CO16, or PBGD-CO24,
[1886] (iii) wherein the ORF is at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical to PBGD-CO5, PBGD-CO9, PBGD-CO10, PBGD-CO12, PBGD-CO15,
PBGD-CO17, PBGD-CO18, PBGD-CO19, PBGD-CO20, PBGD-CO21, or
PBGD-CO22,
[1887] (iv) wherein the ORF is at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical to
PBGD-CO6, PBGD-CO8, PBGD-CO23, or
[1888] (v) wherein the ORF is at least 91%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical to
PBGD-CO4.
[1889] E20. A polynucleotide comprising an ORF,
[1890] (i) wherein the ORF is at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or 100% identical to PBGD-CO3 or PBGD-CO25,
[1891] (ii) wherein the ORF is at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% identical to PBGD-CO1, PBGD-CO2, PBGD-CO7, PBGD-CO11,
PBGD-CO13, PBGD-CO14, PBGD-CO16, or PBGD-CO24,
[1892] (iii) wherein the ORF is at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical to PBGD-CO5, PBGD-CO9, PBGD-CO10, PBGD-CO12, PBGD-CO15,
PBGD-CO17, PBGD-CO18, PBGD-CO19, PBGD-CO20, PBGD-CO21, or
PBGD-CO22,
[1893] (iv) wherein the ORF is at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical to
PBGD-CO6, PBGD-CO8, PBGD-CO23, or
[1894] (v) wherein the ORF is at least 91%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical to
PBGD-CO4.
[1895] E21. The polynucleotide of any one of E1 to E20, wherein the
ORF has at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
sequence identity to a sequence selected from the group consisting
of SEQ ID NOs: 9 to 33.
[1896] E22. The polynucleotide of any one of E1 to E21, wherein the
PBGD polypeptide comprises an amino acid sequence at least about
95%, at least about 96%, at least about 97%, at least about 98%, at
least about 99%, or about 100% identical to (i) the polypeptide
sequence of wild type PBGD, isoform 1 (SEQ ID NO: 1), (ii) the
polypeptide sequence of wild type PBGD, isoform 2 (SEQ ID NO: 3),
the polypeptide sequence of wild type PBGD, isoform 3 (SEQ ID NO:
5), or the polypeptide sequence of wild type PBGD, isoform 4 (SEQ
ID NO: 7), and wherein the PBGD polypeptide has porphobilinogen
deaminase activity.
[1897] E23. The polynucleotide of E22, wherein the PBGD polypeptide
is a variant, derivative, or mutant having a porphobilinogen
deaminase activity.
[1898] E24. The polynucleotide of any one of E1 to E23, wherein the
polynucleotide sequence further comprises a nucleotide sequence
encoding a transit peptide.
[1899] E25. The polynucleotide of any one of E1 to E24, wherein the
polynucleotide is single stranded.
[1900] E26. The polynucleotide of any one of E1 to E24, wherein the
polynucleotide is double stranded.
[1901] E27. The polynucleotide of any one of E1 to E26, wherein the
polynucleotide is DNA.
[1902] E28. The polynucleotide of any one of E1 to E26, wherein the
polynucleotide is RNA.
[1903] E29. The polynucleotide of E28, wherein the polynucleotide
is mRNA.
[1904] E30. The polynucleotide of any one of E1 to E29, wherein the
polynucleotide comprises at least one chemically modified
nucleobase, sugar, backbone, or any combination thereof.
[1905] E31. The polynucleotide of E30, wherein the at least one
chemically modified nucleobase is selected from the group
consisting of pseudouracil (.psi.), N1-methylpseudouracil
(m1.psi.), 2-thiouracil (s2U), 4'-thiouracil, 5-methylcytosine,
5-methyluracil, and any combination thereof.
[1906] E32. The polynucleotide of E30, wherein the at least one
chemically modified nucleobase is 5-methoxyuracil.
[1907] E33. The polynucleotide of E32, wherein at least about 25%,
at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95%, at least about 99%, or 100% of the
uracils are 5-methoxyuracils.
[1908] E34. The polynucleotide of any one of E1 to E33, wherein the
polynucleotide further comprises a miRNA binding site.
[1909] E35. The polynucleotide of E34, wherein the miRNA binding
site comprises one or more nucleotide sequences selected from SEQ
ID NO:36 and SEQ ID NO:38
[1910] E36. The polynucleotide of E34, wherein the miRNA binding
site binds to miR-142.
[1911] E37. The polynucleotide of E35 or E36, wherein the miRNA
binding site binds to miR-142-3p or miR-142-5p.
[1912] E38. The polynucleotide of E36 or E37, wherein the miR142
comprises SEQ ID NO: 34.
[1913] E39. The polynucleotide of any one of E1 to E38, wherein the
polynucleotide further comprises a 5' UTR.
[1914] E40. The polynucleotide of E39, wherein the 5' UTR comprises
a nucleic acid sequence at least 90%, at least about 95%, at least
about 96%, at least about 97%, at least about 98%, at least about
99%, or about 100% identical to a 5'UTR sequence selected from the
group consisting of SEQ ID NO: 39-56, or any combination
thereof.
[1915] E41. The polynucleotide of any one of E1 to E40, wherein the
polynucleotide further comprises a 3' UTR.
[1916] E42. The polynucleotide of E41, wherein the 3' UTR comprises
a nucleic acid sequence at least about 90%, at least about 95%, at
least about 96%, at least about 97%, at least about 98%, at least
about 99%, or about 100% identical to a 3'UTR sequence selected
from the group consisting of SEQ ID NO: 57-81, or any combination
thereof.
[1917] E43. The polynucleotide of E41 or E42, wherein the miRNA
binding site is located within the 3' UTR.
[1918] E44. The polynucleotide of any one of E1 to E43, wherein the
polynucleotide further comprises a 5' terminal cap.
[1919] E45. The polynucleotide of E44, wherein the 5' terminal cap
comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,
2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,
2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5'
methylG cap, or an analog thereof.
[1920] E46. The polynucleotide of any one of E1 to E45, wherein the
polynucleotide further comprises a poly-A region.
[1921] E47. The polynucleotide of E46, wherein the poly-A region is
at least about 10, at least about 20, at least about 30, at least
about 40, at least about 50, at least about 60, at least about 70,
at least about 80, or at least about 90 nucleotides in length.
[1922] E48. The polynucleotide of E47, wherein the poly-A region
has about 10 to about 200, about 20 to about 180, about 50 to about
160, about 70 to about 140, about 80 to about 120 nucleotides in
length.
[1923] E49. The polynucleotide of any one of E1 to E48, wherein the
polynucleotide encodes a PBGD polypeptide that is fused to one or
more heterologous polypeptides.
[1924] E50. The polynucleotide of E49, wherein the one or more
heterologous polypeptides increase a pharmacokinetic property of
the PBGD polypeptide.
[1925] E51. The polynucleotide of any one of E1 to E50, wherein
upon administration to a subject, the polynucleotide has:
[1926] (i) a longer plasma half-life;
[1927] (ii) increased expression of a PBGD polypeptide encoded by
the ORF;
[1928] (iii) a lower frequency of arrested translation resulting in
an expression fragment;
[1929] (iv) greater structural stability; or
[1930] (v) any combination thereof, relative to a corresponding
polynucleotide comprising SEQ ID NO: 2, 4, 6, or 8.
[1931] E52. The polynucleotide of any one of E1 to E51, wherein the
polynucleotide comprises:
[1932] (i) a 5'-terminal cap;
[1933] (ii) a 5'-UTR;
[1934] (iii) an ORF encoding a PBGD polypeptide;
[1935] (iv) a 3'-UTR; and
[1936] (v) a poly-A region.
[1937] E53. The polynucleotide of E52, wherein the 3'-UTR comprises
a miRNA binding site.
[1938] E54. A method of producing the polynucleotide of any one of
E1 to E53, the method comprising modifying an ORF encoding a PBGD
polypeptide by substituting at least one uracil nucleobase with an
adenine, guanine, or cytosine nucleobase, or by substituting at
least one adenine, guanine, or cytosine nucleobase with a uracil
nucleobase, wherein all the substitutions are synonymous
substitutions.
[1939] E55. The method of E54, wherein the method further comprises
replacing at least about 90%, at least about 95%, at least about
99%, or about 100% of uracils with 5-methoxyuracils.
[1940] E56. A composition comprising
[1941] (a) the polynucleotide of any one of E1 to E53; and
[1942] (b) a delivery agent.
[1943] E57. The composition of E56, wherein the delivery agent
comprises a lipidoid, a liposome, a lipoplex, a lipid nanoparticle,
a polymeric compound, a peptide, a protein, a cell, a nanoparticle
mimic, a nanotube, or a conjugate.
[1944] E58. The composition of E56, wherein the delivery agent
comprises a lipid nanoparticle.
[1945] E59. The composition of E58, wherein the lipid nanoparticle
comprises a lipid selected from the group consisting of [1946]
3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine
(KL10), [1947]
N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinedie-
thanamine (KL22), [1948]
14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),
[1949] 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),
[1950] 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA), [1951] heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (DLin-MC3-DMA), [1952]
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), [1953] 1,2-dioleyloxy-N,N-dimethylaminopropane
(DODMA), (13Z,165Z)--N,N-dimethyl-3-nonydocosa-13-16-dien-1-amine
(L608), [1954]
2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-
-octadeca-9,12-di en-1-yloxy]propan-1-amine (Octyl-CLinDMA), [1955]
(2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-
,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA
(2R)), [1956] (2S)-2-({8-[(3
3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-
-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)), and any
combinations thereof.
[1957] E60. The composition of any one of E56 to E59, wherein the
delivery agent comprises a compound having the Formula (I)
##STR00171##
or a salt or stereoisomer thereof, wherein
[1958] R.sub.1 is selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1959] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[1960] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --CH.sub.2).sub.nQ, --CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, and --C(R)N(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5;
[1961] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1962] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1963] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[1964] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1965] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1966] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1967] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1968] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1969] each Y is independently a C.sub.3-6 carbocycle;
[1970] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1971] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
and
[1972] provided when R.sub.4 is --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, or --CQ(R).sub.2, then (i) Q is not
--N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or
7-membered heterocycloalkyl when n is 1 or 2.
[1973] E61. A composition comprising a nucleotide sequence encoding
a PBGD polypeptide and a delivery agent, wherein the delivery agent
comprises a compound having the Formula (I)
##STR00172##
or a salt or stereoisomer thereof, wherein
[1974] R.sub.1 is selected from the group consisting of C.sub.5-20
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[1975] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[1976] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, and --C(R)N(R).sub.2C(O)OR, and each n is
independently selected from 1, 2, 3, 4, and 5;
[1977] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1978] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1979] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
an aryl group, and a heteroaryl group;
[1980] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[1981] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[1982] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[1983] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[1984] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[1985] each Y is independently a C.sub.3-6 carbocycle;
[1986] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[1987] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
and
[1988] provided when R.sub.4 is --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, or --CQ(R).sub.2, then (i) Q is not
--N(R).sub.2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or
7-membered heterocycloalkyl when n is 1 or 2.
[1989] E62. The composition of E60 or E61, wherein the compound is
of Formula (IA):
##STR00173##
or a salt or stereoisomer thereof, wherein
[1990] l is selected from 1, 2, 3, 4, and 5;
[1991] m is selected from 5, 6, 7, 8, and 9;
[1992] M.sub.1 is a bond or M';
[1993] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 1, 2, 3, 4, or 5 and Q is OH,
--NHC(S)N(R).sub.2, or --NHC(O)N(R).sub.2;
[1994] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, an aryl group, and a
heteroaryl group; and
[1995] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[1996] E63. The composition of any one of E60 to E62, wherein m is
5, 7, or 9.
[1997] E64. The composition of any one of E60 to E63, wherein the
compound is of Formula (II):
##STR00174##
or a salt or stereoisomer thereof, wherein
[1998] l is selected from 1, 2, 3, 4, and 5;
[1999] M.sub.1 is a bond or M';
[2000] R.sub.4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which n is 2, 3, or 4 and Q is OH,
--NHC(S)N(R).sub.2, or --NHC(O)N(R).sub.2;
[2001] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, an aryl group, and a
heteroaryl group; and
[2002] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, and C.sub.2-14
alkenyl.
[2003] E65. The composition of any one of E62 to E64, wherein
M.sub.1 is M'.
[2004] E66. The composition of E65, wherein M and M' are
independently --C(O)O-- or --OC(O)--.
[2005] E67. The composition of any one of E62 to E66, wherein l is
1, 3, or 5.
[2006] E68. The composition of E60 or E61, wherein the compound is
selected from the group consisting of Compound 1 to Compound 147,
salts and stereoisomers thereof, and any combination thereof.
[2007] E69. The composition of E60 or E61, wherein the compound is
of the Formula (IIa),
##STR00175##
or a salt or stereoisomer thereof.
[2008] E70. The composition of E60 or E61, wherein the compound is
of the Formula (IIb),
##STR00176##
or a salt or stereoisomer thereof.
[2009] E71. The composition of E60 or E61, wherein the compound is
of the Formula (IIc) or (IIe),
##STR00177##
or a salt or stereoisomer thereof.
[2010] E72. The composition of any one of E69 to E71, wherein
R.sub.4 is selected from --(CH.sub.2).sub.nQ and
--(CH.sub.2).sub.nCHQR.
[2011] E73. The composition of E60 or E61, wherein the compound is
of the Formula (IId),
##STR00178##
or a salt or stereoisomer thereof,
[2012] wherein R.sub.2 and R.sub.3 are independently selected from
the group consisting of C.sub.5-14 alkyl and C.sub.5-14 alkenyl, n
is selected from 2, 3, and 4, and R', R'', R.sub.5, R.sub.6 and m
are as defined in claim 60 or 61.
[2013] E74. The composition of E73, wherein R.sub.2 is C.sub.8
alkyl.
[2014] E75. The composition of E74, wherein R.sub.3 is C.sub.5
alkyl, C.sub.6 alkyl, C.sub.7 alkyl, C.sub.8 alkyl, or C.sub.9
alkyl.
[2015] E76. The composition of any one of E73 to E75, wherein m is
5, 7, or 9.
[2016] E77. The composition of any one of E73 to E76, wherein each
R.sub.5 is H.
[2017] E78. The composition of E77, wherein each R.sub.6 is H.
[2018] E79. The composition of any one of E60 to E78, which is a
nanoparticle composition.
[2019] E80. The composition of E79, wherein the delivery agent
further comprises a phospholipid.
[2020] E81. The composition of E80, wherein the phospholipid is
selected from the group consisting of
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), [2021]
1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), [2022]
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), [2023]
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), [2024]
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), [2025]
1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), [2026]
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), [2027]
1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),
[2028]
1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
(OChemsPC), [2029] 1-hexadecyl-sn-glycero-3-phosphocholine (C16
Lyso PC), [2030] 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,
[2031] 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, [2032]
1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, [2033]
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), [2034]
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE),
[2035] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, [2036]
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, [2037]
1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, [2038]
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, [2039]
1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, [2040]
1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt
(DOPG), sphingomyelin, [2041] and any mixtures thereof.
[2042] E82. The composition of any one of E60 to E81, wherein the
delivery agent further comprises a structural lipid.
[2043] E83. The composition of E82, wherein the structural lipid is
selected from the group consisting of cholesterol, fecosterol,
sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,
tomatidine, ursolic acid, alpha-tocopherol, and any mixtures
thereof.
[2044] E84. The composition of any one of E60 to E83, wherein the
delivery agent further comprises a PEG lipid.
[2045] E85. The composition of E84, wherein the PEG lipid is
selected from the group consisting of a PEG-modified
phosphatidylethanolamine, a PEG-modified phosphatidic acid, a
PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified
diacylglycerol, a PEG-modified dialkylglycerol, and any mixtures
thereof.
[2046] E86. The composition of any one of E60 to E85, wherein the
delivery agent further comprises an ionizable lipid selected from
the group consisting of [2047]
3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine
(KL10), [2048]
N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinedie-
thanamine (KL22), [2049]
14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),
[2050] 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),
[2051] 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA), [2052] heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (DLin-MC3-DMA), [2053]
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), [2054] 1,2-dioleyloxy-N,N-dimethylaminopropane
(DODMA), [2055]
2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-
-octadeca-9,12-di en-1-yloxy]propan-1-amine (Octyl-CLinDMA), [2056]
(2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-
,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA
(2R)), and [2057] (2S)-2-({8-[(3
3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-
-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)).
[2058] E87. The composition of any one of E60 to E86, wherein the
delivery agent further comprises a phospholipid, a structural
lipid, a PEG lipid, or any combination thereof.
[2059] E88. The composition of any one of E60 to E87, wherein the
composition is formulated for in vivo delivery.
[2060] E89. The composition according any one of E60 to E88, which
is formulated for intramuscular, subcutaneous, or intradermal
delivery.
[2061] E90. A host cell comprising the polynucleotide of any one of
E1 to E53.
[2062] E91. The host cell of E90, wherein the host cell is a
eukaryotic cell.
[2063] E92. A vector comprising the polynucleotide of any one of E1
to E53.
[2064] E93. A method of making a polynucleotide comprising
enzymatically or chemically synthesizing the polynucleotide of any
one of E1 to E53.
[2065] E94. A polypeptide encoded by the polynucleotide of any one
of E1 to E53, the composition of any one of E56 to E89, the host
cell of E90 or E91, or the vector of E92 or produced by the method
of E93.
[2066] E95. A method of expressing in vivo an active PBGD
polypeptide in a subject in need thereof comprising administering
to the subject an effective amount of the polynucleotide of any one
of E1 to E53, the composition of any one of E56 to E89, the host
cell of E90 or E91, or the vector of E92.
[2067] E96. A method of treating acute intermittent porphyria (AIP)
in a subject in need thereof comprising administering to the
subject a therapeutically effective amount of the polynucleotide of
any one of E1 to E53, the composition of any one of E56 to E89, the
host cell of E90 or E91, or the vector of E92, wherein the
administration alleviates the signs or symptoms of AIP in the
subject.
[2068] E97. A method to prevent or delay the onset of AIP signs or
symptoms in a subject in need thereof comprising administering to
the subject a prophylactically effective amount of the
polynucleotide of any one of E1 to E53, the composition of any one
of E56 to E89, the host cell of E90 or E91, or the vector of E92
before AIP signs or symptoms manifest, wherein the administration
prevents or delays the onset of AIP signs or symptoms in the
subject.
[2069] E98. A method to ameliorate the signs or symptoms of AIP in
a subject in need thereof comprising administering to the subject a
therapeutically effective amount of the polynucleotide of any one
of E1 to E53, the composition of any one of E56 to E89, the host
cell of E90 or E91, or the vector of E92 before AIP signs or
symptoms manifest, wherein the administration ameliorates AIP signs
or symptoms in the subject.
[2070] E99. A method to increase hepatic PBGD activity in a subject
in need thereof comprising administering to the subject an
effective amount of the polynucleotide of any one of E1 to E53, the
composition of any one of E56 to E89, the host cell of E90 or E91,
or the vector of E92.
[2071] E100. A method to decrease ALA (aminolevulinate) and/or PBG
(porphobilinogen) in a subject in need thereof comprising
administering to the subject an effective amount of the
polynucleotide of any one of E1 to E53, the composition of any one
of E56 to E89, the host cell of E90 or E91, or the vector of
E92.
[2072] E101. The method according to E100, wherein the ALA and/or
PBG levels are urinary excretion levels.
[2073] E102, A method to protect a subject in need thereof against
the increase in heme precursors in an AIP attack comprising
administering to the subject an effective amount of the
polynucleotide of any one of E1 to E53, the composition of any one
of E56 to E89, the host cell of E90 or E91, or the vector of
E92.
[2074] E103. A method to reduce the accumulation of heme precursors
in a subject in need thereof comprising administering to the
subject an effective amount of the polynucleotide of any one of E1
to E53, the composition of any one of E56 to E89, the host cell of
E90 or E91, or the vector of E92.
[2075] E104. The method according to E102 or E103, wherein the heme
precursors are ALA and/or PBG.
[2076] E105. A method to treat, prevent, or ameliorate pain in a
subject in need thereof comprising administering to the subject an
effective amount of the polynucleotide of any one of E1 to E53, the
composition of any one of E56 to E89, the host cell of E90 or E91,
or the vector of E92.
[2077] E106. The method according to E105, wherein the pain is
severe pain.
[2078] E107. The method according to E105, wherein the treatment,
prevention or amelioration in pain results in pain annulment.
[2079] E108. A method to treat, prevent, or ameliorate neuropathy
in a subject in need thereof comprising administering to the
subject an effective amount of the polynucleotide of any one of E1
to E53, the composition of any one of E56 to E89, the host cell of
E90 or E91, or the vector of E92.
[2080] E109. The method according to E108, wherein the treatment,
prevention or amelioration in neuropathy results in neuropathy
annulment.
[2081] E110. The method according to E108 or E109, wherein the
neuropathy is peripheral neuropathy.
[2082] E111. A method to increase survival in a subject in need
thereof comprising administering to the subject an effective amount
of the polynucleotide of any one of E1 to E53, the composition of
any one of E56 to E89, the host cell of E90 or E91, or the vector
of E92.
[2083] E112. The method according to any one of E95 to E111,
wherein the subject is an AIP patient.
[2084] E113. The method according to E112, wherein the AIP patient
is an asymptomatic patient.
[2085] E114. The method according to any one of E95 to E114,
wherein the polynucleotide comprises PBGD-CO3 (SEQ ID NO:11).
31. EQUIVALENTS AND SCOPE
[2086] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[2087] In the claims, articles such as "a," "an," and "the" can
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[2088] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[2089] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[2090] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
can be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they can be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any nucleic acid or protein
encoded thereby; any method of production; any method of use; etc.)
can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[2091] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
[2092] Section and table headings are not intended to be
limiting.
EXAMPLES
Example 1
Chimeric Polynucleotide Synthesis
A. Triphosphate Route
[2093] Two regions or parts of a chimeric polynucleotide can be
joined or ligated using triphosphate chemistry. According to this
method, a first region or part of 100 nucleotides or less can be
chemically synthesized with a 5' monophosphate and terminal 3'desOH
or blocked OH. If the region is longer than 80 nucleotides, it can
be synthesized as two strands for ligation.
[2094] If the first region or part is synthesized as a
non-positionally modified region or part using in vitro
transcription (IVT), conversion the 5'monophosphate with subsequent
capping of the 3' terminus can follow. Monophosphate protecting
groups can be selected from any of those known in the art.
[2095] The second region or part of the chimeric polynucleotide can
be synthesized using either chemical synthesis or IVT methods. IVT
methods can include an RNA polymerase that can utilize a primer
with a modified cap. Alternatively, a cap of up to 80 nucleotides
can be chemically synthesized and coupled to the IVT region or
part.
[2096] It is noted that for ligation methods, ligation with DNA T4
ligase, followed by treatment with DNase should readily avoid
concatenation.
[2097] The entire chimeric polynucleotide need not be manufactured
with a phosphate-sugar backbone. If one of the regions or parts
encodes a polypeptide, then such region or part can comprise a
phosphate-sugar backbone.
[2098] Ligation can then be performed using any known click
chemistry, orthoclick chemistry, solulink, or other bioconjugate
chemistries known to those in the art.
B. Synthetic Route
[2099] The chimeric polynucleotide can be made using a series of
starting segments. Such segments include:
[2100] (a) Capped and protected 5' segment comprising a normal 3'OH
(SEG. 1)
[2101] (b) 5' triphosphate segment which can include the coding
region of a polypeptide and comprising a normal 3'OH (SEG. 2)
[2102] (c) 5' monophosphate segment for the 3' end of the chimeric
polynucleotide (e.g., the tail) comprising cordycepin or no 3'OH
(SEG. 3)
[2103] After synthesis (chemical or IVT), segment 3 (SEG. 3) can be
treated with cordycepin and then with pyrophosphatase to create the
5'monophosphate.
[2104] Segment 2 (SEG. 2) can then be ligated to SEG. 3 using RNA
ligase. The ligated polynucleotide can then be purified and treated
with pyrophosphatase to cleave the diphosphate. The treated
SEG.2-SEG. 3 construct is then purified and SEG. 1 is ligated to
the 5' terminus. A further purification step of the chimeric
polynucleotide can be performed.
[2105] Where the chimeric polynucleotide encodes a polypeptide, the
ligated or joined segments can be represented as: 5'UTR (SEG. 1),
open reading frame or ORF (SEG. 2) and 3'UTR+PolyA (SEG. 3).
[2106] The yields of each step can be as much as 90-95%.
Example 2
PCR for cDNA Production
[2107] PCR procedures for the preparation of cDNA can be performed
using 2.times.KAPA HIFI.TM. HotStart ReadyMix by Kapa Biosystems
(Wobum, Mass.). This system includes 2.times.KAPA ReadyMix 12.5
.mu.l; Forward Primer (10 .mu.M) 0.75 .mu.l; Reverse Primer (10
.mu.M) 0.75 .mu.l; Template cDNA -100 ng; and dH.sub.2O diluted to
25.0 .mu.l. The PCR reaction conditions can be: at 95.degree. C.
for 5 min. and 25 cycles of 98.degree. C. for 20 sec, then
58.degree. C. for 15 sec, then 72.degree. C. for 45 sec, then
72.degree. C. for 5 min. then 4.degree. C. to termination.
[2108] The reverse primer of the instant invention can incorporate
a poly-T.sub.120 for a poly-A.sub.120 in the mRNA. Other reverse
primers with longer or shorter poly(T) tracts can be used to adjust
the length of the poly(A) tail in the polynucleotide mRNA.
[2109] The reaction can be cleaned up using Invitrogen's
PURELINK.TM. PCR Micro Kit (Carlsbad, Calif.) per manufacturer's
instructions (up to 5 .mu.g). Larger reactions will require a
cleanup using a product with a larger capacity. Following the
cleanup, the cDNA can be quantified using the NANODROP.TM. and
analyzed by agarose gel electrophoresis to confirm the cDNA is the
expected size. The cDNA can then be submitted for sequencing
analysis before proceeding to the in vitro transcription
reaction.
Example 3
In Vitro Transcription (IVT)
[2110] The in vitro transcription reactions can generate
polynucleotides containing uniformly modified polynucleotides. Such
uniformly modified polynucleotides can comprise a region or part of
the polynucleotides of the invention. The input nucleotide
triphosphate (NTP) mix can be made using natural and un-natural
NTPs.
[2111] A typical in vitro transcription reaction can include the
following: [2112] 1 Template cDNA--1.0 .mu.g [2113] 2 10.times.
transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl.sub.2, 50
mM DTT, 10 mM Spermidine)--2.0 .mu.l [2114] 3 Custom NTPs (25 mM
each)--7.2 .mu.l [2115] 4 RNase Inhibitor--20 U T7 RNA
polymerase--3000 U [2116] 6 dH.sub.2O--Up to 20.0 .mu.l. and [2117]
7 Incubation at 37.degree. C. for 3 hr-5 hrs.
[2118] The crude IVT mix can be stored at 4.degree. C. overnight
for cleanup the next day. 1 U of RNase-free DNase can then be used
to digest the original template. After 15 minutes of incubation at
37.degree. C., the mRNA can be purified using Ambion's
MEGACLEAR.TM. Kit (Austin, Tex.) following the manufacturer's
instructions. This kit can purify up to 500 .mu.g of RNA. Following
the cleanup, the RNA can be quantified using the NanoDrop and
analyzed by agarose gel electrophoresis to confirm the RNA is the
proper size and that no degradation of the RNA has occurred.
Example 4
Enzymatic Capping
[2119] Capping of a polynucleotide can be performed with a mixture
includes: IVT RNA 60 .mu.g-180 .mu.g and dH.sub.20 up to 72 .mu.l.
The mixture can be incubated at 65.degree. C. for 5 minutes to
denature RNA, and then can be transferred immediately to ice.
[2120] The protocol can then involve the mixing of 10.times.
Capping Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM
MgCl.sub.2) (10.0 .mu.l); 20 mM GTP (5.0 .mu.l); 20 mM S-Adenosyl
Methionine (2.5 .mu.l); RNase Inhibitor (100 U);
2'-O-Methyltransferase (400 U); Vaccinia capping enzyme (Guanylyl
transferase) (40 U); dH.sub.2O (Up to 28 .mu.l); and incubation at
37.degree. C. for 30 minutes for 60 .mu.g RNA or up to 2 hours for
180 .mu.g of RNA.
[2121] The polynucleotide can then be purified using Ambion's
MEGACLEAR.TM. Kit (Austin, Tex.) following the manufacturer's
instructions. Following the cleanup, the RNA can be quantified
using the NANODROP.TM. (ThermoFisher, Waltham, Mass.) and analyzed
by agarose gel electrophoresis to confirm the RNA is the proper
size and that no degradation of the RNA has occurred. The RNA
product can also be sequenced by running a
reverse-transcription-PCR to generate the cDNA for sequencing.
Example 5
PolyA Tailing Reaction
[2122] Without a poly-T in the cDNA, a poly-A tailing reaction must
be performed before cleaning the final product. This can be done by
mixing Capped IVT RNA (100 .mu.l); RNase Inhibitor (20 U);
10.times. Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100
mM MgCl.sub.2)(12.0 .mu.l); 20 mM ATP (6.0 .mu.l); Poly-A
Polymerase (20 U); dH.sub.2O up to 123.5 .mu.l and incubating at
37.degree. C. for 30 min. If the poly-A tail is already in the
transcript, then the tailing reaction can be skipped and proceed
directly to cleanup with Ambion's MEGACLEAR.TM. kit (Austin, Tex.)
(up to 500 rig). Poly-A Polymerase is, in some cases, a recombinant
enzyme expressed in yeast.
[2123] It should be understood that the processivity or integrity
of the polyA tailing reaction does not always result in an exact
size polyA tail. Hence polyA tails of approximately between 40-200
nucleotides, e.g., about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164
or 165 are within the scope of the invention.
Example 6
Natural 5' Caps and 5' Cap Analogues
[2124] 5'-capping of polynucleotides can be completed concomitantly
during the in vitro-transcription reaction using the following
chemical RNA cap analogs to generate the 5'-guanosine cap structure
according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the
ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A;
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). 5'-capping
of modified RNA can be completed post-transcriptionally using a
Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure:
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1
structure can be generated using both Vaccinia Virus Capping Enzyme
and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure can be generated from
the Cap 1 structure followed by the 2'-O-methylation of the
5'-antepenultimate nucleotide using a 2'-O methyl-transferase. Cap
3 structure can be generated from the Cap 2 structure followed by
the 2'-O-methylation of the 5'-preantepenultimate nucleotide using
a 2'-O methyl-transferase. Enzymes can be derived from a
recombinant source.
[2125] When transfected into mammalian cells, the modified mRNAs
can have a stability of between 12-18 hours or more than 18 hours,
e.g., 24, 36, 48, 60, 72 or greater than 72 hours.
Example 7
Capping Assays
A. Protein Expression Assay
[2126] Polynucleotides encoding a polypeptide, containing any of
the caps taught herein, can be transfected into cells at equal
concentrations. After 6, 12, 24 and 36 hours post-transfection, the
amount of protein secreted into the culture medium can be assayed
by ELISA. Synthetic polynucleotides that secrete higher levels of
protein into the medium would correspond to a synthetic
polynucleotide with a higher translationally-competent Cap
structure.
B. Purity Analysis Synthesis
[2127] Polynucleotides encoding a polypeptide, containing any of
the caps taught herein, can be compared for purity using denaturing
Agarose-Urea gel electrophoresis or HPLC analysis. Polynucleotides
with a single, consolidated band by electrophoresis correspond to
the higher purity product compared to polynucleotides with multiple
bands or streaking bands. Synthetic polynucleotides with a single
HPLC peak would also correspond to a higher purity product. The
capping reaction with a higher efficiency would provide a more pure
polynucleotide population.
C. Cytokine Analysis
[2128] Polynucleotides encoding a polypeptide, containing any of
the caps taught herein, can be transfected into cells at multiple
concentrations. After 6, 12, 24 and 36 hours post-transfection the
amount of pro-inflammatory cytokines such as TNF-alpha and IFN-beta
secreted into the culture medium can be assayed by ELISA.
Polynucleotides resulting in the secretion of higher levels of
pro-inflammatory cytokines into the medium would correspond to
polynucleotides containing an immune-activating cap structure.
D. Capping Reaction Efficiency
[2129] Polynucleotides encoding a polypeptide, containing any of
the caps taught herein, can be analyzed for capping reaction
efficiency by LC-MS after nuclease treatment. Nuclease treatment of
capped polynucleotides would yield a mixture of free nucleotides
and the capped 5'-5-triphosphate cap structure detectable by LC-MS.
The amount of capped product on the LC-MS spectra can be expressed
as a percent of total polynucleotide from the reaction and would
correspond to capping reaction efficiency. The cap structure with
higher capping reaction efficiency would have a higher amount of
capped product by LC-MS.
Example 8
Agarose Gel Electrophoresis of Modified RNA or RT PCR Products
[2130] Individual polynucleotides (200-400 ng in a 20 .mu.l volume)
or reverse transcribed PCR products (200-400 ng) can be loaded into
a well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen,
Carlsbad, Calif.) and run for 12-15 minutes according to the
manufacturer protocol.
Example 9
Nanodrop Modified RNA Quantification and UV Spectral Data
[2131] Modified polynucleotides in TE buffer (1 .mu.l) can be used
for Nanodrop UV absorbance readings to quantitate the yield of each
polynucleotide from an chemical synthesis or in vitro transcription
reaction.
Example 10
Formulation of Modified mRNA Using Lipidoids
[2132] Polynucleotides can be formulated for in vitro experiments
by mixing the polynucleotides with the lipidoid at a set ratio
prior to addition to cells. In vivo formulation can require the
addition of extra ingredients to facilitate circulation throughout
the body. To test the ability of these lipidoids to form particles
suitable for in vivo work, a standard formulation process used for
siRNA-lipidoid formulations can be used as a starting point. After
formation of the particle, polynucleotide can be added and allowed
to integrate with the complex. The encapsulation efficiency can be
determined using a standard dye exclusion assays.
Example 11
Method of Screening for Protein Expression
A. Electrospray Ionization
[2133] A biological sample that can contain proteins encoded by a
polynucleotide administered to the subject can be prepared and
analyzed according to the manufacturer protocol for electrospray
ionization (ESI) using 1, 2, 3 or 4 mass analyzers. A biologic
sample can also be analyzed using a tandem ESI mass spectrometry
system.
[2134] Patterns of protein fragments, or whole proteins, can be
compared to known controls for a given protein and identity can be
determined by comparison.
B. Matrix-Assisted Laser Desorption/Ionization
[2135] A biological sample that can contain proteins encoded by one
or more polynucleotides administered to the subject can be prepared
and analyzed according to the manufacturer protocol for
matrix-assisted laser desorption/ionization (MALDI).
[2136] Patterns of protein fragments, or whole proteins, can be
compared to known controls for a given protein and identity can be
determined by comparison.
C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry
[2137] A biological sample, which can contain proteins encoded by
one or more polynucleotides, can be treated with a trypsin enzyme
to digest the proteins contained within. The resulting peptides can
be analyzed by liquid chromatography-mass spectrometry-mass
spectrometry (LC/MS/MS). The peptides can be fragmented in the mass
spectrometer to yield diagnostic patterns that can be matched to
protein sequence databases via computer algorithms. The digested
sample can be diluted to achieve 1 ng or less starting material for
a given protein. Biological samples containing a simple buffer
background (e.g., water or volatile salts) are amenable to direct
in-solution digest; more complex backgrounds (e.g., detergent,
non-volatile salts, glycerol) require an additional clean-up step
to facilitate the sample analysis.
[2138] Patterns of protein fragments, or whole proteins, can be
compared to known controls for a given protein and identity can be
determined by comparison.
Example 12
Synthesis of mRNA Encoding PBGD
[2139] Sequence optimized polynucleotides encoding PBGD
polypeptides, i.e., SEQ ID NOs: 1, 3, 5, or 7, were synthesized and
characterized as described in Examples 1 to 11. mRNAs encoding both
human PBGD isoforms were prepared for the Examples described below,
and were synthesized and characterized as described in Examples 1
to 11.
[2140] An mRNA encoding human PBGD can be constructed, e.g., by
using the ORF sequence provided in SEQ ID NO: 2, 4, 6, or 8. The
mRNA sequence includes both 5' and 3' UTR regions (see, e.g., SEQ
ID NOs: 83 and 84, respectively). In a construct, the 5'UTR and
3'UTR sequences are:
TABLE-US-00008 5' UTR (SEQ ID NO: 83)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAA
TAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 3' UTR (SEQ ID NO: 84)
TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCC
CCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATA
AAGTCTGAGTGGGCGGC or 3' UTR (SEQ ID NO: 149)
TGATAATAGTCCATAAAGTAGGAAACACTACAGCTGGAGCCTCGGTGGCCA
TGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACC
CGTACCCCCCGCATTATTACTCACGGTACGAGTGGTCTTTGAATAAAGTCT GAGTGGGCGGC
[[miR142 + miR126 3' UTR]]
[2141] The PBGD mRNA sequence was prepared as modified mRNA.
Specifically, during in vitro translation, modified mRNA can be
generated using 5-methoxy-UTP to ensure that the mRNAs contain 100%
5-methoxy-uridine instead of uridine. Further, PBGD-mRNA can be
synthesized with a primer that introduces a polyA-tail, and a Cap 1
structure is generated on both mRNAs using Vaccinia Virus Capping
Enzyme and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl.
Example 13
Detecting Endogenous PBGD Expression In Vitro
[2142] PBGD expression is characterized in a variety of cell lines
derived from both mice and human sources. Cells are cultured in
standard conditions and cell extracts are obtained by placing the
cells in lysis buffer. For comparison purposes, appropriate
controls are also prepared. To analyze PBGD expression, lysate
samples are prepared from the tested cells and mixed with lithium
dodecyl sulfate sample loading buffer and subjected to standard
Western blot analysis. For detection of PBGD, the antibody used is
a commercial anti-PBGD antibody. For detection of a load control,
the antibody used is anti-.beta.-Actin (mouse monoclonal; A2228;
Sigma). To examine the localization of endogenous PBGD,
immunofluorescence analysis is performed on cells. PBGD expression
is detected using a commercial anti-PBGD. The location of specific
organelles can be detected with existing commercial products. For
example, mitochondria can be detected using Mitotracker, and the
nucleus can be stained with DAPI. Image analysis is performed on a
Zeiss ELYRA imaging system.
[2143] Endogenous PBGD expression can be used as a base line to
determine changes in PBGD expression resulting from transfection
with mRNAs comprising nucleic acids encoding PBGD.
Example 14
In Vitro Expression of PBGD in HeLa Cells
[2144] To measure in vitro expression of human PBGD in HeLa cells,
those cells are seeded on 12-well plates (BD Biosciences, San Jose,
USA) one day prior to transfection. mRNA formulations comprising
human PBGD or a GFP control are transfected using 800 ng mRNA and 2
.mu.L Lipofectamin 2000 in 60 .mu.L OPTI-MEM per well and
incubated.
[2145] After 24 hours, the cells in each well are lysed using a
consistent amount of lysis buffer. Appropriate controls are used.
Protein concentrations of each are determined using a BCA assay
according to manufacturer's instructions. To analyze PBGD
expression, equal loads of each lysate (50 .mu.g) are prepared in a
loading buffer and subjected to standard Western blot analysis. For
detection of PBGD, a commercial anti-PBGD antibody is used
according to the manufacturer's instructions.
Example 15
In Vitro PBGD Activity in HeLa Cells
[2146] An in vitro PBGD activity assay is performed to determine
whether PBGD exogenously-expressed after introduction of mRNA
comprising a PBGD sequence is active.
A. Expression Assay
[2147] HeLa cells are transfected with mRNA formulations comprising
human PBGD or a GFP control. Cells are transfected with
Lipofectamin 2000 and lysed as described in Example 14 above.
Appropriate controls are also prepared.
B. Activity Assay
[2148] To assess whether exogenous PBGD can function, an in vitro
activity assay is performed using transfected HeLa cell lysates as
the source of enzymatic activity. To begin, lysate is mixed PBGD
substrate. The reaction is stopped by 5% TCA and vortexing. The
reaction tubes are then centrifuged at 2,000 g for 10 min, and the
supernatant is exposed to light for 20 min and analyzed for the
presence of oxidized enzymatic products resulting from the activity
of PBGD using fluorometric quantification.
Example 16
Measuring In Vitro Expression of PBGD in Cells
[2149] Cells from normal subjects and acute intermittent porphyria
patients are examined for their capacity to express exogenous PBGD.
Cells are transfected with mRNA formulations comprising human PBGD,
mouse PBGD, or a GFP control via electroporation using a standard
protocol. Each construct is tested separately. After incubation,
cells are lysed and protein concentration in each lysate is
measured using a suitable assay, e.g., by BCA assay. To analyze
PBGD expression, equal loads of each lysate are prepared in a
loading buffer and subjected to standard Western blot analysis. For
detection of PBGD, an anti-PBGD is used. For detection of a load
control, the antibody used is anti-.beta.-Actin (mouse monoclonal;
A2228; Sigma).
Example 17
Measuring In Vitro PBGD Activity in Lysates
A. Expression
[2150] Cells from normal human subjects and acute intermittent
porphyria patients are cultured. Cells are transfected with mRNA
formulations comprising human PBGD, mouse PBGD, or a GFP control
via electroporation using a standard protocol.
B. Activity Assay
[2151] To assess whether exogenous PBGD function, an in vitro
activity assay is performed using transfected cell lysates as the
source of enzymatic activity. Lysate containing expressed PBGD
protein is incubated with PBGD substrate, and the activity of PBGD
is quantified by measuring the levels of oxidized products
resulting from the enzymatic activity of PBGD.
Example 18
In Vivo PBGD Expression in Animal Models
[2152] To assess the ability of PBGD-containing mRNA's to
facilitate PBGD expression in vivo, mRNA encoding human PBGD is
introduced into CD1 mice. CD1 mice are injected intravenously with
either control mRNA (Luciferase) or human PBGD mRNA. The mRNA is
formulated in lipid nanoparticles for delivery into the mice. Mice
are sacrificed after 24 or 48 hrs. and PBGD protein levels in liver
lysates are determined by capillary electrophoresis (CE).
.beta.-Actin expression is examined for use as a load control. For
control Luciferase injections, 4 mice are tested for each time
point. For human PBGD mRNA injections, 4 mice are tested for each
time point.
Example 19
Human PBGD Mutant and Chimeric Constructs
[2153] A polynucleotide of the present invention can comprise at
least a first region of linked nucleosides encoding human PBGD,
which can be constructed, expressed, and characterized according to
the examples above. Similarly, the polynucleotide sequence can
contain one or more mutations that results in the expression of a
PBGD with increased or decreased activity. Furthermore, the
polynucleotide sequence encoding PBGD can be part of a construct
encoding a chimeric fusion protein.
Example 20
Production of Nanoparticle Compositions
[2154] A. Production of nanoparticle compositions
[2155] Nanoparticles can be made with mixing processes such as
microfluidics and T-junction mixing of two fluid streams, one of
which contains the polynucleotide and the other has the lipid
components.
[2156] Lipid compositions can be prepared by combining an ionizable
amino lipid disclosed herein, e.g., a lipid according to Formula
(I) such as Compound 18 or a lipid according to Formula (III) such
as Compound 236, a phospholipid (such as DOPE or DSPC, obtainable
from Avanti Polar Lipids, Alabaster, Ala.), a PEG lipid (such as
1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known
as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, Ala. or
Compound 428), and a structural lipid (such as cholesterol,
obtainable from Sigma-Aldrich, Taufkirchen, Germany, or a
corticosteroid (such as prednisolone, dexamethasone, prednisone,
and hydrocortisone), or a combination thereof) at concentrations of
about 50 mM in ethanol. Solutions should be refrigerated for
storage at, for example, -20.degree. C. Lipids are combined to
yield desired molar ratios and diluted with water and ethanol to a
final lipid concentration of between about 5.5 mM and about 25
mM.
[2157] Nanoparticle compositions including a polynucleotide and a
lipid composition can be prepared by combining the lipid solution
with a solution including the a polynucleotide at lipid composition
to polynucleotide wt:wt ratios between about 5:1 and about 50:1.
The lipid solution is rapidly injected using aNanoAssemblr
microfluidic based system at flow rates between about 10 ml/min and
about 18 ml/min into the polynucleotide solution to produce a
suspension with a water to ethanol ratio between about 1:1 and
about 4:1.
[2158] For nanoparticle compositions including an RNA, solutions of
the RNA at concentrations of 0.1 mg/ml in deionized water are
diluted in 50 mM sodium citrate buffer at a pH between 3 and 4 to
form a stock solution.
[2159] Nanoparticle compositions can be processed by dialysis to
remove ethanol and achieve buffer exchange. Formulations are
dialyzed twice against phosphate buffered saline (PBS), pH 7.4, at
volumes 200 times that of the primary product using Slide-A-Lyzer
cassettes (Thermo Fisher Scientific Inc., Rockford, Ill.) with a
molecular weight cutoff of 10 kD. The first dialysis is carried out
at room temperature for 3 hours. The formulations are then dialyzed
overnight at 4.degree. C. The resulting nanoparticle suspension is
filtered through 0.2 .mu.m sterile filters (Sarstedt, Ntimbrecht,
Germany) into glass vials and sealed with crimp closures.
Nanoparticle composition solutions of 0.01 mg/ml to 0.10 mg/ml are
generally obtained.
[2160] The method described above induces nano-precipitation and
particle formation. Alternative processes including, but not
limited to, T-junction and direct injection, can be used to achieve
the same nano-precipitation.
B. Characterization of Nanoparticle Compositions
[2161] A Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem,
Worcestershire, UK) can be used to determine the particle size, the
polydispersity index (PDI) and the zeta potential of the
nanoparticle compositions in 1.times.PBS in determining particle
size and 15 mM PBS in determining zeta potential.
[2162] Ultraviolet-visible spectroscopy can be used to determine
the concentration of a polynucleotide (e.g., RNA) in nanoparticle
compositions. 100 .mu.L of the diluted formulation in 1.times.PBS
is added to 900 .mu.L of a 4:1 (v/v) mixture of methanol and
chloroform. After mixing, the absorbance spectrum of the solution
is recorded, for example, between 230 nm and 330 nm on a DU 800
spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,
Calif.). The concentration of polynucleotide in the nanoparticle
composition can be calculated based on the extinction coefficient
of the polynucleotideused in the composition and on the difference
between the absorbance at a wavelength of, for example, 260 nm and
the baseline value at a wavelength of, for example, 330 nm.
[2163] For nanoparticle compositions including an RNA, a
QUANT-IT.TM. RIBOGREEN.RTM. RNA assay (Invitrogen Corporation
Carlsbad, Calif.) can be used to evaluate the encapsulation of an
RNA by the nanoparticle composition. The samples are diluted to a
concentration of approximately 5 .mu.g/mL in a TE buffer solution
(10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 .mu.L of the diluted
samples are transferred to a polystyrene 96 well plate and either
50 .mu.L of TE buffer or 50 .mu.L of a 2% Triton X-100 solution is
added to the wells. The plate is incubated at a temperature of
37.degree. C. for 15 minutes. The RIBOGREEN.RTM. reagent is diluted
1:100 in TE buffer, and 100 .mu.L of this solution is added to each
well. The fluorescence intensity can be measured using a
fluorescence plate reader (Wallac Victor 1420 Multilablel Counter;
Perkin Elmer, Waltham, Mass.) at an excitation wavelength of, for
example, about 480 nm and an emission wavelength of, for example,
about 520 nm. The fluorescence values of the reagent blank are
subtracted from that of each of the samples and the percentage of
free RNA is determined by dividing the fluorescence intensity of
the intact sample (without addition of Triton X-100) by the
fluorescence value of the disrupted sample (caused by the addition
of Triton X-100).
[2164] Exemplary formulations of the nanoparticle compositions are
presented in the TABLE 6 below. The term "Compound" refers to an
ionizable lipid such as MC3, Compound 18, or Compound 236.
"Phospholipid" can be DSPC or DOPE. "PEG-lipid" can be PEG-DMG or
Compound 428.
TABLE-US-00009 TABLE 6 Exemplary Formulations of Nanoparticles
Composition (mol %) Components 40:20:38.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 45:15:38.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 50:10:38.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 55:5:38.5:1.5
Compound:Phospholipid:Chol:PEG-Lipid 60:5:33.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 45:20:33.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 50:20:28.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 55:20:23.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 60:20:18.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 40:15:43.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 50:15:33.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 55:15:28.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 60:15:23.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 40:10:48.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 45:10:43.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 55:10:33.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 60:10:28.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 40:5:53.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 45:5:48.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 50:5:43.5:1.5
Compound:Phospholipid:Chol:PEG-lipid 40:20:40:0
Compound:Phospholipid:Chol:PEG-lipid 45:20:35:0
Compound:Phospholipid:Chol:PEG-lipid 50:20:30:0
Compound:Phospholipid:Chol:PEG-lipid 55:20:25:0
Compound:Phospholipid:Chol:PEG-lipid 60:20:20:0
Compound:Phospholipid:Chol:PEG-lipid 40:15:45:0
Compound:Phospholipid:Chol:PEG-lipid 45:15:40:0
Compound:Phospholipid:Chol:PEG-lipid 50:15:35:0
Compound:Phospholipid:Chol:PEG-lipid 55:15:30:0
Compound:Phospholipid:Chol:PEG-lipid 60:15:25:0
Compound:Phospholipid:Chol:PEG-lipid 40:10:50:0
Compound:Phospholipid:Chol:PEG-lipid 45:10:45:0
Compound:Phospholipid:Chol:PEG-lipid 50:10:40:0
Compound:Phospholipid:Chol:PEG-lipid 55:10:35:0
Compound:Phospholipid:Chol:PEG-lipid 60:10:30:0
Compound:Phospholipid:Chol:PEG-lipid
Example 21
Hepatic PBGD Activity in AIP Mice after Administration of Modified
mRNA Encoding PBGD
[2165] Hepatic PBGD activity was determined in AIP mice
administered mRNA encoding PBGD. AIP mice are compound
heterozygotes of two different disruptions of the PBGD gene: T1
strain [C57BL/6-pbgd.sup.tm1(neo)Uam and T2 strain
(C57BL/6-pbgd.sup.tm2(neo)Uam] (Lindberg R L, et al.
"Porphobilinogen deaminase deficiency in mice causes a neuropathy
resembling that of human hepatic porphyria." Nat Genet. 1996;
12:195-199). These AIP mice develop symptoms that mimic those in
humans with AIP. For example, the AIP mouse phenotype includes,
e.g., overproduction of aminolevulinic acid in response to
phenobarbital treatment, increased urinary excretion of
aminolevulinic acid and porphobilinogen in response to
phenobarbital treatment, slightly increased excretion of
uroporphyrins, pain, ataxia, slower movements, shorter stride
length, skeletal muscle degeneration, and erythruria.
[2166] Sequence modified (1-methyl-pseudouridine) mRNAs comprising
an ORF encoding wild-type PBGD (PBGD COV1 or PBGD COV2), PBGD-SM
(gain of function PBGD mutant with SM mutation), and ApoA1-PBGD-SM
(gain of function PBGD mutant with SM mutation fused to
apolipoprotein A1) at 1 nmol/kg were intravenously administered to
AIP mice (n=4), and hepatic PBGD activity was measured by a
fluorimetric assay (pmol uroporphyrin I/mg protein/hour). The
modified mRNA encoding PBGD-SM included a miR-142 binding site. The
modified mRNAs encoding wild type PBGD, PBGD COV1 (codon optimized
variant 1) and PBGD COV2 (codon optimized variant 2), did not
include a miR-142 binding site. mRNAs were formulated in MC3 lipid
nanoparticles.
[2167] PBGD activity in tissue homogenates was determined by
measuring the conversion of PBG to uroporphyrin. Briefly, 1 g of
tissue was homogenized at 4.degree. C. in 4 vol of KCl solution
1.15%. The homogenate was centrifuged at 12.000 rpm at 4.degree. C.
for 20 minutes and the clear supernatant without any cellular
debris was used the same day for protein determination (Bradford
assay using albumin standard) and PBGD activity. The supernatant
samples were diluted 1:3 with phosphate buffer (pH 7.6), DTT,
Cl.sub.2Mg and Triton X-100; and 100 .mu.l of this mixture were
pre-incubated with 1.8 ml of Tris-HCl 0.1M (pH 8.1) for 3 min at
37.degree. C. Next the mixture was incubated in the dark with 0.5
ml PBG substrate 1 mM 60 min at 37.degree. C. The reaction was
stopped with 350 .mu.l cold TCA 40% and the uroporphyrinogen formed
was oxidised to uroporphyrin after light exposure. Uroporphyrins
were measured quantitatively in a spectrofluorometer with an
excitation peak at 405 nm and window emission peak values between
550-660 nm. PBGD activity was expressed in terms of pmol
uroporphyrin/mg protein/h using appropriate standards.
[2168] The hepatic PBGD activity in AIP mice following
administration of PBGD mRNA is shown in FIG. 13. For comparison,
the 12 units threshold line in FIG. 13 is the level of PBGD
observed in livers of wild-type mice (C57BL/6), and the 3 units
threshold line is the level of PBGD in livers of AIP mice without
treatment.
[2169] The results show that after 24 hours, hepatic PBGD activity
was within the therapeutic window for up to 7 days after
administration. An acute porphyria attack in a human lasts between
5 and 7 days. In humans, the increase from 2 units to 5 units
completely restores the PBGD activity in the liver. Accordingly,
these results suggest that a single administration of mRNA encoding
wild-type PBGD could protect against an acute porphyria attack.
Example 22
Single Dose IV Administration of mRNA Encoding PBGD to AIP Mice
[2170] The ability of PBGD mRNA to reduce symptoms in AIP mice
after multiple phenobarbital induced attacks was tested. 0.5 mg/kg
doses of 1-methyl-pseudouridine modified mRNAs comprising an ORF
encoding wild-type or SM variant of human PBGD or 0.5 mg/kg doses
of control luciferase mRNA were formulated in lipid nanoparticles
(MC3) and administered to AIP mice (n=4) according to the dosing
schedule presented in FIG. 14. The mRNAs were administered as a
single intravenous dose on day 2 of the study. Mice were subjected
to 3 phenobarbital challenges, each of which consisted of 4
separate intraperitoneal injections of phenobarbital. The end
points of the study included (A) urinary excretion of porphyrins
and porphyrin precursors, (B) pain measurements, (C) peripheral
neuropathy using rotarod and footprint analysis, and (D) sciatic
nerve function.
[2171] A. Reduction of Urinary ALA and PBG Excretion:
[2172] Urinary aminolevulinic acid (ALA) excretion and urinary
porphobilinogen (PBG) excretion were measured in the AIP mice
administered PBGD mRNA or luciferase control.
[2173] Urinary excretion of aminolevulinate acid (ALA) and
porphobilinogen (PBG) were quantified using a quantitative ion
exchange column method (BioSystems SA, Barcelona) and measured at
555 nm in an Ultrospc 3000 spectrophotometer (Pharmacia Biotech,
Buckinghamshire, UK). Unzu et al., Mol Ther. 19(2):243-50
(2011).
[2174] Urinary ALA excretion (g ALA/mg creatinine) and urinary PBG
excretion (g PBG/mg creatinine) levels over time are shown in FIG.
15 and FIG. 16, respectively. Both urinary ALA and PBG increased
markedly in AIP mice treated with control luciferase mRNA, reaching
levels of twenty-fold and sixty-fold greater, respectively,
compared to mean baseline values. In contrast, after the first
phenobarbital challenge, full protection against the ALA and PBG
over-excretion was obtained in mice administered PBGD mRNA. After
the administration of PBGD mRNA, ALA and PBG levels were maintained
close to baseline levels. A protective effect was observed in PBGD
mRNA treated mice after the second phenobarbital challenge, which
took place about 2 weeks after mRNA administration. After three
weeks, the protective effect was no longer observed.
[2175] These results indicate that (i) there was a very fast onset
of the protective effect (within one day) by PBGD mRNA
administration, (ii) the observed effect on ALA and PBG urinary
excretion was very potent (close to baseline levels), and (iii) the
observed protective effect was long lasting, indicating that the
half-life of the modified PBGD mRNA is very long (at least two
weeks).
[2176] B. Protection Against Pain:
[2177] 95% of human patients suffering from AIP experience severe
pain. The effect of mRNA encoding wild-type or SM variant of PBGD
on pain in AIP mice was tested. Pain was measured based on
observations of orbital tightening, nose bulge, cheek bulge, ear
position, whisker change, abdominal grooming, and respiratory
distress (Langford et al. Nat Methods. 7(6):447-9 (2010)). The data
presented in FIG. 17 shows that a single administration of mRNA
encoding wild-type or SM variant of human PBGD protected against
pain induced after the first phenobarbital challenge, and extended
protection against recurrent phenobarbital-induced pain was
observed in the AIP mice after the second phenobarbital challenge.
Control luciferase mRNA provided no pain protection in AIP mice
following phenobarbital challenge.
[2178] C. Protection Against Peripheral Neuropathy:
[2179] Peripheral neuropathy was assessed by evaluating rotarod
performance (movement and balance) and gait patterns (stride length
and base of support) after phenobarbital challenge. Motor
coordination was evaluated by the rotarod test, which measures the
time (in seconds) that a mouse can stay on a rotating dowel turning
at increasing speed (Unzu et al., Mol Ther. 19(2):243-50 (2011)).
To assess gait pattern, the stride length and base of support
(i.e., distance between the central pads of the hind feet) was
determined substantially as described in Lindberg R L et al., Nat
Genet, 12: 195-99 (1996). The time spent on the rotarod (seconds)
for each group is shown in FIG. 18A, and stride length (cm) for
each group is shown in FIG. 18B.
[2180] As shown in FIG. 18A, after the each phenobarbital
challenge, the time that AIP mice administered control luciferase
mRNA were able to stay on the rotarod significantly decreased. On
the other hand, after the each phenobarbital challenge, the time
that AIP mice administered PBGD mRNA were able to stay on the
rotarod was comparable to the time observed under baseline
conditions (no phenobarbital challenge).
[2181] As shown in FIG. 18B, AIP mice administered control
luciferase mRNA had decreased stride length compared to animals
administered PBGD mRNA. The gait of AIP mice treated with control
luciferase mRNA deteriorated with each phenobarbital challenge. On
the other hand, the gait maintained in AIP mice treated with PBGD
mRNA. Thus, these results show that the administration of modified
mRNA encoding wild-type or SM variant of human PBGD protected AIP
against peripheral motor disturbance induced by phenobarbital.
[2182] D. Improvement in Sciatic Nerve Dysfunction Caused by
Recurrent Acute Attacks:
[2183] Sciatic nerve function was assessed in AIP mice administered
control luciferase mRNA or PBGD mRNA using substantially the
methods disclosed in Unzu et al., Mol Ther. 19(2):243-50, 2011.
[2184] Axon Measurement.
[2185] Sciatic nerves were fixed overnight at 4.degree. C. in 4%
glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, and
post-fixed for 2.5 h at 4.degree. C. in 1% phosphate-buffered
osmium tetroxide. Samples were dehydrated with ethanol, washed with
propylene oxide and embedded in Epon 812. Semithin (1 mm thick)
sections were cut and stained with 1% toluidine blue for
examination by light microscopy. Axon sections were acquired in a
M1 Zeiss microscope and an INSIGHT AxioCamm ICc3 camera using
AxioVision Zeiss Imaging software. The image analysis software has
been developed using MATLAB v.7.1.0 and DIPlib v1.6 C libraries
under OS Red Hat Linux AS 2.6.18-53.e15. Caliber and density of
axons were quantified by computer-assisted image analysis.
[2186] Neurophysiological Studies.
[2187] Electrophysiological studies included measurements of nerve
conduction velocity and action potential amplitude of the sciatic
nerve. The electrical stimuli were delivered by means of pairs of
needles placed subcutaneously near the exit point of the sciatic
nerve at the hip, with a separation of 0.5 mm and the cathode
located distally. Stimuli were delivered by a Grass S180 stimulator
with a Grass constant current unit (Grass-AstroMed, W. Worwick,
USA), synchronized with the recording equipment. Stimulus intensity
was progressively increased to ensure the maximal response
amplitude. The motor potential was recorded by means of steel
needle electrodes (Viasys Heathcare, UK) located at the center of
the soleus muscle (active) (midpoint between the ankle and the
knee) and near the insertion of the Achilles tendon. The responses
were amplified by a Grass P511 amplifier (Grass Astromed, USA),
digitized by means of a CED power 1401 A/D converter (Cambridge
Electronic Design, Cambridge, UK), and stored in a PC using Signal
3 software (Cambridge Electronic Design, Cambridge, UK).
Stimulation intensity was increased gradually until the amplitude
of the response did not increase any further. The intensity used
for the measured potentials was at least 20% higher than the
minimal value associated with a maximal response. The best response
out of 10 adequate potentials was selected for the analysis. The
response analyzed consisted of a biphasic potential
negative/positive in all cases. Amplitude, latency and duration of
the compound muscle action potential were measured and
double-checked by a different person.
[2188] Increased latency and loss of amplitude correlate with motor
dysfunction and axonal loss developed in AIP mice after repeated
phenobarbital induction of acute attacks (Unzu et al. Mol Ther.
2011). In AIP mice, abnormalities in the nerve conduction
velocities occur with aging and after recurrent acute attacks
induced by increasing doses of phenobarbital. In the human
porphyria, peripheral neuropathy is observed in patients after
maintained high levels of porphyrin precursors over-time.
Peripheral neuropathy can be asymmetric in acute porphyrias.
[2189] The compound-muscle action potentials evoked by proximal
stimulation of the sciatic nerve were measured at day 27
post-injection), 8 hour last phenobarbital challenge in the two
hind legs of AIP mice administered mRNA encoding luciferase or mRNA
encoding wild type PBGD or mRNA encoding PBGD-SM protein variant.
FIG. 19A, FIG. 19B and FIG. 19C show examples of the amplitude over
time (right leg) for control, PBGD mRNA and PGBD-SM mRNA treated
mice, respectively. The control luciferase mRNA group had short
amplitude and long latency measurements and the PBGD mRNA group had
longer amplitude and shorter latency measurements. The latency
after three consecutive phenobarbital challenges (ms) for each
group is shown in FIG. 19D. The amplitude after three consecutive
phenobarbital challenges (mV) for each group is shown in FIG. 19E.
Thus, the administration of a single dose of modified mRNA encoding
wild-type human PBGD protected against increased latency and loss
of amplitude in AIP mice following recurrent acute attacks. The
administration of a single dose of modified mRNA encoding human
PBGD-SM protected against increased latency in AIP mice following
recurrent acute attacks.
[2190] These results show significant therapeutic efficacy of mRNA
encoding PBGD for treatment of AIP signs and symptoms in vivo. The
PBGD mRNA treated AIP mouse, although challenged with
phenobarbital, showed no signs of pain and was able to move around
as actively as a wild type mouse. On the contrary, the luciferase
mRNA treated AIP mouse during phenobarbital induced attack appeared
to be in great pain and could not move freely and showed balancing
problems in the movement.
Example 23
Hepatic Expression of PBGD Protein After Administration of AAV-PBGD
DNA and PBGD mRNA
[2191] Liver tissue from AIP mice injected with an AAV-PBGD vector
or with mRNA comprising an ORF encoding PBGD were analyzed by
immunohistochemistry (IHC) using a rabbit polyclonal anti-PBGD
antibody (Santa cruz # sc-67037) and compared to liver from
untreated AIP mice.
[2192] Histology and PBGD Immunohistochemical Staining.
[2193] Formalin-fixed/paraffin embedded sections (5 .mu.m) of right
and left liver lobes were stained with hematoxylin eosin.
Immunohistochemistry was used to determine the proportion of cells
highly stained with the polyclonal anti-PBGD antibody produced in
rabbit (dilution 1:800). The secondary antibody used was goat
anti-rabbit coupled to a peroxidase-labelled dextran polymer (Dako,
K4003). Peroxidase was visualized using DAB+(Dako, K3468). After
counterstaining with hematoxylin to give a blue background contrast
to brown colour of the positive cells, the percentage of
PBGD-positive cells was calculated in at least 1000 cells from 4-6
fields of vision for each sample under microscope with 40.times.
magnifications. Hepatocytes and non-parenchymal cells were readily
differentiated based upon morphological criteria: hepatocytes have
a polyhedral shape, round nucleus and are larger than the
non-parenchymal cells, which have an irregular shape.
[2194] As shown in FIG. 20A, administration of gene therapy vector
AAV-PBGD in AIP mice induced aggregate staining in the liver
suggesting high PBGD protein levels (>60 U) in some hepatocytes.
A representative liver tissue sample from an untreated AIP mouse is
shown in FIG. 20B, which confirmed essentially very low PBGD
protein level (about 3 U) was detected in the liver of AIP mice. In
the liver of PBGD mRNA treated AIP mice, PBGD protein expression
was homogeneous throughout all hepatocytes and decreased over time.
Increased protein expression was observed at day 1 (about 19U), day
2 (about 14 U), and day 4 (about 4.7 U) after injection of the PBGD
mRNA, as shown in FIG. 20C, FIG. 20D, and FIG. 20E, respectively.
The expression of PBGD protein observed by IHC was consistent with
the PBGD hepatic activity shown in Example 21.
Example 24
Lipid Nanoparticle Delivery Systems for Administering mRNAs
Encoding Wild-Type Human PBGD
[2195] Different formulations of lipid nanoparticles encapsulating
PBGD mRNA were tested for the ability to reduce symptoms in AIP
mice after multiple phenobarbital induced attacks. 0.5 or 0.1 mg/kg
doses of 1-methyl-pseudouridine modified mRNAs comprising an ORF
encoding wild-type human PBGD or 0.5 mg/kg does of control mRNA
comprising an ORF encoding luciferase were formulated in lipid
nanoparticles (either MC3 or Compound 18) and administered to AIP
mice. The mRNAs were administered as a single intravenous dose on
day 2 of the study. Mice were subjected to 3 phenobarbital
challenges, each of which consisted of 4 separate intraperitoneal
injections of phenobarbital. The end points of the study included
(A) urinary excretion of porphyrins and porphyrin precursors, (B)
pain measurements, (C) peripheral neuropathy using rotarod, which
were tested according to the same methods described in Example 22
above. A mouse from the luciferase mRNA-MC3 (control) group, a
mouse from the luciferase mRNA-Compound 18 (control) group, and a
mouse from the PBGD mRNA-MC3 group died after the administration of
the third dose during the second phenobarbital challenge. All three
showed symptoms of an acute porphyria attack before their
deaths.
[2196] A. Reduction of Urinary ALA and PBG Excretion:
[2197] mRNAs encoding wild-type PBGD formulated in either MC3 or
Compound 18 fully protected against the increase of porphyrin
precursors ALA and PBG induced after a first phenobarbital
challenge, and significantly reduced their accumulation in the
second phenobarbital challenge as shown in FIG. 21A and FIG. 21B,
respectively. The administration of the lower 0.1 mg/kg dose of
PBGD mRNA formulated with Compound 18 showed a partial reduction of
porphyrin excretion after the first induction. Control luciferase
mRNA provided no reduction in ALA or PBG increase following
phenobarbital challenge when formulated with either lipid
nanoparticle.
[2198] B. Protection Against Pain:
[2199] The effect of the administration of mRNAs encoding wild-type
PBGD formulated with either MC3 or Compound 18 on pain in AIP mice
was evaluated. mRNA encoding luciferase formulated in MC3 or
Compound 18 was used as a control. As shown in FIG. 22A, mRNA
encoding wild-type PBGD formulated with MC3 at 0.5 mg/kg and
formulated with Compound 18 at 0.5 mg/kg or 0.1 mg/k doses, all
showed a reduction in pain. Administration of 0.1 mg/mg PBGD mRNA
formulated with Compound 18 showed a significant reduction in pain
when the first phenobarbital challenge was applied, but there was
no significant difference with respect to the controls when the
second or third phenobarbital challenge was applied. The pain
reduction with 0.5 mg/kg PBGD mRNA formulated in either MC3 or
Compound 18 was similar after the first and second phenobarbital
inductions. No pain reduction with either formulation was observed
after the third phenobarbital induction. Control luciferase mRNA
provided no protection against pain.
[2200] C. Protection Against Peripheral Neuropathy:
[2201] The time spent on the rotarod (% vs baseline values) for
each group is shown in FIG. 22B. The rotarod results after the
first and second inductions indicated that the 0.5 mg/kg PBGD mRNA
formulation in Compound 18 resulted in longest time spent on the
rotarod. The 0.5 mg/kg PBGD mRNA formulation in MC3 also resulted
in a time spent on the rotarod close to baseline. Control
luciferase treated mice had significantly reduced times on the
rotarod compared to baseline.
Example 25
PBGD Activity in Wild-Type Mice Administered Modified PBGD mRNA
Constructs
[2202] The ability of sequence optimized, chemically modified
PBGD-encoding mRNAs to facilitate PBGD activity in vivo was tested.
Wild-type CD1 mice were injected intravenously with (i) control
1-methyl-pseudouridine modified luciferase mRNA (ORF SEQ ID NO: 86)
(N=3), (ii) 1-methyl-pseudouridine modified mRNA comprising an ORF
without sequence engineering (wt PBGD (ORF SEQ ID NO: 87) in Table
8) encoding human PBGD (N=3); (iii) 1-methyl-pseudouridine modified
mRNA comprising a sequence optimized ORF encoding human PBGD
(constructs PBGD COV2 and PBGD COV1, ORF SEQ ID NO: 88 and 89,
respectively) (N=3); or (iv) 5-methoxyuridine modified mRNA
comprising a sequence optimized ORF encoding human PBGD (constructs
#1 to #26 and #29 to #31 in Table 7) (N=4). The 5-methoxyuridine
modified PBGD mRNA included mir-142 and mir-126 binding sites in
the 3' untranslated region for Constructs #1 to #27 and #29 to #31,
whereas Construct #28 included mir-142 binding site in the 3'
untranslated region (Table 7). The mRNAs were formulated in lipid
nanoparticles (Compound 18) for delivery into each mouse.
TABLE-US-00010 TABLE 7 Modified mRNA constructs including optimized
ORFs encoding human PBGD. Each of constructs #1-#28 comprises a
Cap1 5' terminal cap and a 3' terminal polyA region. 5'UTR PBGD
mRNA SEQ PBGD ORF 3'UTR SEQ construct ID NO Name SEQ ID NO ID NO #1
39 PBGD-CO30 89 149 #2 39 PBGD-CO31 90 149 #3 39 PBGD-CO32 91 149
#4 39 PBGD-CO33 92 149 #5 39 PBGD-CO34 93 149 #6 39 PBGD-CO35 94
149 #7 39 PBGD-CO36 95 149 #8 39 PBGD-CO37 96 149 #9 39 PBGD-CO38
97 149 #10 39 PBGD-CO39 98 149 #11 39 PBGD-CO40A 99 149 #12 39
PBGD-CO41A 100 149 #13 39 PBGD-CO42A 101 149 #14 39 PBGD-CO43A 102
149 #15 39 PBGD-CO44A 103 149 #16 39 PBGD-CO45A 104 149 #17 39
PBGD-CO46A 105 149 #18 39 PBGD-CO47A 106 149 #19 39 PBGD-CO40B 107
149 #20 39 PBGD-CO41B 108 149 #21 39 PBGD-CO42B 109 149 #22 39
PBGD-CO43B 110 149 #23 39 PBGD-CO44B 111 149 #24 39 PBGD-CO45B 112
149 #25 39 PBGD-CO46B 113 149 #26 39 PBGD-CO47B 114 149 #27 39
PBGD-CO45B 112 150 #28 39 PBGD-CO45B 112 151 wt 39 wt ORF 87 81
PBGD COV2 39 PBGD COV2 88 81 PBGD COV1 39 PBGD COV1 89 81 #29 39
PBGD-CO48 115 149 #30 39 PBGD-CO49 116 149 #31 39 PBGD-CO50 117
149
[2203] The hepatic PBGD activity in the wild-type mice at 24 hours
post-injection for sequence optimized, chemically modified PBGD
mRNA Constructs #1-18 formulated in Compound 18 compared to
controls is shown in Table 8.
TABLE-US-00011 TABLE 8 PBGD activity in PBGD mRNA constructs
Average PBGD activity (uroporphyrin I Construct (nM)) StdDev #1
358.4 23.5 #2 242.7 17.9 #3 375.0 76.8 #4 244.0 87.4 #5 266.7 66.9
#6 181.0 24.7 #7 383.9 260.3 #8 265.7 39.9 #9 296.9 96.0 #10 323.9
113.9 #11 401.1 33.8 #12 289.9 39.2 #13 348.0 73.5 #14 501.8 40.6
#15 216.4 52.8 #16 631.7 141.9 #17 400.9 146.8 #18 375.1 67.9 wt
PBGD 272.6 10.4 PBGD COV2 493.6 67.7 PBGD COV1 297.3 110.6
luciferase 103.4 11.6
[2204] The sequence optimized, chemically modified PBGD mRNAs
facilitated increased PBGD activity compared to controls. Notably,
sequence optimized, 5-methoxyuridine modified PBGD mRNA Constructs
#14 and #16 formulated in Compound 18 resulted in PBGD activity
significantly higher than the other 5-methoxyuridine modified
constructs. Sequence optimized, 5-methoxyuridine modified PBGD mRNA
(construct #14) formulated in Compound 18 was used in the further
studies described in Examples 26-27 below. Sequence optimized,
5-methoxyuridine modified PBGD mRNA (construct #16) formulated in
Compound 18 was used in the further studies described in Examples
28-31 below.
[2205] FIGS. 23A and 23B, respectively, show hepatic PBGD activity
and PBGD protein expression in the wild-type mice at 24 hours
post-injection with sequence optimized, chemically modified PBGD
mRNA Constructs #11, #14, #16, #19 to #26, and #29 to #31
formulated in Compound 18. All constructs tested resulted in PBGD
activity and protein expression well above the control luciferase
construct. Notably, sequence optimized, 5-methoxyuridine modified
PBGD mRNAs Constructs #24 and #26 formulated in Compound 18
resulted in PBGD activity and protein expression significantly
higher than the other 5-methoxyuridine modified constructs
tested.
Example 26
Multi-Dose Study in AIP Mice Administered Modified PBGD mRNA
[2206] The ability of multi-dose administration of mRNA encoding
human PBGD formulated in lipid nanoparticle to reduce
disease-associated biomarkers, pain and peripheral neuropathy in
AIP mice after multiple phenobarbital induced attacks was tested.
Three doses (0.5 mg/kg, 0.2 mg/kg, or 0.05 mg/kg) of sequence
optimized, 5-methoxyuridine modified (miR-142 and miR-126) mRNA
encoding PBGD (construct #14 in Table 7) formulated in Compound
18-containing lipid nanoparticles (Compound
18:Cholesterol:DSPC:DMG:PEG) (N=5, each dose) or a 0.5 mg/kg dose
of control 1-methyl-pseudouridine modified (no miR) mRNA encoding
luciferase formulated in Compound 18 (N=5) were administered
intravenously to AIP mice according to the dosing schedule
presented in FIG. 24. The mRNAs were administered as an IV bolus
once every two weeks (i.e., on days 2, 15, and 29) of the study.
AIP mice administered PBS control were included for comparison
(N=4). Mice were subjected to 3 phenobarbital challenges, each of
which consisted of 4 separate intraperitoneal injections of
phenobarbital. The end points of the study included (A) urinary
excretion of porphyrins and porphyrin precursors, (B) pain
measurements, (C) peripheral neuropathy using rotarod and footprint
analysis, (D) sciatic nerve function, (E) liver function test, and
(F) anti-PBGD antibody levels.
[2207] A. Reduction of Urinary ALA, PBG, and Porphyrin
Excretion:
[2208] Urinary excretion levels of ALA, PBG, and porphyrin in AIP
mice following phenobarbital challenge were measured as described
in Example 22 above. The results for urinary excretion of ALA are
shown in FIG. 25, PBG excretion is shown in FIG. 26, and porphyrin
excretion is shown in FIG. 27. Urinary ALA, PBG, and porphyrin
increased in AIP control mice administered control luciferase mRNA
or PBS after phenobarbital challenge. There was a dose-response
effect for AIP mice administered PBGD mRNA. The 0.05 mg/kg doses of
PBGD had no significant effect. Both the 0.2 mg/kg and 0.5 mg/kg
doses of PBGD mRNA reduced biomarker excretion after each
phenobarbital challenge, but the 0.5 mg/kg dose consistently
achieved the greatest reduction throughout all three challenges.
After the administration of the PBGD mRNA at 0.2 mg/kg and 0.5
mg/kg doses, ALA, PBG and prophyrin levels were close to baseline
levels. These results show that (i) there was a very fast onset of
the protective effect of the PBGD mRNA after administration, (ii)
the observed effect on ALA, PBG, and porphyrin urinary excretion
levels was very potent, and (iii) the observed protective effect
was achieved during each attack after multiple dosing.
[2209] B. Protection Against Pain:
[2210] The effect administering multiple 0.5 mg/kg, 0.2 mg/kg, and
0.05 mg/kg doses of PBGD mRNAs on pain in AIP mice after
phenobarbital induction was evaluated. Pain was assessed by
measuring orbital tightening, nose bulge, cheek bulge, ear
position, whisker change, abdominal grooming, and respiratory
distress. Pain measurement results (mean difference score) are
presented in FIG. 28. Data were log transformed prior to a repeated
measures ANOVA analysis to equalize variances and pairwise
comparisons were made using Bonferroni's multiple comparisons.
[2211] The results show that administration of PBGD mRNA at 0.2
mg/kg and 0.5 mg/kg doses protected against the pain induced after
all three phenobarbital challenges.
[2212] C. Protection Against Peripheral Neuropathy:
[2213] Improvement in peripheral neuropathy was assessed by rotarod
and footprint measurements (gait patterns). The rotarod (time spent
in seconds) and gait patterns (stride length in cm) are shown in
FIG. 29 and FIG. 30, respectively. Data are expressed as mean with
SD. In case of the statistical analysis, data were log transformed
prior to repeated measures ANOVA analysis to equalize variances and
comparisons between baseline and marks obtained after each
induction were made using Bonferroni's multiple comparisons. Some
animals increase their mark on the third induction due to training.
These results show that when administered a dose of 0.05 mg/kg the
PBGD mRNA had a weak effect. However, administration of PBGD mRNA
at doses of 0.2 mg/kg and 0.5 mg/kg protected against peripheral
motor disturbance induced after all three phenobarbital
challenges.
[2214] D. Improvement in Sciatic Nerve Dysfunction Caused by
Recurrent Acute Attacks:
[2215] Sciatic nerve function was assessed in AIP mice as described
in Example 22 above. The compound action potentials were measured
in the two hind legs of each animal. The amplitude parameter shown
in FIG. 31 gives an estimation of the total volume of fibers. AIP
mice administered PBS or control luciferase mRNA showed a reduction
in amplitude after three consecutive phenobarbital challenges. The
administration of 0.5 mg/kg of PBGD mRNA showed the most protection
against this loss of amplitude AIP challenged mice.
[2216] E. Evaluation of Serum Transaminases and Bilirubin:
[2217] Levels of serum transaminases (FIG. 32A and FIG. 32B) and
serum bilirubin (FIG. 32C) were assessed in AIP mice at the end of
the study.
[2218] Serum levels of the ALT (alanine transaminase) and AST
(aspartate transaminase) transaminases at sacrifice (six days after
the third administration of modified mRNA encoding wild type PBGD,
and three days after the last phenobarbital dose) showed no
significant variations among the groups and were similar to WT
mouse levels. There was no dose dependent effect.
[2219] Serum bilirubin levels were similar between groups as
well.
[2220] F. Anti-Drug Antibody (ADA) Assay:
[2221] The presence of anti-PBGD antibodies in the serum of the AIP
mice injected with doses of PBGD mRNA used in the experiments
described in this Example was determined using the following
protocol: [2222] 1. Coat with antigen (0.1 .mu.g in 50 .mu.l per
well) in carbonate buffer 0.1 M at 4.degree. C. overnight. [2223]
2. Wash three times with PBS-Tween 0.05% (200 .mu.l per well).
[2224] 3. Block with blocking solution PBS-Tween 0.05% with 5% milk
(200 .mu.l per well), 1 hour at room temperature. [2225] 4. Make 12
dilutions for each serum sample, starting with a 1:20 dilution, in
blocking solution and incubate for 1 hour 30 minutes at 37.degree.
C. (Vf=50 .mu.l). The serum was diluted 1:2, 50 .mu.l serum were
combined with 50 .mu.l assay diluent, and 50 .mu.L were used.
[2226] 5. Wash five times with PBS-Tween 0.05% (200 .mu.l per
well). [2227] 6. Add protein A HPRO in blocking solution with a
dilution 1:5,000, and incubate for 1 hour 30 minutes at 37.degree.
C. [2228] 7. Wash five times with PBS-Tween 0.05% (200 .mu.l per
well). [2229] 8. Add TMB (50 .mu.l per well) and incubate 3 min.
[2230] 9. Stop the reaction with sulfuric acid 2 M (30 .mu.l per
well). [2231] 10. Read the absorbance at 450 nm.
[2232] The ADA assay did not detect the presence of anti-PBGD
antibodies in the serum of the AIP mice (data not shown).
Example 27
Acute Treatment Study in AIP Mice Administered Modified PBGD
mRNA
[2233] In the treatment of acute AIP attacks, it is important to
reduce the very high levels of porphyrin precursors (ALA and PBG)
and porphyrin as fast as possible. Accordingly, a study was
designed to assess the speed of onset to drop urinary levels of
ALA, PBG and prophyrin after the administration of mRNA encoding
human PBGD.
[2234] AIP mice were subjected to a single phenobarbital challenge
to induce an acute AIP episode. After three intraperitoneal
injections of phenobarbital (at days 1, 2 and 3), but 2 hours prior
to the fourth phenobarbital injection in the challenge (at day 4),
sequence optimized, 5-methoxyuridine modified (miR-142/126) mRNA
encoding PBGD (construct #14 in Table 7) formulated in Compound
18-containing lipid nanoparticles (Compound
18:Cholesterol:DSPC:DMG:PEG) was administered intravenously as a
single-dose (0.5 mg/kg) intravenous bolus (N=5). Luciferase mRNA
also formulated in Compound 18-containing lipid nanoparticles
(Compound 18:Cholesterol:DSPC:DMG:PEG) administered with
phenobarbital challenge (N=4) and PBS administered without
phenobarbital challenge (N=3) were included as controls. An
additional fifth phenobarbital injection was given (at day 5) to
sustain the high levels of urinary biomarkers in luciferase
controls at days 4 and 5. The design of the study dosing is
summarized in FIGS. 33A-33C. After each phenobarbital injection,
mice were installed in a metabolic cage to obtain whole urine
during the next 24 hours for measurement of ALA, PBG and porphyrin
levels.
[2235] The results show a rapid lowering of urinary porphyrin
precursors ALA (FIG. 33A) and PBG (FIG. 33B) as well as the levels
of porphyrin (FIG. 33C) against an established acute attack with
administration of 0.5 mg/kg PBGD mRNA. ALA and PBG excretion levels
rapidly returned to basal values within 2 hours at day 4 between
PBGD mRNA and fourth phenobarbital injections, and porphyrin levels
returned to basal level 1 day after administration of PBGD mRNA (at
day 5). In contrast, ALA and PBG levels continued to rise and
porphyrin levels remained elevated at day 5 in mice administered
control luciferase mRNA.
[2236] In this study the modified mRNA was administered late during
the acute AIP episode when the biomarkers had reached peak levels
(as indicated by the values corresponding to the luciferase
control). Under these acute AIP attack conditions, PBGD mRNA
rapidly lowered all the biomarker levels to baseline.
Example 28
Pharmacokinetics Study of Modified mRNA Encoding Wild Type PBGD in
Wild Type CD-1 Mice
[2237] A study was conducted to evaluate the duration and
pharmacokinetics of PBGD mRNA administration in vivo. Sequence
optimized, 5-methoxyuridine modified (miR-142/126) mRNA encoding
PBGD (construct #16 in Table 7) formulated in lipid nanoparticles
(Compound 18) was administered to wild-type mice (CD-1, 9 weeks
old) as a single 0.5 mg/kg intravenous bolus. The time points of
the study were 2 hours, 6 hours, 10 hours, 16 hours, 24 hours, 2
days, 4 days, 7 days, 10 days, and 14 days. Upon sacrifice, livers
and spleens of the animals were perfused. mRNA levels were
determined using bDNA testing. Two samples were collected for bDNA
testing from the left and right medial lobes of the liver of each
animal for each time point (up to 4 days). PBGD activity and
protein levels were measured in liver (1 gram of liver tissue in
3.5 ml KCl solution in 15 ml tubes) and spleen (half spleen, i.e.,
approximately 50-100 mg, in 200 ul KCl solution in 1.5 ml Eppendorf
tubes). Pieces of liver and spleen tissue were fixed in 10%
formalin for histology analysis (both RNAscope and IHC). Liver
samples were taken from the left and right medial lobes and
immediately fixed in 10% formalin. Spleen samples were first cut
cross-sectionally and then fixed in 10% formalin. Small pieces of
liver tissue samples were snap frozen for absolute quantification
by LC/MSMS (n=2, left and right medial lobe sample samples for each
time point).
[2238] The hepatic PBGD activity over time in mice administered
PBGD mRNA compared to mice administered control luciferase mRNA is
shown in FIG. 34. As shown in FIG. 35A, the terminal t1/2 (by
noncompartmental analysis) of mRNA-encoded PBGD activity was 8
days. PBGD activity peaked between 6 and 10 hours, and then decayed
until it reached baseline levels at approximately 7 days post
injection. The half-life of mRNA-encoded PBGD activity was
consistent with the half-life of the protein encoded by the PBGD
construct used in Example 21 (see FIG. 13 and FIG. 35B). A single
dose of the modified mRNA encoding wild type PBGD appears to have
sufficient half-life to treat a full AIP acute attack. PBGD protein
levels in liver (FIG. 36) was consistent with the activity data.
The hepatic levels of human PBGD mRNA also correlated well with the
PBGD activity levels in liver (FIG. 37).
[2239] The PK data of splenic PBGD activity in mice showed no
increase in splenic PBGD (data not shown) indicating that the
increase in PBGD activity was specific to liver tissue. PBGD
protein levels in spleen were consistent with the activity data
(data not shown).
[2240] In the livers of PBGD mRNA treated WT CD1 mice, the PBGD
protein expression observed by IHC was consistent with the PBGD
activity shown in FIG. 34, and was also consistent with the human
PBGD protein levels detected by LC/MSMS shown in FIG. 36. The
expression of exogenous PBGD was homogeneous throughout all
hepatocytes and decreased after 16 hours post-injection. Increased
expression of PBGD protein above the basal level was observed at 2
hours, 6 hours, 10 hours, 16 hours, and 24 hours after injection of
the PBGD mRNA, as shown in FIGS. 38A, 38B, 38C, 38D, and 38E,
respectively. A representative liver tissue sample from a
luciferase mRNA treated WT CD1 mouse is shown in FIG. 38F to assess
the PBGD basal level. No PBGD protein expression was detected in
the luciferase control, which indicated no cross-reaction of the
Novus anti-PBGD antibody used for IHC analysis with mouse
species.
Example 29
Pharmacokinetics Study of Modified mRNA Encoding Wild Type PBGD in
AIP Mice
[2241] A study was conducted to evaluate the duration and
pharmacokinetics of PBGD mRNA administration in vivo. Wild-type
PBGD-encoding 1-methyl-pseudouridine modified mRNA comprising a
codon optimized ORF (PBGD COV1; ORF SEQ ID NO: 89) formulated in
MC3 was administered at 0.5 mg/kg, and sequence optimized,
5-methoxyuridine modified (miR-142 and miR-126) mRNA encoding PBGD
(construct #16 in Table 7) formulated in lipid nanoparticles
(Compound 18) was administered at 0.5 mg/kg and 0.2 mg/kg doses to
AIP mice as a single intravenous bolus. The time points of the
study were 10 hours, 1 day, 2 days, 4 days, 7 days, and 10 days.
The hepatic PBGD activity in AIP mice following administration of
PBGD mRNA is shown in FIG. 39. For comparison, the 12 units
threshold line in FIG. 39 is the level of PBGD observed in livers
of wild-type mice (C57BL/6), and the 3 units threshold line is the
level of PBGD in livers of AIP mice without treatment.
[2242] The results show that hepatic PBGD activity was within the
therapeutic window for up to 7 days after administration. An acute
porphyria attack in a human lasts between 5 and 7 days. In humans,
the increase from 2 units to 5 units completely restores the PBGD
activity in the liver. Accordingly, these results suggests that a
single administration of mRNA encoding wild-type PBGD could protect
against an acute porphyria attack.
Example 30
Blood Pressure in AIP Mice Administered Modified PBGD mRNA
[2243] The ability of administration of PBGD mRNA formulated in
lipid nanoparticle to reduce blood pressure in AIP mice during the
phenobarbital induced attack was tested (FIG. 40A and FIG. 40B).
AIP mice were subjected to 4 intraperitoneal injections of
phenobarbital to induce the acute attack. Wild-type PBGD-encoding
1-methyl-pseudouridine modified mRNA comprising a codon optimized
ORF (PBGD COV1) formulated in MC3 (N=5) and wild-type PBGD-encoding
1-methyl-pseudouridine modified mRNA comprising a codon optimized
ORF (PBGD COV1; ORF SEQ ID NO: 89) formulated in MC3 (N=5) and
wild-type PBGD-encoding 1-methyl-pseudouridine modified mRNA
comprising a codon optimized ORF (PBGD COV2; ORF SEQ ID NO: 88)
formulated in Compound 18-containing lipid nanoparticles (Compound
18:Cholesterol:DSPC:DMG:PEG) (N=6) were administered intravenously
to AIP mice at a dose of 0.5 mg/kg and 1 mg/kg, respectively,
before the first phenobarbital injection. AIP mice subjected to
phenobarbital challenge but with no administration of PBGD mRNA
were included for comparison (N=9). AIP mice (N=1) and wild type
C57BL/6 mice (N=9) without any treatment were included as
control.
[2244] AIP mice have blood pressure readings in the range of wild
type mice. Four phenobarbital injections in AIP mice significantly
increased both systolic and diastolic blood pressure values. Full
protection against hypertension and porphyrin precursors
accumulation induced by phenobarbital challenge was achieved in AIP
mice injected with a therapeutic dose of PBGD mRNA in both MC3 and
compound 18 formulations. These data show for the first time that
porphyrin precursors generated in the liver correlated with blood
hypertension.
Example 31
Pharmacokinetics Study of Modified mRNA Encoding Wild Type PBGD in
Wild-Type Sprague Dawley Rats
[2245] Hepatic PBGD activity was determined in WT Sprague Dayley
rats administered sequence optimized, 5-methoxyuridine modified
PBGD mRNA (construct #16 in Table 7) formulated in lipid
nanoparticles (Compound 18) at a dose of 0.5 mg/kg as a single
intravenous bolus. The hepatic PBGD activity at 24 hours and 48
hours following administration of PBGD mRNA is shown in FIG. 41.
The increase of PBGD activity in rats as a result of PBGD mRNA
injection, compared to the basal PBGD activity level in rats
administered control luciferase mRNA, was to similar extent of that
in WT CD1 mice at 24 hours and 48 hours post administration as
shown in FIG. 34.
[2246] In the livers of PBGD mRNA treated WT Sprague Dawley rats,
the expression level of PBGD protein was assessed by IHC as shown
in FIGS. 42A and 42B. The expression of exogenous PBGD was
homogeneous throughout all hepatocytes. Increased expression of
PBGD protein above the basal level was observed at 24 hours after
injection of the PBGD mRNA formulated in compound 18 at 1 mg/kg as
shown in FIG. 42A. A representative liver tissue sample from a
control PBS injected WT Sprague Dawley rat is shown in FIG. 42B.
Minimal PBGD protein expression (about_U) was detected in the PBS
control, which indicated very low level of cross-reaction of the
Novus anti-PBGD antibody used for IHC analysis with rat
species.
Example 32
PBGD Activity in a Non-human Primate After Administration of a
Modified PBGD mRNA Construct
[2247] Hepatic PBGD activity was determined in Cynomolgus macaque
24 hours after IV-administration of 5-methoxyuridine modified PBGD
mRNA comprising miR142 and miR126 binding sites. The mRNAs were
formulated in lipid nanoparticles comprising Compound 18 or
Compound 236. Animals were administered a single intravenous bolus
of (i) 0.5 mg/kg mRNA formulated with Compound 236 or (ii) 1 mg/kg
mRNA formulated with Compound 18. Control animals were administered
PBS. Hepatic PBGD activity and expression at 24 hours following
administration of PBGD mRNA is shown in FIG. 43 A and 43B,
respectively. Animals administered 0.5 mg/kg mRNA formulated in
lipid nanoparticles comprising Compound 236 showed about 40%
increase in PBGD activity and about 90% increase in PBGD expression
compared to the level seen in control animals administered PBS.
[2248] It is to be appreciated that the Detailed Description
section, and not the Summary and Abstract sections, is intended to
be used to interpret the claims. The Summary and Abstract sections
can set forth one or more but not all exemplary embodiments of the
present invention as contemplated by the inventor(s), and thus, are
not intended to limit the present invention and the appended claims
in any way.
[2249] The present invention has been described above with the aid
of functional building blocks illustrating the implementation of
specified functions and relationships thereof. The boundaries of
these functional building blocks have been arbitrarily defined
herein for the convenience of the description. Alternate boundaries
can be defined so long as the specified functions and relationships
thereof are appropriately performed.
[2250] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art, readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing from
the general concept of the present invention. Therefore, such
adaptations and modifications are intended to be within the meaning
and range of equivalents of the disclosed embodiments, based on the
teaching and guidance presented herein. It is to be understood that
the phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology or
phraseology of the present specification is to be interpreted by
the skilled artisan in light of the teachings and guidance.
[2251] The breadth and scope of the present invention should not be
limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims and
their equivalents.
[2252] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control.
Sequence CWU 1
1
1991361PRTArtificial Sequencepolypeptide sequence of wild type
PBGD, isoform 1 1Met Ser Gly Asn Gly Asn Ala Ala Ala Thr Ala Glu
Glu Asn Ser Pro1 5 10 15Lys Met Arg Val Ile Arg Val Gly Thr Arg Lys
Ser Gln Leu Ala Arg 20 25 30Ile Gln Thr Asp Ser Val Val Ala Thr Leu
Lys Ala Ser Tyr Pro Gly 35 40 45Leu Gln Phe Glu Ile Ile Ala Met Ser
Thr Thr Gly Asp Lys Ile Leu 50 55 60Asp Thr Ala Leu Ser Lys Ile Gly
Glu Lys Ser Leu Phe Thr Lys Glu65 70 75 80Leu Glu His Ala Leu Glu
Lys Asn Glu Val Asp Leu Val Val His Ser 85 90 95Leu Lys Asp Leu Pro
Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala 100 105 110Ile Cys Lys
Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys 115 120 125Phe
Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val Val Gly 130 135
140Thr Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg Lys Phe Pro
His145 150 155 160Leu Glu Phe Arg Ser Ile Arg Gly Asn Leu Asn Thr
Arg Leu Arg Lys 165 170 175Leu Asp Glu Gln Gln Glu Phe Ser Ala Ile
Ile Leu Ala Thr Ala Gly 180 185 190Leu Gln Arg Met Gly Trp His Asn
Arg Val Gly Gln Ile Leu His Pro 195 200 205Glu Glu Cys Met Tyr Ala
Val Gly Gln Gly Ala Leu Gly Val Glu Val 210 215 220Arg Ala Lys Asp
Gln Asp Ile Leu Asp Leu Val Gly Val Leu His Asp225 230 235 240Pro
Glu Thr Leu Leu Arg Cys Ile Ala Glu Arg Ala Phe Leu Arg His 245 250
255Leu Glu Gly Gly Cys Ser Val Pro Val Ala Val His Thr Ala Met Lys
260 265 270Asp Gly Gln Leu Tyr Leu Thr Gly Gly Val Trp Ser Leu Asp
Gly Ser 275 280 285Asp Ser Ile Gln Glu Thr Met Gln Ala Thr Ile His
Val Pro Ala Gln 290 295 300His Glu Asp Gly Pro Glu Asp Asp Pro Gln
Leu Val Gly Ile Thr Ala305 310 315 320Arg Asn Ile Pro Arg Gly Pro
Gln Leu Ala Ala Gln Asn Leu Gly Ile 325 330 335Ser Leu Ala Asn Leu
Leu Leu Ser Lys Gly Ala Lys Asn Ile Leu Asp 340 345 350Val Ala Arg
Gln Leu Asn Asp Ala His 355 36021083DNAArtificial
Sequencenucleotide sequence of wild type PBGD, isoform 1
2atgtctggta acggcaatgc ggctgcaacg gcggaagaaa acagcccaaa gatgagagtg
60attcgcgtgg gtacccgcaa gagccagctt gctcgcatac agacggacag tgtggtggca
120acattgaaag cctcgtaccc tggcctgcag tttgaaatca ttgctatgtc
caccacaggg 180gacaagattc ttgatactgc actctctaag attggagaga
aaagcctgtt taccaaggag 240cttgaacatg ccctggagaa gaatgaagtg
gacctggttg ttcactcctt gaaggacctg 300cccactgtgc ttcctcctgg
cttcaccatc ggagccatct gcaagcggga aaaccctcat 360gatgctgttg
tctttcaccc aaaatttgtt gggaagaccc tagaaaccct gccagagaag
420agtgtggtgg gaaccagctc cctgcgaaga gcagcccagc tgcagagaaa
gttcccgcat 480ctggagttca ggagtattcg gggaaacctc aacacccggc
ttcggaagct ggacgagcag 540caggagttca gtgccatcat cctggcaaca
gctggcctgc agcgcatggg ctggcacaac 600cgggtggggc agatcctgca
ccctgaggaa tgcatgtatg ctgtgggcca gggggccttg 660ggcgtggaag
tgcgagccaa ggaccaggac atcttggatc tggtgggtgt gctgcacgat
720cccgagactc tgcttcgctg catcgctgaa agggccttcc tgaggcacct
ggaaggaggc 780tgcagtgtgc cagtagccgt gcatacagct atgaaggatg
ggcaactgta cctgactgga 840ggagtctgga gcctagacgg ctcagatagc
atacaagaga ccatgcaggc taccatccat 900gtccctgccc agcatgaaga
tggccctgag gatgacccac agttggtagg catcactgct 960cgtaacattc
cacgagggcc ccagttggct gcccagaact tgggcatcag cctggccaac
1020ttgttgctga gcaaaggagc caaaaacatc ctggatgttg cacggcagct
taacgatgcc 1080cat 10833344PRTArtificial Sequencepolypeptide
sequence of wild type PBGD, isoform 2 3Met Arg Val Ile Arg Val Gly
Thr Arg Lys Ser Gln Leu Ala Arg Ile1 5 10 15Gln Thr Asp Ser Val Val
Ala Thr Leu Lys Ala Ser Tyr Pro Gly Leu 20 25 30Gln Phe Glu Ile Ile
Ala Met Ser Thr Thr Gly Asp Lys Ile Leu Asp 35 40 45Thr Ala Leu Ser
Lys Ile Gly Glu Lys Ser Leu Phe Thr Lys Glu Leu 50 55 60Glu His Ala
Leu Glu Lys Asn Glu Val Asp Leu Val Val His Ser Leu65 70 75 80Lys
Asp Leu Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala Ile 85 90
95Cys Lys Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys Phe
100 105 110Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val Val
Gly Thr 115 120 125Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg Lys
Phe Pro His Leu 130 135 140Glu Phe Arg Ser Ile Arg Gly Asn Leu Asn
Thr Arg Leu Arg Lys Leu145 150 155 160Asp Glu Gln Gln Glu Phe Ser
Ala Ile Ile Leu Ala Thr Ala Gly Leu 165 170 175Gln Arg Met Gly Trp
His Asn Arg Val Gly Gln Ile Leu His Pro Glu 180 185 190Glu Cys Met
Tyr Ala Val Gly Gln Gly Ala Leu Gly Val Glu Val Arg 195 200 205Ala
Lys Asp Gln Asp Ile Leu Asp Leu Val Gly Val Leu His Asp Pro 210 215
220Glu Thr Leu Leu Arg Cys Ile Ala Glu Arg Ala Phe Leu Arg His
Leu225 230 235 240Glu Gly Gly Cys Ser Val Pro Val Ala Val His Thr
Ala Met Lys Asp 245 250 255Gly Gln Leu Tyr Leu Thr Gly Gly Val Trp
Ser Leu Asp Gly Ser Asp 260 265 270Ser Ile Gln Glu Thr Met Gln Ala
Thr Ile His Val Pro Ala Gln His 275 280 285Glu Asp Gly Pro Glu Asp
Asp Pro Gln Leu Val Gly Ile Thr Ala Arg 290 295 300Asn Ile Pro Arg
Gly Pro Gln Leu Ala Ala Gln Asn Leu Gly Ile Ser305 310 315 320Leu
Ala Asn Leu Leu Leu Ser Lys Gly Ala Lys Asn Ile Leu Asp Val 325 330
335Ala Arg Gln Leu Asn Asp Ala His 34041032DNAArtificial
Sequencenucleotide sequence of wild type PBGD, isoform 2
4atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt
60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc
120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa
aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg
acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc
ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt
ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga
gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag
420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct
tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag
ctggcctgca gcgcatgggc 540tggcacaacc gggtggggca gatcctgcac
cctgaggaat gcatgtatgc tgtgggccag 600ggggccttgg gcgtggaagt
gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660ctgcacgatc
ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg
720gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg
gcaactgtac 780ctgactggag gagtctggag cctagacggc tcagatagca
tacaagagac catgcaggct 840accatccatg tccctgccca gcatgaagat
ggccctgagg atgacccaca gttggtaggc 900atcactgctc gtaacattcc
acgagggccc cagttggctg cccagaactt gggcatcagc 960ctggccaact
tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcagctt
1020aacgatgccc at 10325321PRTArtificial Sequencepolypeptide
sequence of wild type PBGD, isoform 3 5Met Ser Gly Asn Gly Asn Ala
Ala Ala Thr Ala Glu Glu Asn Ser Pro1 5 10 15Lys Met Arg Val Ile Arg
Val Gly Thr Arg Lys Ser Gln Leu Ala Arg 20 25 30Ile Gln Thr Asp Ser
Val Val Ala Thr Leu Lys Ala Ser Tyr Pro Gly 35 40 45Leu Gln Phe Glu
Ile Ile Ala Met Ser Thr Thr Gly Asp Lys Ile Leu 50 55 60Asp Thr Ala
Leu Ser Lys Ile Gly Glu Lys Ser Leu Phe Thr Lys Glu65 70 75 80Leu
Glu His Ala Leu Glu Lys Asn Glu Val Asp Leu Val Val His Ser 85 90
95Leu Lys Asp Leu Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala
100 105 110Ile Cys Lys Arg Glu Asn Pro His Asp Ala Val Val Phe His
Pro Lys 115 120 125Phe Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys
Ser Val Val Gly 130 135 140Thr Ser Ser Leu Arg Arg Ala Ala Gln Leu
Gln Arg Lys Phe Pro His145 150 155 160Leu Glu Phe Arg Ser Ile Arg
Gly Asn Leu Asn Thr Arg Leu Arg Lys 165 170 175Leu Asp Glu Gln Gln
Glu Phe Ser Ala Ile Ile Leu Ala Thr Ala Gly 180 185 190Leu Gln Arg
Met Gly Trp His Asn Arg Val Gly Gln Ile Leu His Pro 195 200 205Glu
Glu Cys Met Tyr Ala Val Gly Gln Glu Gly Gly Cys Ser Val Pro 210 215
220Val Ala Val His Thr Ala Met Lys Asp Gly Gln Leu Tyr Leu Thr
Gly225 230 235 240Gly Val Trp Ser Leu Asp Gly Ser Asp Ser Ile Gln
Glu Thr Met Gln 245 250 255Ala Thr Ile His Val Pro Ala Gln His Glu
Asp Gly Pro Glu Asp Asp 260 265 270Pro Gln Leu Val Gly Ile Thr Ala
Arg Asn Ile Pro Arg Gly Pro Gln 275 280 285Leu Ala Ala Gln Asn Leu
Gly Ile Ser Leu Ala Asn Leu Leu Leu Ser 290 295 300Lys Gly Ala Lys
Asn Ile Leu Asp Val Ala Arg Gln Leu Asn Asp Ala305 310 315
320His6963DNAArtificial Sequencenucleotide sequence of wild type
PBGD, isoform 3 6atgtctggta acggcaatgc ggctgcaacg gcggaagaaa
acagcccaaa gatgagagtg 60attcgcgtgg gtacccgcaa gagccagctt gctcgcatac
agacggacag tgtggtggca 120acattgaaag cctcgtaccc tggcctgcag
tttgaaatca ttgctatgtc caccacaggg 180gacaagattc ttgatactgc
actctctaag attggagaga aaagcctgtt taccaaggag 240cttgaacatg
ccctggagaa gaatgaagtg gacctggttg ttcactcctt gaaggacctg
300cccactgtgc ttcctcctgg cttcaccatc ggagccatct gcaagcggga
aaaccctcat 360gatgctgttg tctttcaccc aaaatttgtt gggaagaccc
tagaaaccct gccagagaag 420agtgtggtgg gaaccagctc cctgcgaaga
gcagcccagc tgcagagaaa gttcccgcat 480ctggagttca ggagtattcg
gggaaacctc aacacccggc ttcggaagct ggacgagcag 540caggagttca
gtgccatcat cctggcaaca gctggcctgc agcgcatggg ctggcacaac
600cgggtggggc agatcctgca ccctgaggaa tgcatgtatg ctgtgggcca
ggaaggaggc 660tgcagtgtgc cagtagccgt gcatacagct atgaaggatg
ggcaactgta cctgactgga 720ggagtctgga gcctagacgg ctcagatagc
atacaagaga ccatgcaggc taccatccat 780gtccctgccc agcatgaaga
tggccctgag gatgacccac agttggtagg catcactgct 840cgtaacattc
cacgagggcc ccagttggct gcccagaact tgggcatcag cctggccaac
900ttgttgctga gcaaaggagc caaaaacatc ctggatgttg cacggcagct
taacgatgcc 960cat 9637304PRTArtificial Sequencepolypeptide sequence
of wild type PBGD, isoform 4 7Met Arg Val Ile Arg Val Gly Thr Arg
Lys Ser Gln Leu Ala Arg Ile1 5 10 15Gln Thr Asp Ser Val Val Ala Thr
Leu Lys Ala Ser Tyr Pro Gly Leu 20 25 30Gln Phe Glu Ile Ile Ala Met
Ser Thr Thr Gly Asp Lys Ile Leu Asp 35 40 45Thr Ala Leu Ser Lys Ile
Gly Glu Lys Ser Leu Phe Thr Lys Glu Leu 50 55 60Glu His Ala Leu Glu
Lys Asn Glu Val Asp Leu Val Val His Ser Leu65 70 75 80Lys Asp Leu
Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala Ile 85 90 95Cys Lys
Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys Phe 100 105
110Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val Val Gly Thr
115 120 125Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg Lys Phe Pro
His Leu 130 135 140Glu Phe Arg Ser Ile Arg Gly Asn Leu Asn Thr Arg
Leu Arg Lys Leu145 150 155 160Asp Glu Gln Gln Glu Phe Ser Ala Ile
Ile Leu Ala Thr Ala Gly Leu 165 170 175Gln Arg Met Gly Trp His Asn
Arg Val Gly Gln Ile Leu His Pro Glu 180 185 190Glu Cys Met Tyr Ala
Val Gly Gln Glu Gly Gly Cys Ser Val Pro Val 195 200 205Ala Val His
Thr Ala Met Lys Asp Gly Gln Leu Tyr Leu Thr Gly Gly 210 215 220Val
Trp Ser Leu Asp Gly Ser Asp Ser Ile Gln Glu Thr Met Gln Ala225 230
235 240Thr Ile His Val Pro Ala Gln His Glu Asp Gly Pro Glu Asp Asp
Pro 245 250 255Gln Leu Val Gly Ile Thr Ala Arg Asn Ile Pro Arg Gly
Pro Gln Leu 260 265 270Ala Ala Gln Asn Leu Gly Ile Ser Leu Ala Asn
Leu Leu Leu Ser Lys 275 280 285Gly Ala Lys Asn Ile Leu Asp Val Ala
Arg Gln Leu Asn Asp Ala His 290 295 3008912DNAArtificial
Sequencenucleotide sequence of wild type PBGD, isoform 4
8atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt
60gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc
120accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa
aagcctgttt 180accaaggagc ttgaacatgc cctggagaag aatgaagtgg
acctggttgt tcactccttg 240aaggacctgc ccactgtgct tcctcctggc
ttcaccatcg gagccatctg caagcgggaa 300aaccctcatg atgctgttgt
ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360ccagagaaga
gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag
420ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct
tcggaagctg 480gacgagcagc aggagttcag tgccatcatc ctggcaacag
ctggcctgca gcgcatgggc 540tggcacaacc gggtggggca gatcctgcac
cctgaggaat gcatgtatgc tgtgggccag 600gaaggaggct gcagtgtgcc
agtagccgtg catacagcta tgaaggatgg gcaactgtac 660ctgactggag
gagtctggag cctagacggc tcagatagca tacaagagac catgcaggct
720accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca
gttggtaggc 780atcactgctc gtaacattcc acgagggccc cagttggctg
cccagaactt gggcatcagc 840ctggccaact tgttgctgag caaaggagcc
aaaaacatcc tggatgttgc acggcagctt 900aacgatgccc at
91291083RNAArtificial SequencePBGD-CO01 9augagcggga acggcaacgc
cgcagccacc gccgaggaga acagccccaa gaugcggguc 60aucagggugg gcacgcggaa
gucccaguug gcacgcauac agaccgacag cguaguggcg 120acccugaagg
ccuccuaccc cggccuccag uucgagauca ucgccaugag caccaccggg
180gacaagaucc ucgauaccgc ccuguccaag aucggcgaga aguccuuguu
uaccaaggag 240cuggagcacg cccuggagaa gaaugaggug gaccuugugg
ugcacagccu caaggaccug 300cccaccgugc ugccccccgg guucaccaua
ggagccaucu gcaagaggga gaacccgcac 360gacgcagucg uguuccaccc
caaguuugug gguaagacac uggagacccu gcccgagaag 420agcguggugg
guaccuccag ccuccgccga gcagcccagc ugcagaggaa guucccgcac
480cuggaauucc gauccaucag gggcaaucug aacaccagac ugaggaagcu
ggaugagcag 540caggaauuuu ccgcgauaau ccucgcgacc gccgggcugc
agaggauggg cuggcauaac 600agggugggcc aaauccugca ccccgaggag
ugcauguacg ccgugggcca gggcgcccuc 660ggcguggagg ugagggccaa
agaccaagac auccuggacc uggugggggu ccugcacgau 720ccggagacuc
ugcugaggug caucgccgag cgggcguuuc ucaggcaccu cgagggcggc
780ugcuccgugc ccguggccgu ccauaccgcc augaaggacg gccagcugua
ccucaccggc 840ggcgugugga gccuggacgg cagcgacucc auacaggaaa
ccaugcaagc gaccauacau 900guccccgccc agcacgagga cgggcccgag
gaugacccgc agcuggucgg uaucaccgca 960aggaauaucc cccggggccc
acagcuggcc gcccagaauc ugggcaucuc ccuggccaau 1020cugcugcugu
ccaagggcgc caagaacauc cucgacgugg ccaggcaacu gaacgacgca 1080cac
1083101083RNAArtificial SequencePBGD-CO02 10augagcggca acgguaacgc
cgccgccacc gccgaggaga acucgcccaa gaugcggguc 60aucagggugg gaacgaggaa
gucccagcug gccaggaucc agaccgacag cgugguggcg 120acgcugaagg
ccagcuaccc cggccugcag uuugagauaa ucgccauguc aaccaccggc
180gacaagaucc ucgacaccgc gcugagcaag aucggcgaga agucccuguu
caccaaggag 240cuggagcacg ccuuggaaaa gaacgaggug gaucuggucg
ugcacagccu gaaggaccug 300cccaccgugc ugccccccgg cuucaccauc
ggcgccaucu gcaagcggga aaauccccau 360gacgccgugg uguuccaccc
caaguucgug gggaagaccc uggagacccu gccggagaag 420agcguggugg
gaaccagcuc acugcggcgg gccgcccagc ugcaacguaa guucccccau
480cuggaguucc gauccauccg aggcaaucug aacaccaggc ugagaaagcu
ggaugaacaa 540caggaguuca gcgccaucau ccuggcaacc gccggucugc
agaggauggg cuggcacaac 600agggugggcc aaauccugca ccccgaggag
ugcauguaug caguggggca gggcgcgcug 660ggugucgagg ugcgagcgaa
ggaccaggac auccucgacc uggugggagu gcugcacgac 720cccgagaccc
ugcugcggug caucgccgag agggccuucc ugcgccaccu cgagggcggc
780ugcucagugc ccguggccgu gcauaccgcc augaaggacg gccagcugua
ccugaccggc 840ggugugugga gccucgacgg cagcgacucc auccaggaaa
cgaugcaggc gacuauccac 900gugcccgcac agcacgagga cggcccggaa
gacgaccccc agcuggucgg gaucaccgcc 960aggaacaucc cccgagggcc
ccagcuggcg gcccaaaacc ucggcauauc ccuggccaac 1020cugcugcugu
ccaagggcgc caagaacauc cuggaugugg cccgccaacu gaacgacgcc 1080cac
1083111083RNAArtificial SequencePBGD-CO03 11augagcggua acggcaacgc
cgccgccaca gccgaagaga acucccccaa gaugagggug 60auuagggugg guacccgaaa
gagccagcug gcccggaucc agaccgacuc cgugguggcc 120acccucaagg
caagcuaccc cggccugcag uucgagauca ucgccaugag caccaccggg
180gacaaaauuc ucgacaccgc ccugagcaag aucggcgaga aguccuuguu
caccaaggag 240cuagagcacg cccucgaaaa gaacgaggug gaucuggucg
ugcacucccu gaaggaccug 300cccaccgugc ugccccccgg cuucacgauc
ggcgcgaucu gcaagaggga gaacccgcau 360gacgccgugg uguuucaucc
caaauuugug ggaaaaaccc uggagacucu ccccgagaag 420agcgucgucg
ggaccuccuc ccugcggcgg gccgcccagc ugcagcgcaa guucccgcac
480cuggaguucc gcagcaucag gggaaaccug aacacccggc ugaggaagcu
ggacgagcag 540caggaguuca gcgcuaucau ccucgccacc gccggccucc
agcgaauggg cuggcauaac 600cgggugggcc agauccugca cccggaagaa
uguauguacg cggugggcca gggcgcgcug 660ggcguggagg ugagggccaa
ggaccaggac auccuggauc ucgugggcgu gcugcacgac 720cccgagaccc
ugcugagaug caucgcagaa cgggccuucc ugaggcaccu ggagggcggc
780ugcuccgugc cgguggccgu acauaccgca augaaagacg gccagcucua
ccugacgggg 840ggggugugga gccucgacgg cuccgacucc auccaggaga
ccaugcaggc gacgauccau 900guacccgccc agcacgagga cggccccgag
gaugaucccc aacucguggg caucaccgcc 960cgcaacaucc cgaggggccc
ccagcuggcc gcacagaacc ugggaauauc acuggccaac 1020cuccugcuca
gcaagggcgc caagaauauc cucgacgugg ccaggcagcu gaaugacgcc 1080cac
1083121083RNAArtificial SequencePBGD-CO04 12augagcggca acggcaacgc
cgccgccacc gccgaggaga acagccccaa aaugcgcgug 60aucagggugg gcaccaggaa
gagccagcug gcccgcaucc agaccgacag ugucguggcc 120acccucaagg
ccagcuaucc gggccugcaa uucgagauca ucgccaugag caccaccggc
180gacaagaucc ucgacaccgc ccucagcaag aucggcgaga agucccuguu
caccaaggag 240cuggagcacg cccuggagaa gaacgaggug gaccuggugg
ugcacagccu caaggaccug 300cccaccgugc ugccccccgg cuucaccauc
ggcgccaucu gcaagcggga gaacccccac 360gacgccgugg uguuccaucc
caaguucgug ggcaagaccc uggagacccu gcccgagaag 420agcguggucg
gcaccagcuc ccucaggaga gccgcccagc ugcagcgaaa guucccccac
480cuggaauucc gcagcauccg ggggaaccug aacacccgcc ugaggaagcu
ggacgagcag 540caagaauuca gcgccaucau ccuggccacc gcgggccugc
agaggauggg guggcacaac 600cgcgucggcc agauccugca cccagaggag
ugcauguacg cgguggggca gggggccuug 660gggguggaag ugcgcgccaa
ggaccaggac auccuggacc uggucggcgu gcugcaugac 720cccgagaccc
ugcucaggug caucgccgag agggccuuuc ugcgccaccu cgaggggggu
780ugcagcgugc cggucgccgu ccauacggcc augaaggacg ggcagcugua
ccugaccggc 840ggcguguggu cccuggacgg cuccgacagc auccaggaga
ccaugcaggc caccauccac 900gugcccgccc aacacgagga cggccccgag
gacgacccgc agcucgucgg caucaccgcc 960aggaauauac cccguggccc
ccagcuggcc gcccagaauc ucggcaucag ccuggccaau 1020cugcugcuga
gcaagggggc caagaacauc cuggacgugg ccaggcagcu gaaugaugcc 1080cac
1083131083RNAArtificial SequencePBGD-CO05 13augagcggca acggaaacgc
cgccgcgacg gccgaggaga acucccccaa aaugaggguc 60aucagggugg gcacccggaa
gagccagcug gcgcggaucc agaccgacag cgucguggcc 120acccucaaag
ccagcuaccc aggccugcag uucgagauca ucgccauguc gaccaccggg
180gacaagaucc uggauaccgc ccucagcaag aucggcgaga agucccuguu
caccaaggag 240cucgagcacg cccuggagaa gaaugaggug gaucuggugg
ugcauucccu gaaagaccug 300ccgaccgucc ugccccccgg cuucacgauc
ggagccaucu gcaaacggga gaacccccac 360gacgcugugg uguuccaccc
gaaguucgug gguaagaccc uggaaacacu gcccgagaag 420uccguggugg
gcaccagcag ccugaggcgg gcggcccaac ugcagaggaa guucccccac
480cucgaguuca gaagcauccg ggggaaccug aauacccggc ugcgcaagcu
ggacgagcag 540caggaguuca gcgccaucau ccuggccacc gcuggccugc
agaggauggg auggcacaac 600cgugugggcc agauccugca cccggaggag
ugcauguacg ccguggggca gggcgcgcug 660ggcguggagg ugcgcgcgaa
ggaccaggac auccuggacc uugucggcgu gcugcacgau 720cccgagacgc
ugcugaggug caucgccgag agggccuucc ugaggcaccu ggaagggggc
780uguagcgucc ccguggcugu ccacaccgcc augaaggacg gccagcugua
ccugaccggc 840ggcguguggu cccuggacgg cagcgacagc auccaggaga
ccaugcaggc caccauccac 900guccccgccc aacacgaaga cggcccggag
gacgaccccc agcugguagg cauaacugcc 960cgcaauaucc ccagggggcc
ccaacuggcc gcccagaacc ugggcauaag ccuggcgaac 1020cugcugcugu
ccaagggcgc caagaacauc cuggacgucg cccgccagcu gaacgacgcc 1080cac
1083141083RNAArtificial SequencePBGD-CO06 14augagcggca acggcaacgc
cgccgccacc gccgaggaga auucccccaa aaugagggug 60auccgcgugg gcacgaggaa
gagccagcug gcccggaucc agacggacag cgugguggcc 120acccugaagg
ccucguaucc cgggcugcag uucgagauca ucgccaugag caccaccggc
180gacaagaucc ucgacaccgc ccuguccaag aucggcgaga aaagccuguu
caccaaggaa 240cuggagcacg cacuggaaaa gaacgaggua gaccucgugg
ugcauucccu gaaggaccug 300cccaccgugc ugcccccagg cuucaccauc
ggcgccaucu gcaagcgaga gaacccccau 360gaugccgugg uguuccaccc
gaaguuuguu ggcaagacgc uggagacccu gcccgagaag 420agcguagugg
gcaccagcuc gcugcggagg gccgcccagc ugcagaggaa guucccccac
480cuggaguuua ggagcauuag gggcaaccug aacaccaggc ucaggaagcu
ggacgagcag 540caggaguucu ccgcuauaau ccuggccacc gccgggcugc
agaggauggg guggcacaac 600aggguggggc aaauccugca ccccgaagag
ugcauguacg ccgucggcca gggggcccug 660ggcguugagg uucgcgccaa
ggaucaggac auccuggacc uggugggggu gcugcaugac 720cccgagaccc
ugcugaggug caucgcggaa agggccuucc ugcgccaccu ggagggcggc
780ugcagcgugc ccguggccgu gcacaccgcg augaaggacg gccagcugua
ccugaccggg 840ggggucuggu cccuggacgg cuccgacagc auccaggaga
cgaugcaggc cacgauccac 900gugcccgccc aacacgagga cggucccgaa
gaugaucccc agcuggucgg caucaccgca 960aggaacaucc ccaggggccc
ccagcuggcc gcacagaacc ugggcaucuc gcuggccaac 1020cugcugcuga
gcaagggggc caagaacauc cucgacgugg ccaggcagcu aaacgacgcc 1080cac
1083151083RNAArtificial SequencePBGD-CO07 15auguccggca acggcaacgc
ugccgccacg gccgaggaga acagccccaa gaugagggug 60auccgggugg gcaccagaaa
gucccagcuc gccaggaucc agacugauag cguggucgcc 120acccucaaag
ccagcuaccc gggccugcag uuugagauca ucgccauguc cacaaccggc
180gacaagaucc uggacacagc gcugucgaag aucggcgaaa aaagccucuu
caccaaggag 240cuggagcacg cccuggagaa gaaugaggug gaccuggugg
ugcauucccu gaaggaccug 300cccaccgugc ugccccccgg cuucaccauc
ggcgccaucu gcaagaggga gaacccacac 360gacgccgugg ucuuccaccc
caaguucgug gggaaaaccc ucgaaacccu ccccgaaaag 420uccguggugg
gcaccuccag ccugaggcgg gcggcccagc ugcagaggaa guucccacac
480cucgaguucc gcagcauccg gggcaaucug aacaccaggc ucaggaagcu
ggacgagcag 540caggaguuca gcgccaucau ccuggccacc gcgggccucc
agaggauggg cuggcacaac 600agggugggcc agauccucca ccccgaggaa
uguauguacg ccguggggca aggcgcccug 660ggcguggagg uccgggcgaa
ggaccaggac auccuggacc uggucggcgu gcugcacgau 720cccgagacgc
ugcucaggug caucgccgaa cgcgccuucc uccggcaccu cgaggggggc
780ugcagcgugc cgguggccgu ccacaccgcc augaaggacg gccagcugua
ccugaccggc 840ggcgucugga gccuggacgg cuccgacucc auccaggaga
cgaugcaggc caccauccau 900gugccagccc agcaugagga cggcccggaa
gacgaccccc aacugguggg caucaccgcc 960cggaacaucc ccaggggccc
ucagcucgcc gcacagaauc uuggcaucag ccucgccaau 1020cuccugcuga
gcaagggagc caagaacauc cucgacgugg ccaggcagcu gaacgacgcc 1080cau
1083161083RNAArtificial SequencePBGD-CO08 16augagcggca acggcaacgc
cgccgccacc gccgaggaga auagccccaa gaugcgggug 60auccgcgugg gcacccgcaa
gagccagcug gcccggaucc agaccgacuc cgucguggcc 120acccugaagg
ccagcuaccc cggacuccag uucgagauca ucgccauguc gaccacgggc
180gacaagaucc ucgacaccgc ccucagcaaa aucggagaga agucccuguu
caccaaggag 240cucgaacacg cccuggaaaa gaacgaggug gaccuggugg
ugcacucccu gaaggaccug 300cccaccgugc ucccccccgg auuuaccauc
ggagccaucu gcaagcggga gaacccucac 360gacgccgugg uguuccaucc
caaguucgug gggaagacgc uggaaacccu gccggagaag 420agcguagugg
gcaccagcag ccugcggagg gccgcccagc ugcagaggaa guucccccac
480cuggaguuuc guagcauccg cggcaaucug aacacgcggc ugcgcaagcu
cgacgagcag 540caggaguucu ccgcgaucau acuggccacc gccggccugc
agcggauggg guggcacaac 600cgggucggcc agauccugca ccccgaggag
ugcauguacg ccgugggcca gggggccuug 660ggcguggagg ugagggccaa
ggaccaggac auccuggacc ucgugggcgu cuugcacgac 720cccgaaaccc
ugcugaggug caucgccgag cgggccuuuc ugaggcaccu ggagggcggg
780ugcagcgugc ccguggccgu ccacaccgca augaaggacg ggcagcugua
ccugaccggc 840ggggucugga gccuggacgg cagcgauucg auacaggaga
caaugcaagc cacgauccac 900guccccgccc agcacgagga cgggcccgag
gacgaccccc aacuggucgg gaucaccgcc 960cggaacaucc ccagggggcc
gcagcuggcc gcccagaacc ugggcaucuc ccuggccaac 1020cugcugcuga
gcaaaggcgc caagaacauu cuggaugugg cccgccagcu gaacgaugcc 1080cac
1083171083RNAArtificial SequencePBGD-CO09 17augagcggga acggcaacgc
cgccgccacc gccgaggaga acaguccgaa gaugagggua 60aucagggucg ggacccguaa
gagccagcuc gccaggaucc agaccgacag cgugguggcc 120acccugaagg
ccagcuaucc agggcugcag uucgagauca ucgccauguc caccaccggg
180gacaaaaucc uggacaccgc ccugagcaag auaggcgaga agagccuguu
caccaaggaa 240cuggagcaug cccuggaaaa gaacgaggug gaccuggucg
ugcacagccu gaaggaccug 300cccaccgugc ugccccccgg cuucaccauc
ggcgcgaucu gcaaaaggga gaacccccac 360gacgcugugg ucuuccaccc
aaaauucgug gggaagacuc ucgagacccu gcccgagaag 420agcguggugg
guacuuccag ccugaggagg gccgcccagc ugcaaaggaa guucccccau
480cuggaguuca ggagcauccg gggcaaccug aauaccaggc ugcgcaagcu
ggaugagcag 540caggaauuca gcgccaucau ccuggccacc gccggccugc
agaggauggg cuggcacaac 600agggucggcc agauccugca ccccgaggag
ugcauguaug ccgugggcca gggugcccug 660ggaguggagg ucagggcgaa
ggaccaggac auccuugacc uugucggcgu gcugcacgac 720cccgagaccc
ugcucaggug caucgccgag agggccuucc ugaggcaucu ggaggggggg
780uguagcgugc ccgucgcagu gcacaccgcc augaaggacg gccaacugua
ccugaccggc 840ggcgugugga gccuggacgg cucugauucg auccaagaga
ccaugcaagc cacuauacac 900gugcccgccc agcacgagga cgggcccgag
gaugacccac agcuggucgg aaucaccgcc 960cggaacaucc ccaggggacc
ccagcuggcc gcgcagaauc ugggcaucuc acuggccaac 1020cugcugcucu
ccaagggggc caagaacauc cuggacgugg cuaggcagcu gaacgacgcc 1080cac
1083181083RNAArtificial SequencePBGD-CO10 18augagcggca acggaaacgc
cgccgccacc gccgaggaaa acucccccaa aaugcggguc 60aucagggugg gcaccaggaa
gucccagcuc gccaggauac aaaccgacuc cgugguagcc 120acccugaagg
ccagcuaccc cgggcuccag uucgagauca ucgccaugag cacaaccggu
180gacaagauuc uugacaccgc ccucagcaag auaggcgaga agagccuguu
caccaaggag 240cuggaacaug cccuggaaaa gaacgagguc gaccucgucg
ugcacagccu gaaggaccug 300cccacggugc ugccacccgg cuucacgauc
ggcgccaucu gcaaaagaga gaauccccau 360gacgcggugg uguuucaccc
caaguuugug gggaagaccc uggagacccu gcccgagaag 420agcguggugg
ggacgagcag ccugcgcagg gccgcccagc ugcagcggaa guucccccau
480cuggaguuua ggagcaucag ggggaaccug aacacgaggc ugaggaagcu
ggacgagcag 540caggaauuca gcgccaucau ccuggcgacc gccggccugc
agcggauggg cuggcauaac 600cgcgugggac agauacugca ccccgaggaa
ugcauguacg cggucggcca aggcgcgcug 660ggcguggagg ugagggccaa
agaucaggau auccuagacc ucgugggcgu gcuucacgac 720cccgagacac
ugcugaggug caucgccgag agggcguuuc ugcggcaccu ggagggcggg
780ugcagcgugc cgguggccgu gcauaccgcc augaaggacg gccagcugua
ccugacgggg 840ggggugugga gccucgacgg cucggacagc auccaggaga
cgaugcaggc cacgauacac 900gugcccgccc agcacgaaga cggacccgag
gaugaccccc agcugguggg gaucacagcc 960aggaacaucc ccaggggccc
ccaacucgcc gcccagaacc uggggauuag ccuggccaac 1020cugcuccucu
ccaagggcgc caagaacauc cuggacgugg ccaggcagcu gaacgaugcc 1080cau
1083191083RNAArtificial SequencePBGD-CO011 19auguccggca acgggaacgc
agccgcgacc gccgaggaga acucccccaa gaugcgcgug 60auuagggucg ggacgaggaa
gagccagcug gccaggauac agaccgauuc cgugguggcc 120acccucaagg
ccagcuaccc ugggcugcaa uucgagauca uagccaugag caccaccggc
180gauaagauac uggacaccgc ccucagcaag aucggugaga agucgcuguu
uaccaaggaa 240cuggagcacg cccuggagaa gaaugaagug gaucuggucg
uacacagcuu gaaggaccug 300cccaccgugc ugccccccgg auucaccauc
ggggccaucu gcaagaggga gaacccccac 360gacgccgugg uguuccaccc
caaauucguc ggcaagaccc uggaaacccu gcccgaaaag 420agcguggugg
gcaccuccag ccugcgcagg gccgcccagc ugcagaggaa guucccccac
480cuggaguuca ggagcauacg gggcaaccug aacacccgac ugaggaagcu
ggacgaacag 540caagaguuca gcgccaucau ccuggcaacc gccggccugc
agaggauggg cuggcacaac 600agggugggcc agauccugca ccccgaggag
uguauguacg ccguaggcca gggcgcccuc 660ggaguggagg ucagggccaa
ggaccaggac auccucgacc uggugggugu gcugcacgac 720cccgagacac
ugcugaggug caucgccgag agggccuuuc ugaggcaccu cgaaggcggc
780ugcuccgugc ccguggccgu gcacaccgcc augaaagacg gucagcugua
cuugaccggu 840ggcgugugga gccucgacgg cagcgacagc auccaggaga
caaugcaggc caccauccac 900guccccgcac agcacgaaga cgggccugag
gacgaccccc agcugguggg aaucaccgcc 960aggaauaucc cccgaggccc
ccagcuggcg gcccagaacc ugggcauuuc ccuggccaac 1020cuccugcuga
gcaaaggugc caagaauauc cuggacgugg cccggcagcu gaaugacgcc 1080cac
1083201083RNAArtificial SequencePBGD-CO012 20augagcggga acgggaacgc
cgccgccacc gccgaggaga auucccccaa gaugagggug 60auaaggguag ggaccaggaa
gagccagcug gccaggaucc agacugacag cgugguugcc 120acccugaagg
ccagcuaccc cggucugcag uucgagauca ucgccauguc caccaccggu
180gacaagauuc uggacaccgc ccuguccaag aucggagaaa agagccuguu
caccaaggaa 240cucgaacacg cccuugagaa gaaugaagug gaccuggugg
ugcacucccu caaggaccua 300cccaccgugc ugccccccgg cuuuaccauc
ggcgccaucu gcaagaggga gaacccccau 360gacgcggugg uguuucaccc
caaauucgug ggaaagaccc uggagacacu gcccgagaag 420uccguggugg
gcaccagcag ccugaggagg gccgcccagc ugcagaggaa guucccccau
480cuggaguuua ggagcaucag gggcaaccug aacacccggc ugaggaagcu
ggaugaacag 540caggaguuca gcgccaucau ccuggccacg gcgggccugc
agcggauggg guggcauaac 600cggguggggc agauccugca ccccgaagaa
ugcauguaug cgguagggca aggggcccug 660ggcgucgagg ugcgggccaa
ggaccaggac auacuggacc uggugggggu gcugcaugau 720cccgaaaccc
ugcuccggug caucgccgag agggccuucc ucaggcaccu ggagggcgga
780ugcucggugc ccgucgccgu gcauaccgcc augaaggacg gccagcucua
ccucaccggc 840ggcguuuggu cccuggaugg aagcgacagc auucaggaaa
ccaugcaggc caccauccac 900gugccagcgc agcacgagga cggccccgag
gacgaccccc agcuaguggg caucaccgcc 960aggaacaucc cccggggccc
ccagcuggcc gcacagaacc uaggcaucag ccuggccaac 1020cuccugcuga
gcaagggcgc caagaacauc cuggaugugg ccaggcagcu gaacgacgcc 1080cac
1083211083RNAArtificial SequencePBGD-CO013 21auguccggua acggcaacgc
ggccgccacc gccgaggaga acagccccaa gaugagggug 60auccgcgugg gcaccaggaa
gagccagcug gccaggaucc aaaccgacag cguggucgcc 120acgcugaagg
ccagcuaccc aggucugcaa uucgagauca ucgccaugag caccaccggg
180gacaaaaucc uggauaccgc ccuguccaag aucggcgaga agagccuguu
caccaaggag 240cuggaacacg cccuggagaa gaacgaggug gaccuggucg
ugcacagccu gaaggaccug 300cccaccgucc ugccccccgg guucaccauc
ggcgccauuu gcaagcgaga gaacccacac 360gacgccgucg uguuccaccc
caaguucguc ggcaagacgc ucgagacccu gcccgagaag 420agcguggucg
ggacgagcuc ccuccggcgc gccgcccagc uccagcgcaa auuuccccau
480uuagaguucc guagcauccg uggcaaccug aacacgcguc ugaggaagcu
ggaugaacaa 540caggaguuca gcgccaucau ccuggccacc gccggccugc
agaggauggg cuggcacaac 600cgugugggcc agauccuuca ucccgaggag
ugcauguacg cugucggcca gggggcucug 660gggguggaag ucagggccaa
ggaccaggac auccuggauc uggucggcgu gcuccacgac 720cccgagacuc
ugcugaggug uaucgcagag agggcguucc ucaggcaccu cgagggcggg
780ugcagcgucc cuguggcggu gcacaccgcc augaaggacg ggcagcugua
ccucaccggc 840ggcguguggu cccuggaugg cagcgacagc auccaggaga
cgaugcaggc caccauccac 900gugcccgccc agcacgagga cggccccgag
gacgaccccc aacuggucgg caucaccgcc 960cgcaacaucc ccagggggcc
ccaacuggcc gcccagaauc ugggcaucuc ccuggccaac 1020cugcuccuga
gcaagggugc caagaacauc cucgacgugg ccaggcagcu caacgacgcc 1080cac
1083221083RNAArtificial SequencePBGD-CO014 22augagcggca acggcaacgc
cgcagccacc gccgaggaga acucccccaa gaugagggug 60aucagggugg gcaccaggaa
gucccaacug gcgcggaucc agaccgacag cgugguggcc 120acccugaagg
cguccuaccc cggccuccag uucgagauua ucgccaugag caccaccggc
180gacaagaucc uggauaccgc ccugagcaag aucggggaaa aaagccuguu
caccaaggag 240cuggagcacg cccuggagaa gaacgaggug gaucucgugg
uacacucccu gaaggauuug 300cccaccgugc ugccccccgg guucaccauc
ggggccaucu guaaaaggga gaauccccac 360gacgccgugg uguuccaccc
caaguucgug ggcaagaccc uggagacccu ccccgagaag 420agcguggucg
gaaccagcag ccugcgccgc gccgcucagc uacaacgcaa guucccccac
480cuggaguucc ggagcaucag ggggaaccug aacacccguc ugcggaagcu
cgacgagcag 540caggaguuca gcgcuaucau ccuggcuacg gccgggcugc
agaggauggg auggcacaac 600agggucgggc agauccugca ucccgaggag
ugcauguacg ccgugggcca aggcgcccuc 660ggaguggaag ucagggccaa
agaccaagac auccuggacc uagucggcgu gcugcaugac 720cccgagaccc
ugcuccgcug caucgccgag agggcguucc ugcgccaucu ggaggggggc
780ugcagcgugc cgguggccgu gcauacagcu augaaagaug gccaacugua
ccucaccggc 840ggcgucugga gccuggacgg cucggacucc auucaagaga
cgaugcaggc cacgauccac 900gugcccgcgc agcacgagga ugggcccgag
gacgaucccc agcugguagg caucaccgcc 960cggaacaucc cccggggccc
gcagcuggcc gcccagaacc uggggaucuc ccuggccaac 1020cuccugcugu
cgaagggggc caagaacauc cuggaugucg ccaggcagcu gaacgacgcc 1080cac
1083231083RNAArtificial SequencePBGD-CO015 23auguccggga acgggaacgc
ggccgccacg gcggaggaga acucccccaa aaugagggug 60auaagggugg gcacccggaa
aagccagcug gcucgaaucc agaccgacag cgugguggcc 120acccugaaag
ccuccuaccc cggccuccag uuugagauca ucgccaugag caccaccggc
180gacaagaucc ucgacaccgc ccugagcaag aucggugaaa agucgcuguu
cacgaaggag 240cuggagcacg cccuagagaa gaacgagguc gaccuggugg
uccacagccu gaaggaccug 300cccaccgucc ucccccccgg auuuaccauu
ggagccauau gcaaacggga gaacccccau 360gacgccgugg ucuuccaccc
caaguucguc ggcaagaccc uggagacccu cccggagaag 420uccguggugg
ggaccagcag ccugcgcagg gccgcccagc ugcagaggaa auucccccau
480cuggaauuca gguccaucag ggggaaccuc aacacccggc ugcggaagcu
cgacgaacaa 540caggaauuca gcgccaucau ccuggccacc gcggggcugc
agaggauggg guggcacaac 600agggugggac agauccugca ccccgaggag
uguauguacg ccguggggca gggagcccug 660ggcgucgagg uaagggcgaa
ggaucaggac auccuggauc
uggugggggu gcugcacgac 720cccgagaccc ugcuccggug cauugccgag
agggccuucc ugaggcaccu ggagggcggc 780uguagcgugc ccguggccgu
gcacacagcg augaaagacg gccagcugua ucugaccggg 840ggcguguggu
cccucgaugg cucagacucc auccaggaga cgaugcaggc caccauccau
900gugcccgccc agcacgagga cggccccgag gacgaccccc agcucguggg
gaucaccgcc 960cguaacauac ccaggggccc ccagcuggcc gcccagaauc
uggggauuag ccucgccaac 1020cugcugcuga gcaagggcgc caagaacauc
cuggacgugg cccggcagcu gaacgacgcc 1080cau 1083241083RNAArtificial
SequencePBGD-CO016 24auguccggca acgggaacgc cgccgccacu gccgaggaga
acagccccaa gaugagggug 60auuagggugg gcacccguaa gagccagcuc gccaggaucc
agaccgacag cguggucgcg 120acccucaagg ccuccuaccc cggccugcag
uucgaaauca ucgccaugag caccacgggg 180gacaaaaucc uggauaccgc
ccugagcaag auaggugaga agagccuguu caccaaggaa 240cuggagcacg
cccuggaaaa gaacgaaguc gaccucgugg ugcacucccu gaaggaccuc
300cccaccgugc ugccgcccgg cuucaccauc ggagccaucu gcaagaggga
gaacccccac 360gacgccgugg uauuucaccc caaguucguc ggcaagaccc
uggagacccu gcccgagaag 420agcgugguag gcaccagcuc ccuccgcagg
gccgcccaac uccaaaggaa guucccccac 480cucgaguucc ggagcaucag
gggaaaucug aacaccaggc ugaggaagcu cgaugagcag 540caggaguucu
ccgccaucau ccuggccacc gccggacugc aaaggauggg guggcacaac
600cgcguugggc aaauccugca ccccgaagag ugcauguacg ccguggggca
gggcgcacuc 660gggguggagg ugagggccaa ggaccaggac auccuggacc
ucguaggcgu acugcacgac 720cccgagaccc ugcugcgcug uaucgcugag
agggccuuuc ugcggcaccu ggagggcggc 780ugcagcgugc ccgucgccgu
gcacaccgcc augaaagacg gccagcucua ucugacgggg 840ggggugugga
gccuggacgg cagcgacagc auccaggaga caaugcaggc caccauccac
900gugcccgccc agcacgagga cgggcccgaa gacgaccccc agcucguggg
caucacggcc 960cguaacaucc ccaggggccc gcaacuggcc gcgcagaacc
ugggcaucuc gcuggccaau 1020cugcugcuga gcaagggugc caagaacauc
cuggauguug cccgacagcu gaaugacgcc 1080cac 1083251083RNAArtificial
SequencePBGD-CO017 25augagcggca acgguaacgc cgccgcgacc gcggaggaga
acucacccaa gaugcggguc 60aucaggguag gcaccaggaa gagccagcug gccaggauac
agaccgacag cgugguggcc 120acgcugaagg cguccuaucc gggccugcaa
uucgaaauua ucgccaugag cacgaccggg 180gacaagaucc uggacaccgc
gcugagcaaa aucggcgaga agagccucuu caccaaagag 240cuggagcaug
cccuggagaa gaacgaggug gaccuggucg ugcauucacu gaaggaucuc
300cccaccgugc ugcccccugg cuuuaccauc ggagccaucu gcaagcgcga
aaacccccac 360gaugccgugg ucuuccaccc caaguucgua gggaagaccc
uugagacccu gccggagaag 420ucgguggucg ggaccagcag ccugcggagg
gccgcccagc uccagaggaa guucccgcac 480cuugaguuca ggagcauccg
gggcaaucug aauaccaggc ugaggaagcu ggacgagcag 540caagaguuca
gugccaucau ccucgccacc gccggccugc agaggauggg guggcacaac
600agggugggac agauccugca ucccgaggag ugcauguacg ccgugggcca
gggcgcccuc 660ggcguggaag ugcgggccaa ggaccaggac auucuggacc
uggugggcgu gcugcacgau 720cccgagaccc ugcugcgaug caucgccgag
cgcgccuucu uaaggcaccu cgaggggggu 780ugcagcgugc ccguggcagu
gcacaccgcc augaaggacg gccagcugua ccugaccggg 840ggcgugugga
gccuggacgg cucggacagc auccaggaga caaugcaggc caccauccac
900gugccagcuc aacaugagga uggccccgag gacgauccgc agcugguggg
aaucaccgcc 960cggaacaucc cuaggggccc ccaacuggca gcccaaaacc
ugggcauaag ccuggccaac 1020cugcugcucu ccaagggcgc caagaacauu
cuggacgucg cgaggcagcu gaacgacgcc 1080cac 1083261083RNAArtificial
SequencePBGD-CO018 26augagcggca acggcaacgc cgccgcgacc gccgaggaga
auagccccaa aaugagggug 60auccgggugg gcacccggaa gucccagcug gcccggaucc
agaccgacag cgugguggcc 120acccucaagg ccagcuaccc cggccugcaa
uucgaaauca ucgccaugag caccaccggc 180gacaagaucc uggauaccgc
ccugagcaag aucggcgaga aaagccucuu cacuaaggag 240cuggagcacg
cccuggagaa aaacgaagug gaccuggugg uccauagccu uaaggaccug
300cccaccgugc ugccccccgg cuucaccauc ggcgccaucu guaagcguga
gaacccgcac 360gacgccgugg uguuccaccc caaguuugug gggaagaccc
ucgagacucu ccccgagaaa 420agcguggugg gcacuagcuc ucugaggagg
gcagcccagc uccagaggaa auucccccac 480cucgaguuca ggagcaucag
gggcaaccug aacacccggc ugaggaagcu ggacgagcag 540caggaguucu
ccgcgaucau ccuggccacc gcgggacugc agcgaauggg cuggcacaac
600cgggugggcc aaauccugca ccccgaggag ugcauguacg ccgugggcca
gggggcacua 660gggguggagg ugcgggccaa ggaucaggac auccuggacc
uggucggggu gcugcacgac 720cccgagaccc ugcucagaug caucgccgag
cgggcguucc uccggcauuu ggaggguggc 780uguagcgugc ccgucgccgu
gcacaccgcc augaaggacg gucagcugua ccucaccggg 840ggcguguggu
cccuggacgg cagcgacagc auccaggaga ccaugcaggc caccauccau
900gugcccgccc agcacgagga cggccccgag gacgaccccc agcuggucgg
caucaccgcc 960aggaacaucc cgagggggcc ucagcuggcc gcucaaaacc
uaggcaucag ccuggccaac 1020cuccugcuca gcaagggcgc caagaauauc
cuggacgucg ccaggcagcu gaacgacgcg 1080cau 1083271083RNAArtificial
SequencePBGD-CO019 27augagcggca acgggaacgc cgccgccacc gccgaggaga
acagccccaa gaugagggug 60auccgggucg guaccaggaa gagccagcug gcccgaaucc
agaccgauag cgugguggcc 120acgcugaagg ccagcuaccc cggacugcag
uucgagauaa ucgcgaugag cacaacgggg 180gacaagaucc uggacaccgc
ccucuccaag aucggcgaga aaagccucuu caccaaagag 240cuggagcaug
cccuggagaa gaacgaggua gaccucguag uccauagccu gaaggaccug
300cccaccgugc ugccccccgg auucaccauc ggcgccaucu guaagcgcga
gaacccccac 360gacgccgugg ucuuccaccc caaguucguc ggaaagacac
uggagacccu gcccgaaaag 420agcguggugg gcaccagcag ccugaggagg
gccgcccagc ugcagcgcaa guucccccac 480cuagaauuca ggagcauccg
gggcaacuua aacaccaggc ugaggaagcu ggaugagcag 540caggaauuca
gcgccaucau ccucgccacg gccgggcugc agaggauggg cuggcacaac
600cgggugggcc agauccugca uccggaagag ugcauguacg ccgugggcca
gggagcccug 660ggcguggagg uccgggccaa ggaucaagau auccuggauc
uggugggggu gcugcacgac 720cccgagaccc ugcugcgcug caucgccgag
agggccuuuc ugcgccaucu ggaggggggc 780ugcagcgugc ccguggcggu
gcauaccgcc augaaggacg gccagcucua ucugaccggc 840ggggucugga
gccuggacgg gagcgacagc auccaggaga ccaugcaggc gaccauccac
900gugcccgccc agcacgagga cggccccgaa gaugaccccc agcugguugg
gaucaccgcg 960aggaacaucc ccaggggccc ccagcuagcc gcccagaacc
uuggcaucuc ccuggccaac 1020cugcuacuca gcaagggugc caagaauauc
cuggaugucg ccaggcagcu gaacgacgcc 1080cau 1083281083RNAArtificial
SequencePBGD-CO020 28auguccggca acggcaacgc cgccgccacc gccgaggaaa
auagccccaa gaugcgggug 60auccgggugg gaacacgaaa gucccagcuc gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc uggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggauacggc
ccugagcaag aucggggaga aaucccucuu caccaaggaa 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg uccacucccu gaaggaccug
300cccacagugc ucccacccgg cuucaccauc ggcgccaucu gcaaacgcga
gaacccccac 360gacgccgugg ucuuucaccc caaguucgug gguaagaccc
uggagacccu gcccgaaaag 420agcguggugg gcaccuccag ccugaggcgg
gccgcccagc ugcagaggaa auuuccccac 480cuggaguucc ggagcaucag
gggcaaccuc aacaccaggc ugaggaagcu ggacgagcag 540caggaauuua
gcgcgaucau ccuggccacc gccggccugc agaggauggg guggcacaac
600agggugggac aaauccugca ucccgaggag ugcauguacg ccgugggcca
aggagcucug 660ggcguggaag ugagggccaa ggaccaggac auccuggacc
ucgugggcgu acugcacgac 720cccgagaccc ugcugcggug cauugccgag
cgggccuucc uccgucaccu cgagggcggc 780uguuccgugc ccguggccgu
gcauacggcc augaaggaug gacagcugua ucugacgggc 840ggcguguggu
cacuggacgg gucggacucc auccaggaga ccaugcaggc caccauccac
900gugcccgccc agcacgagga cggcccagaa gacgaccccc agcucgucgg
gauaaccgcc 960cgcaauaucc cuaggggccc ccagcuggcc gcccagaacc
ucgggaucag ccuggccaac 1020cuccugcugu ccaagggcgc caagaacaua
cuggacgucg cccggcagcu gaacgaugcc 1080cau 1083291083RNAArtificial
SequencePBGD-CO021 29augagcggca acgggaacgc cgccgcaacc gccgaggaga
acucaccaaa gaugagggug 60auccgagugg gcaccaggaa gucgcagcug gccaggaucc
agaccgacag cgugguggcg 120acccucaagg ccuccuaccc cggccugcag
uucgagauaa ucgccauguc caccaccggc 180gacaagaucc uggauaccgc
ccucagcaag aucggcgaaa agagccuguu caccaaggag 240cucgagcacg
cucuggagaa gaacgaggug gaccuggucg ugcacucccu gaaagaccug
300cccaccgugc ugcccccggg cuucaccaua ggggccaucu gcaagcggga
gaacccccac 360gacgccgugg uguuccaucc gaaguucgug ggcaagaccc
uggagacccu gcccgagaag 420agcguggugg gcacgagcag ccuaagaagg
gccgcacagc uccagaggaa guucccccac 480cucgaguucc gcagcaucag
aggcaaccug aacaccaggc ugcggaagcu cgacgagcag 540caggaguuua
gcgccaucau ccucgccacc gccggccugc agaggauggg auggcauaac
600cggguggggc agauccugca ccccgaggag uguauguacg ccguggggca
gggggcccug 660ggcguggagg ugcgggccaa ggaucaggac auccucgacc
uggugggcgu gcuucacgau 720cccgagaccc uucugcggug caucgccgag
cgggccuuuc ugaggcaucu ggagggcggg 780uguagugugc caguggcagu
ucacaccgcc augaaggacg gccagcugua ccugaccggu 840gggguuugga
gccuggacgg guccgacagc auccaggaga ccaugcaggc cacgauccau
900gugcccgcuc agcaugagga cggaccagaa gacgacccgc agcugguggg
caucacggcc 960cggaacauac ccagggggcc ccagcuggcg gcccagaacc
ugggcaucag ccuggccaac 1020cugcugcuga gcaagggcgc caagaacauc
cuggaugugg cgcggcagcu gaaugaugcc 1080cac 1083301083RNAArtificial
SequencePBGD-CO022 30augucgggca acggcaacgc ggccgccacc gcggaggaga
auagccccaa gaugcgggug 60aucagggugg gcaccaggaa gagccagcug gccaggaucc
agaccgacag ugugguggcc 120acccugaagg ccagcuaccc agggcugcag
uucgagauca uugccauguc uaccaccggc 180gacaagaucc ucgacaccgc
ccugagcaaa aucggggaga agagccuguu uaccaaagag 240cuggagcacg
cccuggagaa gaaugaggug gaccuggucg ugcacucccu gaaggaccug
300cccaccgugc ugccacccgg cuucaccauc ggcgccaucu guaagaggga
gaauccccac 360gacgcaguug uguuccaccc caaguucgug ggcaagaccc
uggagacccu gcccgagaag 420agcguggugg gcacgucgag ccugcgacgg
gccgcucagc ugcagcggaa auucccccac 480cucgaguucc ggucuauuag
gggcaaccuc aacacccgcc ucaggaaacu ggacgagcag 540caggaguucu
cggccaucau ccucgccacc gccggacugc aaaggauggg guggcacaac
600cgcgugggac agauccugca ucccgaggag ugcauguacg ccgugggcca
gggcgcgcug 660ggaguggagg ucagggcuaa agaccaggac auacucgacc
ucgugggcgu gcugcacgac 720ccggagaccc ugcugcggug cauagccgag
cgggccuucc uuaggcaccu ggagggcggc 780ugcuccgugc ccguggccgu
gcacaccgcc augaaggacg gccaacugua ccugaccggc 840gggguguggu
cccuggacgg cucagacagc auacaggaga ccaugcaagc gaccauccac
900gugcccgccc agcacgagga cggccccgag gaugaccccc agcugguggg
gaucacagcc 960cgcaacaucc ccaggggccc ccagcuggcc gcccagaacu
ugggcauuag ccuggcaaac 1020cuccugcugu ccaagggagc caaaaauauc
cuggacgugg ccaggcagcu gaacgacgcc 1080cau 1083311083RNAArtificial
SequencePBGD-CO023 31augagcggca acggcaacgc cgcggccacc gcggaggaga
acagccccaa gaugagggug 60aucagggugg gcaccaggaa gucccaacug gcuaggaucc
agaccgacag cgugguggcc 120acccugaaag cauccuaccc cggccuccag
uucgagauaa ucgccaugag caccacaggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agucgcuguu uaccaaggag 240cuggagcacg
cgcuggagaa aaacgaggug gaccuggucg ugcacagccu gaaggaucug
300cccacagugc uccccccagg cuucaccauc ggagccaucu gcaagaggga
gaauccccac 360gacgcggugg uguuucaucc caaguucgug gggaagaccc
uggagacccu gccggagaag 420agcguggugg ggaccagcag ccugaggcgg
gccgcccagc ugcagaggaa auucccccau 480cuggaguuca gaagcaucag
gggcaaccug aacaccaggc ugaggaagcu cgacgagcag 540caggaauuca
gcgccaucau ccucgccacg gccgggcugc agaggauggg cuggcauaac
600aggguggggc agauccucca ccccgaagag ugcauguacg ccgugggcca
gggcgcccuc 660ggcguggagg ugagggccaa ggaccaggac auccuggacu
uggugggcgu ccugcaugac 720cccgagaccc ugcugcggug caucgccgag
cgggccuucc ucaggcaccu ggagggcggc 780ugcagcgugc ccguggccgu
acacaccgcc augaaagaug gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cuccgacagc auccaggaga ccaugcaggc caccauccau
900gugcccgccc agcacgagga cgggccagag gacgaucccc agcuuguggg
aaucaccgcc 960aggaacaucc cuaggggccc acagcuggcc gcccaaaauc
ucgggauaag ccuggcuaac 1020cugcugcuga gcaagggcgc gaagaacauc
cuggacgugg cgcgacagcu gaacgacgcc 1080cau 1083321083RNAArtificial
SequencePBGD-CO024 32augagcggca acggcaacgc ggcggccacg gccgaggaga
acagccccaa aaugaggguu 60aucagggugg gcacccgcaa gucacagcug gccaggaucc
agacagauuc cgugguggcc 120accuugaagg ccuccuaccc cggccugcag
uucgagauca uagccauguc aacgaccggg 180gacaagaucc ucgacaccgc
ccugagcaag aucggggaga agucacuguu cacaaaggaa 240cuggaacacg
cccuugagaa gaacgagguu gaccuggugg uccacucccu gaaggaccug
300ccaaccgugc ugccgccggg cuucaccauc ggggccaucu gcaagcggga
gaacccccac 360gacgccgucg uguuccaccc gaaauucgug gggaaaaccc
ucgagacccu gcccgagaag 420agcguggugg gcacguccag ccugcggagg
gccgcccagc ugcagcggaa guucccccac 480cuggaguuca gguccauccg
ggggaaccug aauacgaggc ugaggaagcu ggacgaacag 540caggaguuca
gcgccaucau ccuggcgacc gccggccugc agcggauggg cuggcacaau
600cgggugggcc agauccugca cccggaggag ugcauguacg ccguggggca
gggcgcccuc 660ggcgucgagg ugcgggccaa ggaccaggau auccuggacc
ugguaggagu ccugcacgau 720cccgaaaccc ugcuccggug uauagccgag
cgugccuucc ugaggcaucu ggaaggcgga 780ugcuccgucc ccguggccgu
gcacaccgca augaaagacg gccagcugua ccugaccggc 840ggcgucugga
gccuggacgg cucagacagc auccaagaga ccaugcaggc caccauccac
900gucccugccc agcacgagga cggccccgaa gacgacccgc agcugguggg
caucacagcc 960aggaacaucc cccggggccc gcagcuggcc gcccagaauc
ugggaauuag ccuggccaac 1020cuccugcuga gcaagggagc caagaacauc
cuggacgugg cccggcagcu gaacgacgcc 1080cac 1083331083RNAArtificial
SequencePBGD-CO025 33augagcggca acggcaacgc ggccgccacc gccgaagaga
acagccccaa gaugagggug 60aucagggugg gcacgaggaa gagccagcug gccaggaucc
agaccgacag cgugguggcg 120acccugaagg ccucuuaccc uggccugcag
uuugagauca ucgccaugag caccaccggg 180gauaagaucc ucgacaccgc
ccuguccaag auuggagaaa aaucgcuguu caccaaggaa 240cuggagcacg
cccuggagaa gaacgaggua gaccuggugg ugcauagccu caaggaucug
300ccaaccgugc ugccccccgg guuuaccauc ggcgccauau gcaagaggga
gaacccccac 360gacgcggugg uguuccaccc caaguucgug ggcaaaaccc
ucgagacgcu gcccgagaag 420agcgucguug gcaccuccag ccugaggcgg
gccgcccagc ugcagaggaa guucccccac 480cucgaguuca gaagcaucag
gggcaaccug aacaccaggc ugaggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggccacc gccggccugc agaggauggg auggcacaau
600agggugggcc agauccugca ccccgaggag ugcauguacg ccgucggaca
gggcgcgcug 660ggcguggagg ucagggcgaa ggaccaggac auccuggacc
uggucggggu ccugcacgac 720ccggagaccc ugcugaggug caucgccgag
cgggccuucc uccggcaccu ggagggcggu 780ugcagcgugc caguggccgu
gcacaccgcc augaaggaug ggcagcugua ucugaccggc 840ggagugugga
gccucgacgg uagcgacucc auccaggaaa ccaugcaggc cacuauccac
900gugcccgccc agcacgagga uggccccgag gacgaucccc agcuggucgg
gauuacggcc 960cggaacaucc ccaggggccc gcagcucgcc gcacagaacc
ucggcaucuc ccuggccaac 1020cugcugcucu ccaagggcgc caagaacauc
cucgacgugg cccggcaacu gaacgacgcc 1080cac 10833487RNAArtificial
SequencemiR-142 34gacagugcag ucacccauaa aguagaaagc acuacuaaca
gcacuggagg guguaguguu 60uccuacuuua uggaugagug uacugug
873523RNAArtificial SequencemiR 142-3p sequence 35uguaguguuu
ccuacuuuau gga 233623RNAArtificial SequencemiR 142-3p binding site
36uccauaaagu aggaaacacu aca 233721RNAArtificial SequencemiR 142-5p
sequence 37cauaaaguag aaagcacuac u 213821RNAArtificial
SequencemiR-142-5p binding site 38aguagugcuu ucuacuuuau g
213947RNAArtificial Sequence5'UTR-001 (Upstream UTR) 39gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccacc 474047RNAArtificial
Sequence5'UTR-002 (Upstream UTR) 40gggagaucag agagaaaaga agaguaagaa
gaaauauaag agccacc 4741145RNAArtificial Sequence5'UTR-003 (Upstream
UTR) 41ggaauaaaag ucucaacaca acauauacaa aacaaacgaa ucucaagcaa
ucaagcauuc 60uacuucuauu gcagcaauuu aaaucauuuc uuuuaaagca aaagcaauuu
ucugaaaauu 120uucaccauuu acgaacgaua gcaac 1454242RNAArtificial
Sequence5'UTR-004 (Upstream UTR) 42gggagacaag cuuggcauuc cgguacuguu
gguaaagcca cc 424347RNAArtificial Sequence5'UTR-005 (Upstream UTR)
43gggagaucag agagaaaaga agaguaagaa gaaauauaag agccacc
4744145RNAArtificial Sequence5'UTR-006 (Upstream UTR) 44ggaauaaaag
ucucaacaca acauauacaa aacaaacgaa ucucaagcaa ucaagcauuc 60uacuucuauu
gcagcaauuu aaaucauuuc uuuuaaagca aaagcaauuu ucugaaaauu
120uucaccauuu acgaacgaua gcaac 1454542RNAArtificial
Sequence5'UTR-007 (Upstream UTR) 45gggagacaag cuuggcauuc cgguacuguu
gguaaagcca cc 424647RNAArtificial Sequence5'UTR-008 (Upstream UTR)
46gggaauuaac agagaaaaga agaguaagaa gaaauauaag agccacc
474747RNAArtificial Sequence5'UTR-009 (Upstream UTR) 47gggaaauuag
acagaaaaga agaguaagaa gaaauauaag agccacc 474847RNAArtificial
Sequence5'UTR-010 (Upstream UTR) 48gggaaauaag agaguaaaga acaguaagaa
gaaauauaag agccacc 474947RNAArtificial Sequence5'UTR-011 (Upstream
UTR) 49gggaaaaaag agagaaaaga agacuaagaa gaaauauaag agccacc
475047RNAArtificial Sequence5'UTR-012 (Upstream UTR) 50gggaaauaag
agagaaaaga agaguaagaa gauauauaag agccacc 475147RNAArtificial
Sequence5'UTR-013 (Upstream UTR) 51gggaaauaag agacaaaaca agaguaagaa
gaaauauaag agccacc 475247RNAArtificial Sequence5'UTR-014 (Upstream
UTR) 52gggaaauuag agaguaaaga acaguaagua gaauuaaaag agccacc
475347RNAArtificial Sequence5'UTR-015 (Upstream UTR) 53gggaaauaag
agagaauaga agaguaagaa gaaauauaag agccacc 475447RNAArtificial
Sequence5'UTR-016 (Upstream UTR) 54gggaaauaag agagaaaaga agaguaagaa
gaaaauuaag agccacc 475547RNAArtificial Sequence5'UTR-017 (Upstream
UTR) 55gggaaauaag agagaaaaga agaguaagaa gaaauuuaag agccacc
475692RNAArtificial Sequence5'UTR-018 (Upstream UTR) 56ucaagcuuuu
ggacccucgu acagaagcua auacgacuca cuauagggaa auaagagaga 60aaagaagagu
aagaagaaau auaagagcca cc 9257142RNAArtificial Sequence142-3p 3'UTR
(UTR including miR142-3p binding site) 57ugauaauagu ccauaaagua
ggaaacacua cagcuggagc cucgguggcc augcuucuug 60ccccuugggc cuccccccag
ccccuccucc ccuuccugca cccguacccc cguggucuuu 120gaauaaaguc
ugagugggcg gc 14258142RNAArtificial Sequence142-3p 3'UTR (UTR
including miR142-3p binding site) 58ugauaauagg cuggagccuc
gguggcucca uaaaguagga aacacuacac augcuucuug 60ccccuugggc cuccccccag
ccccuccucc ccuuccugca cccguacccc cguggucuuu 120gaauaaaguc
ugagugggcg gc 14259142RNAArtificial Sequence142-3p 3'UTR (UTR
including miR142-3p binding site) 59ugauaauagg cuggagccuc
gguggccaug cuucuugccc cuuccauaaa guaggaaaca 60cuacaugggc cuccccccag
ccccuccucc ccuuccugca cccguacccc cguggucuuu 120gaauaaaguc
ugagugggcg gc 14260142RNAArtificial Sequence142-3p 3'UTR (UTR
including miR142-3p binding site) 60ugauaauagg cuggagccuc
gguggccaug cuucuugccc cuugggccuc cccccagucc 60auaaaguagg aaacacuaca
ccccuccucc ccuuccugca cccguacccc cguggucuuu 120gaauaaaguc
ugagugggcg gc 14261142RNAArtificial Sequence142-3p 3'UTR (UTR
including miR142-3p binding site) 61ugauaauagg cuggagccuc
gguggccaug cuucuugccc cuugggccuc cccccagccc 60cuccuccccu ucuccauaaa
guaggaaaca cuacacugca cccguacccc cguggucuuu 120gaauaaaguc
ugagugggcg gc 14262142RNAArtificial Sequence142-3p 3'UTR (UTR
including miR142-3p binding site) 62ugauaauagg cuggagccuc
gguggccaug cuucuugccc cuugggccuc cccccagccc 60cuccuccccu uccugcaccc
guacccccuc cauaaaguag gaaacacuac aguggucuuu 120gaauaaaguc
ugagugggcg gc 14263142RNAArtificial Sequence142-3p 3'UTR (UTR
including miR142-3p binding site) 63ugauaauagg cuggagccuc
gguggccaug cuucuugccc cuugggccuc cccccagccc 60cuccuccccu uccugcaccc
guacccccgu ggucuuugaa uaaaguucca uaaaguagga 120aacacuacac
ugagugggcg gc 14264371RNAArtificial Sequence3'UTR-001 (Creatine
Kinase UTR) 64gcgccugccc accugccacc gacugcugga acccagccag
ugggagggcc uggcccacca 60gaguccugcu cccucacucc ucgccccgcc cccuguccca
gagucccacc ugggggcucu 120cuccacccuu cucagaguuc caguuucaac
cagaguucca accaaugggc uccauccucu 180ggauucuggc caaugaaaua
ucucccuggc aggguccucu ucuuuuccca gagcuccacc 240ccaaccagga
gcucuaguua auggagagcu cccagcacac ucggagcuug ugcuuugucu
300ccacgcaaag cgauaaauaa aagcauuggu ggccuuuggu cuuugaauaa
agccugagua 360ggaagucuag a 37165568RNAArtificial Sequence3'UTR-002
(Myoglobin UTR) 65gccccugccg cucccacccc cacccaucug ggccccgggu
ucaagagaga gcggggucug 60aucucgugua gccauauaga guuugcuucu gagugucugc
uuuguuuagu agaggugggc 120aggaggagcu gaggggcugg ggcuggggug
uugaaguugg cuuugcaugc ccagcgaugc 180gccucccugu gggaugucau
cacccuggga accgggagug gcccuuggcu cacuguguuc 240ugcaugguuu
ggaucugaau uaauuguccu uucuucuaaa ucccaaccga acuucuucca
300accuccaaac uggcuguaac cccaaaucca agccauuaac uacaccugac
aguagcaauu 360gucugauuaa ucacuggccc cuugaagaca gcagaauguc
ccuuugcaau gaggaggaga 420ucugggcugg gcgggccagc uggggaagca
uuugacuauc uggaacuugu gugugccucc 480ucagguaugg cagugacuca
ccugguuuua auaaaacaac cugcaacauc ucauggucuu 540ugaauaaagc
cugaguagga agucuaga 56866289RNAArtificial Sequence3'UTR-003
(alpha-actin UTR) 66acacacucca ccuccagcac gcgacuucuc aggacgacga
aucuucucaa ugggggggcg 60gcugagcucc agccaccccg cagucacuuu cuuuguaaca
acuuccguug cugccaucgu 120aaacugacac aguguuuaua acguguacau
acauuaacuu auuaccucau uuuguuauuu 180uucgaaacaa agcccugugg
aagaaaaugg aaaacuugaa gaagcauuaa agucauucug 240uuaagcugcg
uaaauggucu uugaauaaag ccugaguagg aagucuaga 28967379RNAArtificial
Sequence3'UTR-004 (Albumin UTR) 67caucacauuu aaaagcaucu cagccuacca
ugagaauaag agaaagaaaa ugaagaucaa 60aagcuuauuc aucuguuuuu cuuuuucguu
gguguaaagc caacacccug ucuaaaaaac 120auaaauuucu uuaaucauuu
ugccucuuuu cucugugcuu caauuaauaa aaaauggaaa 180gaaucuaaua
gagugguaca gcacuguuau uuuucaaaga uguguugcua uccugaaaau
240ucuguagguu cuguggaagu uccaguguuc ucucuuauuc cacuucggua
gaggauuucu 300aguuucuugu gggcuaauua aauaaaucau uaauacucuu
cuaauggucu uugaauaaag 360ccugaguagg aagucuaga 37968118RNAArtificial
Sequence3'UTR-005 (alpha-globin UTR) 68gcugccuucu gcggggcuug
ccuucuggcc augcccuucu ucucucccuu gcaccuguac 60cucuuggucu uugaauaaag
ccugaguagg aaggcggccg cucgagcaug caucuaga 11869908RNAArtificial
Sequence3'UTR-006 (G-CSF UTR) 69gccaagcccu ccccauccca uguauuuauc
ucuauuuaau auuuaugucu auuuaagccu 60cauauuuaaa gacagggaag agcagaacgg
agccccaggc cucugugucc uucccugcau 120uucugaguuu cauucuccug
ccuguagcag ugagaaaaag cuccuguccu cccauccccu 180ggacugggag
guagauaggu aaauaccaag uauuuauuac uaugacugcu ccccagcccu
240ggcucugcaa ugggcacugg gaugagccgc ugugagcccc ugguccugag
gguccccacc 300ugggacccuu gagaguauca ggucucccac gugggagaca
agaaaucccu guuuaauauu 360uaaacagcag uguuccccau cuggguccuu
gcaccccuca cucuggccuc agccgacugc 420acagcggccc cugcaucccc
uuggcuguga ggccccugga caagcagagg uggccagagc 480ugggaggcau
ggcccugggg ucccacgaau uugcugggga aucucguuuu ucuucuuaag
540acuuuuggga caugguuuga cucccgaaca ucaccgacgc gucuccuguu
uuucugggug 600gccucgggac accugcccug cccccacgag ggucaggacu
gugacucuuu uuagggccag 660gcaggugccu ggacauuugc cuugcuggac
ggggacuggg gaugugggag ggagcagaca 720ggaggaauca ugucaggccu
gugugugaaa ggaagcucca cugucacccu ccaccucuuc 780accccccacu
caccaguguc cccuccacug ucacauugua acugaacuuc aggauaauaa
840aguguuugcc uccauggucu uugaauaaag ccugaguagg aaggcggccg
cucgagcaug 900caucuaga 90870835RNAArtificial Sequence3'UTR-007
(Col1a2; collagen, type I, alpha 2 UTR) 70acucaaucua aauuaaaaaa
gaaagaaauu ugaaaaaacu uucucuuugc cauuucuucu 60ucuucuuuuu uaacugaaag
cugaauccuu ccauuucuuc ugcacaucua cuugcuuaaa 120uugugggcaa
aagagaaaaa gaaggauuga ucagagcauu gugcaauaca guuucauuaa
180cuccuucccc cgcuccccca aaaauuugaa uuuuuuuuuc aacacucuua
caccuguuau 240ggaaaauguc aaccuuugua agaaaaccaa aauaaaaauu
gaaaaauaaa aaccauaaac 300auuugcacca cuuguggcuu uugaauaucu
uccacagagg gaaguuuaaa acccaaacuu 360ccaaagguuu aaacuaccuc
aaaacacuuu cccaugagug ugauccacau uguuaggugc 420ugaccuagac
agagaugaac ugagguccuu guuuuguuuu guucauaaua caaaggugcu
480aauuaauagu auuucagaua cuugaagaau guugauggug cuagaagaau
uugagaagaa 540auacuccugu auugaguugu aucguguggu guauuuuuua
aaaaauuuga uuuagcauuc 600auauuuucca ucuuauuccc aauuaaaagu
augcagauua uuugcccaaa ucuucuucag 660auucagcauu uguucuuugc
cagucucauu uucaucuucu uccaugguuc cacagaagcu 720uuguuucuug
ggcaagcaga aaaauuaaau uguaccuauu uuguauaugu gagauguuua
780aauaaauugu gaaaaaaaug aaauaaagca uguuugguuu uccaaaagaa cauau
83571297RNAArtificial Sequence3'UTR-008 (Col6a2; collagen, type VI,
alpha 2 UTR) 71cgccgccgcc cgggccccgc agucgagggu cgugagccca
ccccguccau ggugcuaagc 60gggcccgggu cccacacggc cagcaccgcu gcucacucgg
acgacgcccu gggccugcac 120cucuccagcu ccucccacgg gguccccgua
gccccggccc ccgcccagcc ccaggucucc 180ccaggcccuc cgcaggcugc
ccggccuccc ucccccugca gccaucccaa ggcuccugac 240cuaccuggcc
ccugagcucu ggagcaagcc cugacccaau aaaggcuuug aacccau
29772602RNAArtificial Sequence3'UTR-009 (RPN1; ribophorin I UTR)
72ggggcuagag cccucuccgc acagcgugga gacggggcaa ggaggggggu uauuaggauu
60ggugguuuug uuuugcuuug uuuaaagccg ugggaaaaug gcacaacuuu accucugugg
120gagaugcaac acugagagcc aagggguggg aguugggaua auuuuuauau
aaaagaaguu 180uuuccacuuu gaauugcuaa aaguggcauu uuuccuaugu
gcagucacuc cucucauuuc 240uaaaauaggg acguggccag gcacgguggc
ucaugccugu aaucccagca cuuugggagg 300ccgaggcagg cggcucacga
ggucaggaga ucgagacuau ccuggcuaac acgguaaaac 360ccugucucua
cuaaaaguac aaaaaauuag cugggcgugg uggugggcac cuguaguccc
420agcuacucgg gaggcugagg caggagaaag gcaugaaucc aagaggcaga
gcuugcagug 480agcugagauc acgccauugc acuccagccu gggcaacagu
guuaagacuc ugucucaaau 540auaaauaaau aaauaaauaa auaaauaaau
aaauaaaaau aaagcgagau guugcccuca 600aa 60273785RNAArtificial
Sequence3'UTR-010 (LRP1; low density lipoprotein receptor-related
protein 1 UTR) 73ggcccugccc cgucggacug cccccagaaa gccuccugcc
cccugccagu gaaguccuuc 60agugagcccc uccccagcca gcccuucccu ggccccgccg
gauguauaaa uguaaaaaug 120aaggaauuac auuuuauaug ugagcgagca
agccggcaag cgagcacagu auuauuucuc 180cauccccucc cugccugcuc
cuuggcaccc ccaugcugcc uucagggaga caggcaggga 240gggcuugggg
cugcaccucc uacccuccca ccagaacgca ccccacuggg agagcuggug
300gugcagccuu ccccucccug uauaagacac uuugccaagg cucuccccuc
ucgccccauc 360ccugcuugcc cgcucccaca gcuuccugag ggcuaauucu
gggaagggag aguucuuugc 420ugccccuguc uggaagacgu ggcucugggu
gagguaggcg ggaaaggaug gaguguuuua 480guucuugggg gaggccaccc
caaaccccag ccccaacucc aggggcaccu augagauggc 540caugcucaac
cccccuccca gacaggcccu cccugucucc agggccccca ccgagguucc
600cagggcugga gacuuccucu gguaaacauu ccuccagccu ccccuccccu
ggggacgcca 660aggagguggg ccacacccag gaagggaaag cgggcagccc
cguuuugggg acgugaacgu 720uuuaauaauu uuugcugaau uccuuuacaa
cuaaauaaca cagauauugu uauaaauaaa 780auugu 785743001RNAArtificial
Sequence3'UTR-011 (Nnt1; cardiotrophin-like cytokine factor 1 UTR)
74auauuaagga ucaagcuguu agcuaauaau gccaccucug caguuuuggg aacaggcaaa
60uaaaguauca guauacaugg ugauguacau cuguagcaaa gcucuuggag aaaaugaaga
120cugaagaaag caaagcaaaa acuguauaga gagauuuuuc aaaagcagua
aucccucaau 180uuuaaaaaag gauugaaaau ucuaaauguc uuucugugca
uauuuuuugu guuaggaauc 240aaaaguauuu uauaaaagga gaaagaacag
ccucauuuua gauguagucc uguuggauuu 300uuuaugccuc cucaguaacc
agaaauguuu uaaaaaacua aguguuuagg auuucaagac 360aacauuauac
auggcucuga aauaucugac acaauguaaa cauugcaggc accugcauuu
420uauguuuuuu uuuucaacaa augugacuaa uuugaaacuu uuaugaacuu
cugagcuguc 480cccuugcaau ucaaccgcag uuugaauuaa ucauaucaaa
ucaguuuuaa uuuuuuaaau 540uguacuucag agucuauauu ucaagggcac
auuuucucac uacuauuuua auacauuaaa 600ggacuaaaua aucuuucaga
gaugcuggaa acaaaucauu ugcuuuauau guuucauuag 660aauaccaaug
aaacauacaa cuugaaaauu aguaauagua uuuuugaaga ucccauuucu
720aauuggagau cucuuuaauu ucgaucaacu uauaaugugu aguacuauau
uaagugcacu 780ugaguggaau ucaacauuug acuaauaaaa ugaguucauc
auguuggcaa gugauguggc 840aauuaucucu ggugacaaaa gaguaaaauc
aaauauuucu gccuguuaca aauaucaagg 900aagaccugcu acuaugaaau
agaugacauu aaucugucuu cacuguuuau aauacggaug 960gauuuuuuuu
caaaucagug uguguuuuga ggucuuaugu aauugaugac auuugagaga
1020aaugguggcu uuuuuuagcu accucuuugu ucauuuaagc accaguaaag
aucaugucuu 1080uuuauagaag uguagauuuu cuuugugacu uugcuaucgu
gccuaaagcu cuaaauauag 1140gugaaugugu gaugaauacu cagauuauuu
gucucucuau auaauuaguu ugguacuaag 1200uuucucaaaa aauuauuaac
acaugaaaga caaucucuaa accagaaaaa gaaguaguac 1260aaauuuuguu
acuguaaugc ucgcguuuag ugaguuuaaa acacacagua ucuuuugguu
1320uuauaaucag uuucuauuuu gcugugccug agauuaagau cuguguaugu
gugugugugu 1380gugugugcgu uuguguguua aagcagaaaa gacuuuuuua
aaaguuuuaa gugauaaaug 1440caauuuguua auugaucuua gaucacuagu
aaacucaggg cugaauuaua ccauguauau 1500ucuauuagaa gaaaguaaac
accaucuuua uuccugcccu uuuucuucuc ucaaaguagu 1560uguaguuaua
ucuagaaaga agcaauuuug auuucuugaa aagguaguuc cugcacucag
1620uuuaaacuaa aaauaaucau acuuggauuu uauuuauuuu ugucauagua
aaaauuuuaa 1680uuuauauaua uuuuuauuua guauuaucuu auucuuugcu
auuugccaau ccuuugucau 1740caauuguguu aaaugaauug aaaauucaug
cccuguucau uuuauuuuac uuuauugguu 1800aggauauuua aaggauuuuu
guauauauaa uuucuuaaau uaauauucca aaagguuagu 1860ggacuuagau
uauaaauuau ggcaaaaauc uaaaaacaac aaaaaugauu uuuauacauu
1920cuauuucauu auuccucuuu uuccaauaag ucauacaauu gguagauaug
acuuauuuua 1980uuuuuguauu auucacuaua ucuuuaugau auuuaaguau
aaauaauuaa aaaaauuuau 2040uguaccuuau agucugucac caaaaaaaaa
aaauuaucug uagguaguga aaugcuaaug 2100uugauuuguc uuuaagggcu
uguuaacuau ccuuuauuuu cucauuuguc uuaaauuagg 2160aguuuguguu
uaaauuacuc aucuaagcaa aaaauguaua uaaaucccau uacuggguau
2220auacccaaag gauuauaaau caugcugcua uaaagacaca ugcacacgua
uguuuauugc 2280agcacuauuc acaauagcaa agacuuggaa ccaacccaaa
uguccaucaa ugauagacuu 2340gauuaagaaa augugcacau auacaccaug
gaauacuaug cagccauaaa aaaggaugag 2400uucauguccu uuguagggac
auggauaaag cuggaaacca ucauucugag caaacuauug 2460caaggacaga
aaaccaaaca cugcauguuc ucacucauag gugggaauug aacaaugaga
2520acacuuggac acaagguggg gaacaccaca caccagggcc ugucaugggg
uggggggagu 2580ggggagggau agcauuagga gauauaccua auguaaauga
ugaguuaaug ggugcagcac 2640accaacaugg cacauguaua cauauguagc
aaaccugcac guugugcaca uguacccuag 2700aacuuaaagu auaauuaaaa
aaaaaaagaa aacagaagcu auuuauaaag aaguuauuug 2760cugaaauaaa
ugugaucuuu cccauuaaaa aaauaaagaa auuuuggggu aaaaaaacac
2820aauauauugu auucuugaaa aauucuaaga gaguggaugu gaaguguucu
caccacaaaa 2880gugauaacua auugagguaa ugcacauauu aauuagaaag
auuuugucau uccacaaugu 2940auauauacuu aaaaauaugu uauacacaau
aaauacauac auuaaaaaau aaguaaaugu 3000a 3001751037RNAArtificial
Sequence3'UTR-012 (Col6a1; collagen, type VI, alpha 1 UTR)
75cccacccugc acgccggcac caaacccugu ccucccaccc cuccccacuc aucacuaaac
60agaguaaaau gugaugcgaa uuuucccgac caaccugauu cgcuagauuu uuuuuaagga
120aaagcuugga aagccaggac acaacgcugc ugccugcuuu gugcaggguc
cuccggggcu 180cagcccugag uuggcaucac cugcgcaggg cccucugggg
cucagcccug agcuaguguc 240accugcacag ggcccucuga ggcucagccc
ugagcuggcg ucaccugugc agggcccucu 300ggggcucagc ccugagcugg
ccucaccugg guuccccacc ccgggcucuc cugcccugcc 360cuccugcccg
cccucccucc ugccugcgca gcuccuuccc uaggcaccuc ugugcugcau
420cccaccagcc ugagcaagac gcccucucgg ggccugugcc gcacuagccu
cccucuccuc 480uguccccaua gcugguuuuu cccaccaauc cucaccuaac
aguuacuuua caauuaaacu 540caaagcaagc ucuucuccuc agcuuggggc
agccauuggc cucugucucg uuuugggaaa 600ccaaggucag gaggccguug
cagacauaaa ucucggcgac ucggccccgu cuccugaggg 660uccugcuggu
gaccggccug gaccuuggcc cuacagcccu ggaggccgcu gcugaccagc
720acugaccccg accucagaga guacucgcag gggcgcuggc ugcacucaag
acccucgaga 780uuaacggugc uaaccccguc ugcuccuccc ucccgcagag
acuggggccu ggacuggaca 840ugagagcccc uuggugccac agagggcugu
gucuuacuag aaacaacgca aaccucuccu 900uccucagaau agugaugugu
ucgacguuuu aucaaaggcc cccuuucuau guucauguua 960guuuugcucc
uucuguguuu uuuucugaac cauauccaug uugcugacuu uuccaaauaa
1020agguuuucac uccucuc 103776577RNAArtificial Sequence3'UTR-013
(Calr; calreticulin UTR) 76agaggccugc cuccagggcu ggacugaggc
cugagcgcuc cugccgcaga gcuggccgcg 60ccaaauaaug ucucugugag acucgagaac
uuucauuuuu uuccaggcug guucggauuu 120gggguggauu uugguuuugu
uccccuccuc cacucucccc cacccccucc ccgcccuuuu 180uuuuuuuuuu
uuuuaaacug guauuuuauc uuugauucuc cuucagcccu caccccuggu
240ucucaucuuu cuugaucaac aucuuuucuu gccucugucc ccuucucuca
ucucuuagcu 300ccccuccaac cuggggggca guggugugga gaagccacag
gccugagauu ucaucugcuc 360uccuuccugg agcccagagg agggcagcag
aagggggugg ugucuccaac cccccagcac 420ugaggaagaa cggggcucuu
cucauuucac cccucccuuu cuccccugcc cccaggacug 480ggccacuucu
ggguggggca guggguccca gauuggcuca cacugagaau guaagaacua
540caaacaaaau uucuauuaaa uuaaauuuug ugucucc 577772212RNAArtificial
Sequence3'UTR-014 (Col1a1; collagen, type I, alpha 1 UTR)
77cucccuccau cccaaccugg cucccuccca cccaaccaac uuucccccca acccggaaac
60agacaagcaa cccaaacuga acccccucaa aagccaaaaa augggagaca auuucacaug
120gacuuuggaa aauauuuuuu uccuuugcau ucaucucuca aacuuaguuu
uuaucuuuga 180ccaaccgaac augaccaaaa accaaaagug cauucaaccu
uaccaaaaaa aaaaaaaaaa 240aaagaauaaa uaaauaacuu uuuaaaaaag
gaagcuuggu ccacuugcuu gaagacccau 300gcggggguaa gucccuuucu
gcccguuggg cuuaugaaac cccaaugcug cccuuucugc 360uccuuucucc
acaccccccu uggggccucc ccuccacucc uucccaaauc ugucucccca
420gaagacacag gaaacaaugu auugucugcc cagcaaucaa aggcaaugcu
caaacaccca 480aguggccccc acccucagcc cgcuccugcc cgcccagcac
ccccaggccc ugggggaccu 540gggguucuca gacugccaaa gaagccuugc
caucuggcgc ucccauggcu cuugcaacau 600cuccccuucg uuuuugaggg
ggucaugccg ggggagccac cagccccuca cuggguucgg 660aggagaguca
ggaagggcca cgacaaagca gaaacaucgg auuuggggaa cgcgugucaa
720ucccuugugc cgcagggcug ggcgggagag acuguucugu uccuugugua
acuguguugc 780ugaaagacua ccucguucuu gucuugaugu gucaccgggg
caacugccug ggggcgggga 840ugggggcagg guggaagcgg cuccccauuu
uauaccaaag gugcuacauc uaugugaugg 900gugggguggg gagggaauca
cuggugcuau agaaauugag augccccccc aggccagcaa 960auguuccuuu
uuguucaaag ucuauuuuua uuccuugaua uuuuucuuuu uuuuuuuuuu
1020uuuuugugga uggggacuug ugaauuuuuc uaaaggugcu auuuaacaug
ggaggagagc 1080gugugcggcu ccagcccagc ccgcugcuca cuuuccaccc
ucucuccacc ugccucuggc 1140uucucaggcc ucugcucucc gaccucucuc
cucugaaacc cuccuccaca gcugcagccc 1200auccucccgg cucccuccua
gucuguccug cguccucugu ccccggguuu cagagacaac 1260uucccaaagc
acaaagcagu uuuucccccu agggguggga ggaagcaaaa gacucuguac
1320cuauuuugua uguguauaau aauuugagau guuuuuaauu auuuugauug
cuggaauaaa 1380gcauguggaa augacccaaa cauaauccgc aguggccucc
uaauuuccuu cuuuggaguu 1440gggggagggg uagacauggg gaaggggcuu
uggggugaug ggcuugccuu ccauuccugc 1500ccuuucccuc cccacuauuc
ucuucuagau cccuccauaa ccccacuccc cuuucucuca 1560cccuucuuau
accgcaaacc uuucuacuuc cucuuucauu uucuauucuu gcaauuuccu
1620ugcaccuuuu ccaaauccuc uucuccccug caauaccaua caggcaaucc
acgugcacaa 1680cacacacaca cacucuucac aucugggguu guccaaaccu
cauacccacu ccccuucaag 1740cccauccacu cuccaccccc uggaugcccu
gcacuuggug gcggugggau gcucauggau 1800acugggaggg ugaggggagu
ggaacccgug aggaggaccu gggggccucu ccuugaacug 1860acaugaaggg
ucaucuggcc ucugcucccu ucucacccac gcugaccucc ugccgaagga
1920gcaacgcaac aggagagggg ucugcugagc cuggcgaggg ucugggaggg
accaggagga 1980aggcgugcuc ccugcucgcu guccuggccc
ugggggagug agggagacag acaccuggga 2040gagcuguggg gaaggcacuc
gcaccgugcu cuugggaagg aaggagaccu ggcccugcuc 2100accacggacu
gggugccucg accuccugaa uccccagaac acaacccccc ugggcugggg
2160uggucugggg aaccaucgug cccccgccuc ccgccuacuc cuuuuuaagc uu
221278729RNAArtificial Sequence3'UTR-015 (Plod1;
procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 UTR)
78uuggccaggc cugacccucu uggaccuuuc uucuuugccg acaaccacug cccagcagcc
60ucugggaccu cgggguccca gggaacccag uccagccucc uggcuguuga cuucccauug
120cucuuggagc caccaaucaa agagauucaa agagauuccu gcaggccaga
ggcggaacac 180accuuuaugg cuggggcucu ccgugguguu cuggacccag
ccccuggaga caccauucac 240uuuuacugcu uuguagugac ucgugcucuc
caaccugucu uccugaaaaa ccaaggcccc 300cuucccccac cucuuccaug
gggugagacu ugagcagaac aggggcuucc ccaaguugcc 360cagaaagacu
gucuggguga gaagccaugg ccagagcuuc ucccaggcac agguguugca
420ccagggacuu cugcuucaag uuuuggggua aagacaccug gaucagacuc
caagggcugc 480ccugagucug ggacuucugc cuccauggcu ggucaugaga
gcaaaccgua guccccugga 540gacagcgacu ccagagaacc ucuugggaga
cagaagaggc aucugugcac agcucgaucu 600ucuacuugcc uguggggagg
ggagugacag guccacacac cacacugggu cacccugucc 660uggaugccuc
ugaagagagg gacagaccgu cagaaacugg agaguuucua uuaaagguca 720uuuaaacca
72979847RNAArtificial Sequence3'UTR-016 (Nucb1; nucleobindin 1 UTR)
79uccuccggga ccccagcccu caggauuccu gaugcuccaa ggcgacugau gggcgcugga
60ugaaguggca cagucagcuu cccugggggc uggugucaug uugggcuccu ggggcggggg
120cacggccugg cauuucacgc auugcugcca ccccaggucc accugucucc
acuuucacag 180ccuccaaguc uguggcucuu cccuucuguc cuccgagggg
cuugccuucu cucgugucca 240gugaggugcu cagugaucgg cuuaacuuag
agaagcccgc ccccuccccu ucuccgucug 300ucccaagagg gucugcucug
agccugcguu ccuagguggc ucggccucag cugccugggu 360uguggccgcc
cuagcauccu guaugcccac agcuacugga auccccgcug cugcuccggg
420ccaagcuucu gguugauuaa ugagggcaug gggugguccc ucaagaccuu
ccccuaccuu 480uuguggaacc agugaugccu caaagacagu guccccucca
cagcugggug ccaggggcag 540gggauccuca guauagccgg ugaacccuga
uaccaggagc cugggccucc cugaaccccu 600ggcuuccagc caucucaucg
ccagccuccu ccuggaccuc uuggccccca gccccuuccc 660cacacagccc
cagaaggguc ccagagcuga ccccacucca ggaccuaggc ccagccccuc
720agccucaucu ggagccccug aagaccaguc ccacccaccu uucuggccuc
aucugacacu 780gcuccgcauc cugcugugug uccuguucca uguuccgguu
ccauccaaau acacuuucug 840gaacaaa 84780110RNAArtificial
Sequence3'UTR-017 (alpha-globin) 80gcuggagccu cgguggccau gcuucuugcc
ccuugggccu ccccccagcc ccuccucccc 60uuccugcacc cguacccccg uggucuuuga
auaaagucug agugggcggc 11081119RNAArtificial Sequence3'UTR-018
81ugauaauagg cuggagccuc gguggccaug cuucuugccc cuugggccuc cccccagccc
60cuccuccccu uccugcaccc guacccccgu ggucuuugaa uaaagucuga gugggcggc
1198218DNAArtificial SequenceSyn5 promoter 82attgggcacc cgtaaggg
188392DNAArtificial Sequence5'UTR of mRNA encoding human PBGD
83tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga
60aaagaagagt aagaagaaat ataagagcca cc 9284119DNAArtificial
Sequence3'UTR of mRNA encoding human PBGD 84tgataatagg ctggagcctc
ggtggccatg cttcttgccc cttgggcctc cccccagccc 60ctcctcccct tcctgcaccc
gtacccccgt ggtctttgaa taaagtctga gtgggcggc 1198519PRTArtificial
Sequencehuman specific peptide 85Ala Ser Tyr Pro Gly Leu Gln Phe
Glu Ile Ile Ala Met Ser Thr Thr1 5 10 15Gly Asp
Lys861650RNAArtificial SequencePBGD-CO27 86auggaagaug cgaagaacau
caagaaggga ccugccccgu uuuacccuuu ggaggacggu 60acagcaggag aacagcucca
caaggcgaug aaacgcuacg cccugguccc cggaacgauu 120gcguuuaccg
augcacauau ugagguagac aucacauacg cagaauacuu cgaaaugucg
180gugaggcugg cggaagcgau gaagagauau ggucuuaaca cuaaucaccg
caucguggug 240uguucggaga acucauugca guuuuucaug ccgguccuug
gagcacuuuu caucgggguc 300gcagucgcgc cagcgaacga caucuacaau
gagcgggaac ucuugaauag caugggaauc 360ucccagccga cggucguguu
ugucuccaaa aaggggcugc agaaaauccu caacgugcag 420aagaagcucc
ccauuauuca aaagaucauc auuauggaua gcaagacaga uuaccaaggg
480uuccagucga uguauaccuu ugugacaucg cauuugccgc caggguuuaa
cgaguaugac 540uucguccccg agucauuuga cagagauaaa accaucgcgc
ugauuaugaa uuccucgggu 600agcaccgguu ugccaaaggg gguggcguug
ccccaccgca cugcuugugu gcgguucucg 660cacgcuaggg auccuaucuu
ugguaaucag aucauucccg acacagcaau ccuguccgug 720guaccuuuuc
aucacgguuu uggcauguuc acgacucucg gcuauuugau uugcgguuuc
780agggucguac uuauguaucg guucgaggaa gaacuguuuu ugagauccuu
gcaagauuac 840aagauccagu cggcccuccu ugugccaacg cuuuucucau
ucuuugcgaa aucgacacuu 900auugauaagu augaccuuuc caaucugcau
gagauugccu cagggggagc gccgcuuagc 960aaggaagucg gggaggcagu
ggccaagcgc uuccaccuuc ccggaauucg gcagggauac 1020gggcucacgg
agacaacauc cgcgauccuu aucacgcccg agggugacga uaagccggga
1080gccgucggaa aagugguccc cuucuuugaa gccaaggucg uagaccucga
cacgggaaaa 1140acccucggag ugaaccagag gggcgagcuc ugcgugagag
ggccgaugau caugucaggu 1200uacgugaaua acccugaagc gacgaaugcg
cugaucgaca aggaugggug guugcauucg 1260ggagacauug ccuauuggga
ugaggaugag cacuucuuua ucguagaucg acuuaagagc 1320uugaucaaau
acaaaggcua ucagguagcg ccugccgagc ucgagucaau ccugcuccag
1380caccccaaca uuuucgacgc cggaguggcc ggguugcccg augacgacgc
gggugagcug 1440ccagcggccg ugguaguccu cgaacauggg aaaacaauga
ccgaaaagga gaucguggac 1500uacguagcau cacaagugac gacugcgaag
aaacugaggg gagggguagu cuuuguggac 1560gaggucccga aaggcuugac
ugggaagcuu gacgcucgca aaauccggga aauccugauu 1620aaggcaaaga
aaggcgggaa aaucgcuguc 1650871083RNAArtificial SequencePBGD-CO26
87augucuggua acggcaaugc ggcugcaacg gcggaagaaa acagcccaaa gaugagagug
60auucgcgugg guacccgcaa gagccagcuu gcucgcauac agacggacag ugugguggca
120acauugaaag ccucguaccc uggccugcag uuugaaauca uugcuauguc
caccacaggg 180gacaagauuc uugauacugc acucucuaag auuggagaga
aaagccuguu uaccaaggag 240cuugaacaug cccuggagaa gaaugaagug
gaccugguug uucacuccuu gaaggaccug 300cccacugugc uuccuccugg
cuucaccauc ggagccaucu gcaagcggga aaacccucau 360gaugcuguug
ucuuucaccc aaaauuuguu gggaagaccc uagaaacccu gccagagaag
420aguguggugg gaaccagcuc ccugcgaaga gcagcccagc ugcagagaaa
guucccgcau 480cuggaguuca ggaguauucg gggaaaccuc aacacccggc
uucggaagcu ggacgagcag 540caggaguuca gugccaucau ccuggcaaca
gcuggccugc agcgcauggg cuggcacaac 600cggguggggc agauccugca
cccugaggaa ugcauguaug cugugggcca gggggccuug 660ggcguggaag
ugcgagccaa ggaccaggac aucuuggauc uggugggugu gcugcacgau
720cccgagacuc ugcuucgcug caucgcugaa agggccuucc ugaggcaccu
ggaaggaggc 780ugcagugugc caguagccgu gcauacagcu augaaggaug
ggcaacugua ccugacugga 840ggagucugga gccuagacgg cucagauagc
auacaagaga ccaugcaggc uaccauccau 900gucccugccc agcaugaaga
uggcccugag gaugacccac aguugguagg caucacugcu 960cguaacauuc
cacgagggcc ccaguuggcu gcccagaacu ugggcaucag ccuggccaac
1020uuguugcuga gcaaaggagc caaaaacauc cuggauguug cacggcagcu
uaacgaugcc 1080cau 1083881083RNAArtificial SequencePBGD-CO28
88augaguggca acggaaacgc cgccgcuaca gcagaggaga acuccccgaa gaugcgcgug
60auuaggguag gcaccagaaa gucucagcug gccagaaucc aaaccgauag cguuguggcc
120acauugaagg cuagcuaucc cggccugcag uucgagauca ucgccaugag
caccaccggc 180gacaagauac ucgacaccgc ucugaguaaa aucggcgaga
agagccuguu uaccaaggag 240cuggagcacg cccuggaaaa gaacgaagug
gaccuggugg ugcauagucu gaaagaccuu 300cccaccgucc uuccaccagg
cuucacuauc ggcgccaucu gcaagaggga gaauccucac 360gaugccgucg
uguuucaucc caaguucgug ggcaagaccu uagaaacccu gccagagaaa
420agcgucguug ggaccuccuc ccugcgacgg gccgcccagc ugcagagaaa
guucccccac 480uuggaauuca gauccaucag agggaaucug aauacucgcc
ugagaaagcu ggacgagcag 540caggaguuua gcgcuaucau ccuggccacg
gcugguuugc agagaauggg cuggcacaac 600cgggugggac agauccugca
ccccgaggag ugcauguaug caguaggcca gggggcccug 660gggguggagg
ucagagccaa agaucaggac auccuggacc uggucggcgu gcugcacgau
720cccgaaacac ugcugcggug uaucgccgag agggcuuucc uccggcacuu
agagggcggc 780ugcuccgucc ccguggccgu ucacacugcc augaaagacg
ggcagcugua ccugacgggc 840ggcguguggu cccuggacgg cucagacucc
auucaggaga ccaugcaagc uaccauccac 900gucccugccc aacacgaaga
uggccccgag gacgaccccc agcugguggg caucaccgcc 960aggaauaucc
caagaggccc ccaguuggcc gcccagaacc ugggcaucag ucuggccaac
1020cugcugcuga guaagggcgc caaaaacauc cuggacgugg cucggcagcu
gaaugacgcc 1080cac 1083891083RNAArtificial SequencePBGD-CO30
89augagcggca acggcaacgc cgcagccacc gccgaggaaa acagccccaa gaugcgggug
60aucagagugg gcacccggaa gagccagcug gcccggaucc agaccgacag cgugguggcc
120acccugaagg ccuccuaccc cggccugcag uucgagauca uugccaugag
caccaccggc 180gacaagaucc uggacaccgc ccugagcaag aucggcgaga
agagccuguu cacaaaagag 240cuggaacacg cccuggaaaa gaacgaggug
gaccuggugg ugcacagccu gaaggaccug 300cccaccgugc ugcccccugg
cuucaccauc ggcgccaucu gcaagagaga gaacccccac 360gacgccgugg
uguuccaccc uaaguucgug ggcaagacac uggaaacccu gcccgagaag
420uccguggugg gcaccagcag ccugcggaga gccgcccagc ugcagcggaa
guucccccac 480cuggaauuuc ggagcauccg gggcaaccug aacacccggc
ugcggaagcu ggacgagcag 540caggaauuuu ccgcuaucau ccuggccaca
gccggacugc agcggauggg cuggcacaac 600agagugggcc agauccugca
ccccgaggaa ugcauguacg ccgugggcca gggagcccug 660ggcguggaag
ugcgggccaa ggaccaggac auccuggauc uggugggcgu gcugcaugac
720cccgagacac ugcugcggug uaucgccgag cgggccuucc ugcggcaccu
ggaaggcggc 780ugcagcgugc ccguggccgu gcacaccgcc augaaggacg
gacagcugua ccugacaggc 840ggcgugugga gccuggacgg cagcgacagc
auccaggaga ccaugcaggc caccauccac 900gugcccgccc agcacgagga
cggccccgag gacgacccuc agcuggucgg caucaccgcc 960cggaacaucc
ccagaggccc ccagcuggcc gcccagaacc ugggcaucag ccuggccaac
1020cugcugcugu ccaagggcgc caagaacauc cuggacgugg cccggcagcu
gaacgacgcc 1080cac 1083901083RNAArtificial SequencePBGD-CO31
90augagcggca acggcaacgc cgccgcuacc gccgaagaga acagcccaaa gaugcgcgug
60aucagggucg gcacgcgcaa gucccagcuc gcccggaucc aaaccgauag cgugguggcc
120acgcucaagg cgagcuaucc gggcuuacag uucgagauca ucgccaugag
caccaccggc 180gauaagauac uggacaccgc ccuguccaag aucggcgaaa
agagccuguu caccaaggaa 240cuggagcacg cgcuggagaa gaacgaggug
gaccuggugg ugcacagccu gaaggaccug 300ccgaccgugc ugccgccggg
auucaccauc ggcgccaucu gcaagaggga gaauccgcac 360gaugccgugg
uguuccaccc aaaguucgug ggcaagaccu uggaaacccu gccagagaag
420ucuguggucg gcaccuccag ccugcggcga gccgcccagc ugcagcgaaa
guucccgcac 480cuggaguuca gguccauccg cggaaaucug aacaccaggc
ugcgcaagcu cgacgagcag 540caggaguucu ccgccaucau ccuggccacc
gcaggccucc aaagaauggg cuggcauaac 600cgagucggcc agauccucca
cccggaggag ugcauguacg cagugggcca aggcgcccug 660ggcgucgagg
ugcgugccaa ggaccaggac auccuggacc uggugggcgu gcuccacgau
720ccagagacac ugcugagaug caucgcggag cgcgccuucc ugcgccaucu
ggagggaggc 780ugcuccgucc cgguggccgu acauaccgcc augaaggacg
gucagcugua ccucaccggc 840ggcguauggu cccucgacgg uagcgacagc
auacaggaga cgaugcaggc caccauccac 900gugccggcgc agcacgagga
uggaccagag gacgacccgc agcugguggg uaucaccgcc 960aggaauaucc
cgcggggacc ucagcuggcc gcccagaacc ugggcaucuc ccucgccaac
1020cuccugcuga gcaagggcgc caagaacauc cuggacgugg ccaggcagcu
caacgaugcc 1080cau 1083911083RNAArtificial SequencePBGD-CO32
91augagcggca acggcaacgc cgccgccacc gccgaggaaa acagcccgaa gaugcgggug
60aucagggugg gcaccaggaa gucccagcuc gcccggaucc agaccgacag cguggucgcc
120accuugaagg ccuccuaccc gggccuccag uucgagauca ucgccauguc
cacaaccggc 180gacaagaucc uggauaccgc ccucagcaag aucggcgaga
agucccuguu caccaaggag 240cuggagcacg cccuggagaa gaaugaggug
gaccuggugg ugcacagccu gaaggaccug 300ccuaccgugc ugccaccagg
cuucacaauc ggcgccaucu gcaagagaga gaacccgcac 360gacgccgugg
uguuccaucc gaaguucgug ggcaagaccc uggaaacccu gccggagaag
420uccguagugg gaaccucaag ccugaggcgc gccgcccagc uccagaggaa
guucccucac 480cuggaauucc gguccaucag gggcaaccug aacacgcgcc
ugcggaagcu cgacgagcag 540caggaguucu ccgccaucau ccuggccaca
gccggccuuc agcgcauggg cuggcacaac 600agggugggcc agauccugca
cccggaagaa ugcauguacg ccgugggcca aggcgcccuc 660ggcguggaag
ugcgugccaa ggaccaggac auccuggacc uggugggcgu gcugcacgac
720ccugagacgc ugcucaggug caucgccgaa cgcgcguucc ugcggcaccu
ggagggaggc 780ugcagcgucc cgguggccgu ccacaccgcc augaaggacg
gccagcucua ccugacuggc 840ggcgugugga gccuggacgg cagcgacagc
auucaggaaa ccaugcaggc caccauccac 900gugccugccc agcacgagga
cggcccggag gacgacccuc aacugguggg cauuacugcg 960cgaaacaucc
cgcgcggacc ucagcuggcc gcccagaacc ugggcaucag ccuggccaac
1020cugcuccugu ccaagggcgc caagaacauc cucgacgugg ccaggcagcu
gaacgacgcg 1080cac 1083921083RNAArtificial SequencePBGD-CO33
92augagcggca acggaaacgc cgccgcgacc gcggaggaga acucgccuaa gaugagagug
60auaaggguag gcacccggaa gucucaacuc gccaggaucc agaccgacag cgugguggcc
120acccucaagg ccagcuaucc aggacuccag uucgaaauca ucgccauguc
caccacaggc 180gauaagaucc uggacaccgc ccuguccaag aucggcgaga
agucccucuu caccaaggaa 240cuggagcacg cccuggagaa gaacgagguc
gaucuggucg ugcacagccu gaaggaucug 300ccuaccgugc ucccgccggg
cuucaccauc ggcgccaucu gcaagaggga gaauccucac 360gacgccgugg
uguuccaccc gaaguucgug ggcaagaccc uggagacacu gccagaaaag
420ucgguggugg gcaccagcag ccugcggcgg gcggcccagc ugcagcggaa
guucccacac 480cuggaguuca gguccauccg uggcaaucug aacacccggc
ugcguaagcu ggacgagcag 540caggaauuca gcgcgaucau ccuggcaacc
gccggucugc aaaggauggg cuggcacaac 600agggugggcc agauccugca
cccugaggag ugcauguacg ccgugggcca gggagcccug 660ggcguggaag
ugcgggccaa ggaccaggac auccuggacc uggugggugu gcuccacgac
720ccugaaaccc ugcugcggug caucgccgaa agggccuucc ugaggcaccu
cgagggcggc 780ugcagcgugc cggucgccgu gcacaccgcc augaaggacg
gccagcugua ccugaccgga 840ggagugugga gccuggacgg cuccgacucc
auccaggaga cuaugcaggc caccauucau 900gugccggccc agcaugagga
cgguccggag gacgauccac agcuggucgg caucaccgcg 960cggaacaucc
caagaggccc gcaacuggcc gcucagaacc ugggcauauc ccuggccaac
1020cugcuccuga gcaagggcgc caagaacauc cuggacgugg ccaggcagcu
gaaugacgcc 1080cac 1083931083RNAArtificial SequencePBGD-CO34
93auguccggca acggcaacgc cgccgcuacc gccgaggaga acuccccuaa gaugcggguc
60aucagggugg gcacccgaaa gucccaacuu gcccggaucc agaccgacuc cgucguggcc
120acccucaagg cuagcuaucc aggccuccag uucgaaauca ucgccaugag
caccaccggc 180gacaagauuc uggacaccgc ccuguccaag aucggcgaga
agagucuguu cacgaaggag 240cucgagcacg cccuggaaaa gaacgaggug
gaccuggugg ugcauucccu gaaggaccug 300ccaaccgugc ugccgccggg
cuucacuaua ggagccaucu gcaagcggga gaacccgcac 360gacgcggugg
uguuccaucc gaaguucgug ggcaagacuc uggaaacccu gccggagaag
420uccguggugg gaacuagcuc ccugcggcgg gccgcccagc ugcagaggaa
guucccgcac 480cuggaguuca ggagcauacg cggcaaccug aacacccgcc
ugcguaagcu cgacgagcag 540caggaauuca gugccaucau ccuggccacg
gcgggccugc agcggauggg cuggcacaac 600agggugggcc agauccucca
cccggaggaa uguauguacg ccgugggcca gggcgcacug 660ggcguggagg
uccgcgccaa ggaccaagac auccuggacc uggucggcgu gcugcacgac
720ccugaaaccc ugcugaggug cauugccgag agagccuucc ugaggcaucu
ggagggcggc 780ugcagcgugc cuguggccgu gcacacagcc augaaggacg
gucagcugua ccugaccggc 840ggcgugugga gccuggacgg cagcgacucc
auccaggaga caaugcaggc caccauccac 900gucccggccc aacacgagga
cggaccugag gacgauccuc agcugguggg caucaccgcc 960aggaacaucc
cucggggccc gcagcuggcc gcccagaacc ugggcaucuc ccucgccaac
1020cugcugcugu ccaagggcgc caagaacauc cucgacgugg ccagacagcu
gaacgacgcc 1080cac 1083941083RNAArtificial SequencePBGD-CO35
94augagcggca acggcaacgc cgccgccacc gccgaggaga acagcccgaa gaugagggug
60auaagggugg gcacacggaa gucccagcuc gcccgcaucc aaaccgacuc cgugguggcc
120acccucaagg ccagcuaccc gggccuccaa uucgagauca ucgccaugag
caccaccggc 180gacaagaucc uggacaccgc ccugucuaag auaggcgaaa
agagccuguu caccaaggag 240cuggagcaug cccuggagaa gaacgaggug
gaccuggugg uccacagucu caaggaccug 300ccaaccgugc ugccgccagg
cuucaccauc ggcgccaucu gcaagcguga gaacccgcac 360gaugcugugg
uguuccaccc uaaguucgug ggaaagaccc uggagacgcu gccggaaaag
420agcguggucg gcaccuccag ccugcggagg gccgcccaac uccagaggaa
guucccgcac 480cuggaguuca ggagcauccg cggcaaccug aacaccaggc
ugcgaaagcu ggacgagcag 540caggaauucu cggccaucau ccucgccacc
gccggcuugc aaagaauggg cuggcauaau 600cgcgugggcc agauccugca
cccugaggag ugcauguacg ccgugggcca gggugcucug 660ggaguggagg
ugcgggccaa ggaccaggau auccuggacc uggucggcgu gcuucaugac
720ccggagacgc uccugaggug caucgccgag cgggccuucc ugagacaccu
ggagggcggc 780ugcuccgugc caguggccgu gcacaccgcc augaaggacg
gacagcugua ccugaccggc 840ggcgugugga gccuggacgg aagcgacagc
auccaagaaa ccaugcaggc gaccauucac 900gucccugccc agcacgagga
uggaccagag gacgacccgc agcugguggg caucaccgcc 960cgcaacaucc
cuagaggccc acagcuggcc gcccagaauc ugggcaucag ccuggccaac
1020cugcugcugu cuaagggagc caagaacauc cuggacgugg ccaggcagcu
gaacgacgcc 1080cau 1083951083RNAArtificial SequencePBGD-CO36
95auguccggca acggcaacgc cgcagccacc gccgaggaga auuccccgaa gaugcgggug
60auccgggugg gcaccagaaa gagccagcuc gcccgcaucc aaaccgacuc cgugguggcc
120acccucaagg ccuccuaccc aggcuugcag uucgaaauca ucgccaugag
caccaccggc 180gacaagaucc uggacaccgc ccugagcaag auuggcgaga
agucccuguu caccaaggag 240cuggagcaug cucuggagaa gaacgaggug
gaccucgugg ugcacucccu gaaggaccug 300ccgacugugc ugccgccugg
cuucacgauc ggcgccauau gcaagcggga aaacccacac 360gacgccgugg
ucuuccaccc aaaguucgug ggcaagaccc uggaaacccu gccggaaaag
420agcguggucg gcacaagcuc ccugaggaga gccgcccaac ugcaaaggaa
guucccucac 480cucgaguuca gguccauccg gggcaaccug aacaccaggc
ugagaaagcu cgacgaacag 540caggaguuca gcgccaucau ccuggccacg
gccggccugc agaggauggg auggcauaac 600agggugggcc agauccugca
cccggaggag ugcauguacg ccgugggcca gggagcccuc 660ggcguggagg
ucagggccaa ggaucaggau auccuggacc uggugggcgu gcugcacgau
720ccugagacgc ugcugaggug caucgccgag cgggccuucc ugcggcaccu
agagggcgga 780ugcagcgugc cggucgcggu ccacaccgcg augaaggacg
gccagcugua ccugaccggc
840ggcguguggu cccuggacgg cagcgauuca auccaggaga cgaugcaggc
caccauccac 900gugccagccc agcacgagga uggcccggag gacgacccgc
agcugguggg cauuacagcc 960aggaacaucc cucggggccc gcagcuggcc
gcccagaauc ugggcaucag ccuggcgaac 1020cugcugcuca gcaagggagc
gaagaacauc cuggacgugg cccgccagcu gaacgaugcc 1080cac
1083961083RNAArtificial SequencePBGD-CO37 96augagcggca acggcaacgc
cgccgccacc gccgaggaga acagcccaaa gaugcgggug 60aucagggugg gcacccgcaa
gagccaacuc gccagaaucc agaccgacag cgugguggcc 120accuugaagg
ccagcuaccc gggccuccag uucgagauca ucgcuauguc caccaccggc
180gacaagaucc uggacaccgc gcuguccaag aucggcgaaa agagccuguu
caccaaggaa 240cuggagcacg cccucgagaa gaacgaggug gaccuggugg
ugcacucccu gaaggaccug 300ccgacggucc ugccgccggg cuucaccauc
ggcgccaucu gcaagcggga aaacccgcac 360gacgcugugg uguuccaccc
aaaguucgug ggcaagaccc uggaaacccu gccagaaaag 420agcguggugg
gcaccagcag ccucaggaga gccgcccagc ugcagaggaa guucccgcac
480cuggaguuca ggagcaucag gggcaaccug aacaccaggc ugcguaagcu
ggacgagcag 540caggaguucu ccgccaucau ccucgccaca gccggccucc
agaggauggg uuggcacaac 600agggugggcc agauccugca cccggaagag
ugcauguacg cagugggcca gggcgcccuu 660ggcguggaag ugcgagccaa
ggaucaggau auccuggacc uggugggcgu gcugcacgac 720ccggaaacuc
ugcugcggug caucgccgaa agggccuucc ugcgccaccu cgaaggcggc
780uguagcgugc cgguggccgu gcacaccgcc augaaggacg gccagcugua
ccugaccggc 840ggcgugugga gccucgacgg cagcgacagc auccaggaga
caaugcaggc caccauccac 900gugccggccc agcaugagga uggcccggag
gacgacccuc agcugguggg caucaccgcc 960cgcaacaucc caagaggacc
gcaacuggcc gcccagaacc ugggcaucuc ccuggccaac 1020cugcuccuga
gcaagggcgc gaagaacauc cucgacgucg cacggcagcu gaacgacgcc 1080cac
1083971083RNAArtificial SequencePBGD-CO38 97augagcggca acggcaacgc
cgccgcgacg gccgaggaaa auagcccgaa gaugcgggug 60aucagggugg gcaccaggaa
gucccagcuc gccaggaucc agaccgacag cgugguggcc 120acccucaagg
ccuccuaccc gggccuccaa uucgagauca ucgccauguc caccaccggc
180gacaagaucc ucgacaccgc ccugagcaag aucggcgaaa agucgcuguu
caccaaggag 240cuggagcacg cccucgagaa gaacgaggug gaccugguag
ugcacucccu aaaggaccug 300ccgaccgugc ugccgccggg cuucacgauc
ggcgccaucu gcaagcgcga gaacccgcau 360gaugccgucg uuuuccaccc
uaaguucgug ggcaagaccc uggagacgcu gccggagaag 420ucgguggugg
gaaccagcag ccugaggagg gccgcacaac ugcagaggaa guucccgcau
480cuggaguucc gcagcauucg aggcaaccug aacacgcgcc ugagaaagcu
cgaugaacag 540caggaguuca gcgccaucau ucuggccacu gccggacugc
agcggauggg cuggcacaac 600agagugggcc agauccugca uccggaagag
uguauguacg ccgugggcca gggugcccug 660ggcguggagg ugcgggccaa
ggaccaggau auacuggauc uggucggcgu gcuccacgac 720ccagaaacac
uccugaggug caucgcugag agagccuucc uccggcaccu cgagggcggc
780uguuccgugc cgguggccgu ccauaccgcc augaaggacg gucagcugua
ccugaccgga 840ggcguuuggu cccuggacgg cagcgacagc auccaggaaa
ccaugcaggc caccauccac 900gugccggcgc agcacgagga cggcccggaa
gacgacccgc agcuggucgg caucacggcc 960agaaacaucc cgcggggccc
gcagcuggcg gcccagaacc ugggaaucuc ccuggccaac 1020cugcugcuga
gcaagggcgc gaagaacauc cuggacgugg ccaggcagcu gaacgaugcc 1080cac
1083981083RNAArtificial SequencePBGD-CO39 98augagcggua acggcaacgc
cgccgccacc gccgaggaga acuccccgaa gaugcgcgug 60auucgggucg gcacaagaaa
gucucaacuc gcccgaaucc aaacggacag cgugguggcc 120acccucaagg
cgagcuaccc gggccuccag uucgaaauca ucgccaugag caccaccggc
180gacaagaucc uggacaccgc ccugucgaag auuggcgaaa agucccuguu
caccaaggag 240cuggagcacg cccuggagaa gaacgaaguc gaccuggucg
ugcacagccu gaaggaccug 300ccgaccguuc ugccgccggg cuucaccauc
ggagccaucu gcaagcggga gaauccgcac 360gacgccgugg ucuuccaccc
aaaguucgug ggaaagaccc ucgagacacu gccggagaag 420uccguggugg
gaaccuccuc ccugcggagg gccgcccaac ugcagcggaa guucccacac
480cuggaauucc gguccaucag aggcaaccuc aacaccaggc ugaggaagcu
cgaugagcag 540caggaguuca gcgccaucau ccuggccaca gccggacugc
agcgcauggg cuggcauaac 600agagugggcc agauccucca cccggaggag
ugcauguacg ccgugggaca aggcgcgcug 660ggcguggaag uucgggccaa
ggaccaggau auccuggacc uggugggcgu gcuccacgac 720ccagagacgc
ugcugcggug caucgccgag cgcgccuucc ugcggcaccu cgagggcggc
780ugcagcgugc cggucgcugu gcacacagcc augaaggacg gccagcugua
ccugaccggc 840ggcgugugga gucuggacgg cagcgacucc auccaggaga
cuaugcaagc caccauccau 900gugccggccc aacaugagga cggcccggag
gacgacccgc aacugguggg caucaccgcc 960cggaacaucc cgaggggccc
gcagcuggcc gcccagaacc ugggcauuag ccuggccaac 1020cugcuccuga
gcaagggcgc uaagaacauc cuggacgucg ccagacagcu gaacgacgcc 1080cac
1083991083RNAArtificial SequencePBGD-CO40A 99augagcggca acggcaacgc
cgccgccacc gccgaggaga acagccccaa gaugcgggug 60auccgggugg gcacccguaa
gagccagcug gcccggaucc agaccgacag cgugguggcc 120acccugaagg
ccagcuaccc cggccugcag uucgagauca ucgccaugag caccaccggc
180gacaagaucc uggacaccgc ccugagcaag aucggcgaga agucccuguu
caccaaggag 240cuggagcacg cccuggagaa gaacgaggug gaccuggugg
ugcacagccu gaaggaccug 300cccaccgugc ugccuccagg cuucaccauc
ggcgccaucu gcaagcggga gaacccccac 360gacgccgugg uguuccaccc
caaguucgug ggcaagaccc uggaaacccu gccugagaag 420uccgucguag
gaaccagcag ccugcggcgg gccgcccagc ugcagcggaa guucccccac
480cuggaguucc ggagcauccg gggcaaccug aacacccggc ugcggaagcu
ggacgagcag 540caggaguuca gcgccaucau ccuggcuacc gccggucugc
aacgaauggg cuggcacaau 600agggugggcc agauccugca ccccgaggag
ugcauguacg cggugggaca gggcgcccug 660ggcguggagg ugcgggccaa
ggaccaggac auccuugauc uggugggcgu gcugcacgac 720cccgagacgc
ugcugcggug caucgccgag cgggccuucc ugcggcauuu ggagggcgga
780ugcagcgugc ccguggccgu gcacaccgcc augaaggacg gccagcugua
ccugaccggc 840ggcgugugga gccuggacgg cagcgacagc auccaggaaa
ccaugcaggc caccauccac 900gugccugcuc agcacgaaga cggcccagag
gacgaccccc agcugguagg caucaccgcc 960cggaacaucc cccggggccc
ucagcucgcc gcacagaacc uuggaaucag ccuggccaac 1020cugcuguugu
caaagggcgc caagaauauc cucgacgugg cccggcagcu gaacgacgcc 1080cac
10831001083RNAArtificial SequencePBGD-CO41A 100augagcggca
acggcaacgc cgccgccacc gccgaggaga acagccccaa gaugcgggug 60auccgggugg
gcaccagaaa gagccagcug gcccggaucc agaccgacag cgugguggcc
120acccugaagg ccagcuaccc cggccugcag uucgagauca ucgccaugag
caccaccggc 180gacaagaucc uggacaccgc ccugagcaag aucggcgaga
agagucuguu caccaaggag 240cuggagcacg cccuggagaa gaacgaggug
gaccuggugg ugcacagccu gaaggaccug 300cccaccgugc ugccgccugg
cuucaccauc ggcgccaucu gcaagcggga gaacccccac 360gacgccgugg
uguuccaccc caaguucgug ggcaagacuc uggaaacccu gccugagaag
420uccguggucg gaacaagcag ccugcggcgg gccgcccagc ugcagcggaa
guucccccac 480cuggaguucc ggagcauccg gggcaaccug aacacccggc
ugcggaagcu ggacgagcag 540caggaguuca gcgccaucau ccuggccaca
gccggccuuc agaggauggg cuggcacaau 600cggguaggcc agauccugca
ccccgaggag ugcauguacg cgguagguca gggcgcccug 660ggcguggagg
ugcgggccaa ggaccaggac aucuuagauc ugguuggcgu gcugcacgac
720cccgaaacac ugcugcggug caucgccgag cgggccuucc ugcggcaccu
cgagggcggc 780ugcagugugc ccguggccgu gcacaccgcc augaaggacg
gccagcugua ccugaccggc 840ggcgugugga gccuggacgg cagcgacagc
auccaggaga caaugcaggc caccauccac 900gugccagcuc agcacgaaga
cggaccagag gacgaccccc aguuaguggg aaucaccgcc 960cggaacaucc
cccggggccc ucagcucgcg gcccagaacc ugggcaucag ccuggccaac
1020cugcugcugu cuaagggcgc caagaacauc cuagacgugg cccggcagcu
gaacgacgcc 1080cac 10831011083RNAArtificial SequencePBGD-CO42A
101augagcggca acggcaacgc cgccgccacc gccgaggaga acagccccaa
gaugcgggug 60auccgggugg gcaccaggaa gucacagcug gcccggaucc agaccgacag
cgugguggcc 120acccugaagg ccagcuaccc cggccugcag uucgagauca
ucgccaugag caccaccggc 180gacaagaucc uggacaccgc ccugagcaag
aucggcgaga agucccuguu caccaaggag 240cuggagcacg cccuggagaa
gaacgaggug gaccuggugg ugcacagccu gaaggaccug 300cccaccgugc
ugccgccagg cuucaccauc ggcgccaucu gcaagcggga gaacccccac
360gacgccgugg uguuccaccc caaguucgug ggcaagaccc uugaaacccu
gccagagaag 420ucuguggucg gcaccagcag ccugcggcgg gccgcccagc
ugcagcggaa guucccccac 480cuggaguucc ggagcauccg gggcaaccug
aacacccggc ugcggaagcu ggacgagcag 540caggaguuca gcgccaucau
ccuggccacc gcuggccuac agcggauggg cuggcacaau 600agaguugguc
agauccugca ccccgaggag ugcauguacg ccgucggcca gggcgcccug
660ggcguggagg ugcgggccaa ggaccaggac auacuagacc ucgugggcgu
gcugcacgac 720cccgaaaccu ugcugcggug caucgccgag cgggccuucc
ugcggcaccu ggaaggcggu 780ugcagcgugc ccguggccgu gcacaccgcc
augaaggacg gccagcugua ccugaccggc 840ggcgugugga gccuggacgg
cagcgacagc auccaggaga caaugcaggc caccauccac 900gugccggccc
aacacgagga cggaccugag gacgaccccc agcuuguggg aaucaccgcc
960cggaacaucc cccggggccc ucaacuggca gcccagaacu uaggcauaag
ccuggccaac 1020cugcugcugu ccaagggcgc caagaacauc cucgaugugg
cccggcagcu gaacgacgcc 1080cac 10831021083RNAArtificial
SequencePBGD-CO43A 102augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccgcaa gagucagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agagucuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccaccugg cuucaccauc ggcgccaucu gcaagcggga
gaacccccac 360gacgccgugg uguuccaccc caaguucgug ggcaagacac
uggaaacccu gccggagaag 420uccgugguag gcaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccccac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggcaaca gccggcuuac agcguauggg cuggcacaac
600agggugggac agauccugca ccccgaggag ugcauguacg cugugggcca
gggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac aucuuagauc
ucgucggcgu gcugcacgac 720cccgaaaccu ugcugcggug caucgccgag
cgggccuucc ugcggcaucu ugagggcgga 780ugcuccgugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaga cuaugcaggc caccauccac
900gugccagccc agcacgaaga cggcccagag gacgaccccc agcugguggg
aaucaccgcc 960cggaacaucc cccggggccc ucaacuggcc gcacagaacc
uaggcaucag ccuggccaac 1020cugcugcuca gcaagggcgc caagaauauc
uuggacgugg cccggcagcu gaacgacgcc 1080cac 10831031083RNAArtificial
SequencePBGD-CO44A 103augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcaccagaaa gagccagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agucucuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccuccugg cuucaccauc ggcgccaucu gcaagcggga
gaacccccac 360gacgccgugg uguuccaccc caaguucgug ggcaagaccc
uugagacucu gccagagaag 420ucuguagugg gaaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccccac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggccacg gccggauuac agagaauggg cuggcacaac
600cgagugggac agauccugca ccccgaggag ugcauguaug ccguuggcca
aggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac auccucgauc
ucgugggcgu gcugcacgac 720cccgagacuu ugcugcggug caucgccgag
cgggccuucc ugcggcaccu agagggcggc 780ugcucggugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaga caaugcaggc caccauccac
900gugccagccc agcacgagga uggcccugaa gacgaccccc agcugguggg
caucaccgcc 960cggaacaucc cccggggccc acaauuggcc gcucagaacu
uaggcauuag ccuggccaac 1020cugcugcugu cuaagggcgc caagaacaua
cuggacgugg cccggcagcu gaacgacgcc 1080cac 10831041083RNAArtificial
SequencePBGD-CO45A 104augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccggaa gagccagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agagccuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccccccgg cuucaccauc ggcgccaucu gcaagcggga
gaacccccac 360gacgccgugg uguuccaccc caaguucgug ggcaagaccc
uggagacccu gcccgagaag 420agcguggugg gcaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccccac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggccacc gccggccugc agcggauggg cuggcacaac
600cgggugggcc agauccugca ccccgaggag ugcauguacg ccgugggcca
gggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac auccuggacc
uggugggcgu gcugcacgac 720cccgagaccc ugcugcggug caucgccgag
cgggccuucc ugcggcaccu ggagggcggc 780ugcagcgugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaga ccaugcaggc caccauccac
900gugcccgccc agcacgagga cggccccgag gacgaccccc agcugguggg
caucaccgcc 960cggaacaucc cccggggccc ccagcuggcc gcccagaacc
ugggcaucag ccuggccaac 1020cugcugcuga gcaagggcgc caagaacauc
cuggacgugg cccggcagcu gaacgacgcc 1080cac 10831051083RNAArtificial
SequencePBGD-CO46A 105augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccggaa gagccagcug gcgaggaucc
agacggacag cgugguggcg 120acgcugaagg cgagcuaccc ggggcugcag
uucgagauca ucgcgaugag cacgacgggg 180gacaagaucc uggacacggc
gcugagcaag aucggggaga agagccuguu cacgaaggag 240cuggagcacg
cgcuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300ccgacggugc ugccgccggg guucacgauc ggggcgaucu gcaagaggga
gaacccgcac 360gacgcggugg uguuccaccc gaaguucgug gggaagacgc
uggagacgcu gccggagaag 420agcguggugg ggacgagcag ccugaggagg
gcggcgcagc ugcagaggaa guucccgcac 480cuggaguuca ggagcaucag
ggggaaccug aacacgaggc ugaggaagcu ggacgagcag 540caggaguuca
gcgcgaucau ccuggcgacg gcggggcugc agaggauggg guggcacaac
600aggguggggc agauccugca cccggaggag ugcauguacg cgguggggca
gggggcgcug 660gggguggagg ugagggcgaa ggaccaggac auccuggacc
uggugggggu gcugcacgac 720ccggagacgc ugcugaggug caucgcggag
agggcguucc ugaggcaccu ggaggggggg 780ugcagcgugc cgguggcggu
gcacacggcg augaaggacg ggcagcugua ccugacgggg 840ggggugugga
gccuggacgg gagcgacagc auccaggaga cgaugcaggc gacgauccac
900gugccggcgc agcacgagga cgggccggag gacgacccgc agcugguggg
gaucacggcg 960aggaacaucc cgagggggcc gcagcuggcg gcgcagaacc
uggggaucag ccuggcgaac 1020cugcugcuga gcaagggggc gaagaacauc
cuggacgugg cgaggcagcu gaacgacgcg 1080cac 10831061083RNAArtificial
SequencePBGD-CO47A 106augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccggaa gagccagcug gcccgcaucc
agaccgacuc cgucgucgcc 120acccucaagg ccuccuaccc cggccuccag
uucgagauca ucgccauguc caccaccggc 180gacaagaucc ucgacaccgc
ccucuccaag aucggcgaga agucccucuu caccaaggag 240cucgagcacg
cccucgagaa gaacgagguc gaccucgucg uccacucccu caaggaccuc
300cccaccgucc ucccccccgg cuucaccauc ggcgccaucu gcaagcgcga
gaacccccac 360gacgccgucg ucuuccaccc caaguucguc ggcaagaccc
ucgagacccu ccccgagaag 420uccgucgucg gcaccuccuc ccuccgccgc
gccgcccagc uccagcgcaa guucccccac 480cucgaguucc gcuccauccg
cggcaaccuc aacacccgcc uccgcaagcu cgacgagcag 540caggaguucu
ccgccaucau ccucgccacc gccggccucc agcgcauggg cuggcacaac
600cgcgucggcc agauccucca ccccgaggag ugcauguacg ccgucggcca
gggcgcccuc 660ggcgucgagg uccgcgccaa ggaccaggac auccucgacc
ucgucggcgu ccuccacgac 720cccgagaccc uccuccgcug caucgccgag
cgcgccuucc uccgccaccu cgagggcggc 780ugcuccgucc ccgucgccgu
ccacaccgcc augaaggacg gccagcucua ccucaccggc 840ggcgucuggu
cccucgacgg cuccgacucc auccaggaga ccaugcaggc caccauccac
900guccccgccc agcacgagga cggccccgag gacgaccccc agcucgucgg
caucaccgcc 960cgcaacaucc cccgcggccc ccagcucgcc gcccagaacc
ucggcaucuc ccucgccaac 1020cuccuccucu ccaagggcgc caagaacauc
cucgacgucg cccgccagcu caacgacgcc 1080cac 10831071083RNAArtificial
SequencePBGD-CO40B 107augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccguaa gagccagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agucccuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccuccagg cuucaccauc ggcgccaucu gcaagcggga
gaacccucac 360gacgccgugg uguuccaccc caaguucgug ggcaagaccc
uggaaacccu gccugagaag 420uccgucguag gaaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccacac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggcuacc gccggucugc aacgaauggg cuggcacaau
600agggugggcc agauccugca ccccgaggag ugcauguacg cggugggaca
gggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac auccuugauc
uggugggcgu gcugcacgac 720cccgagacgc ugcugcggug caucgccgag
cgggccuucc ugcggcauuu ggagggcgga 780ugcagcgugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaaa ccaugcaggc caccauccac
900gugccugcuc agcacgaaga cggcccagag gacgacccuc agcugguagg
caucaccgcc 960cggaacaucc cucggggccc ucagcucgcc gcacagaacc
uuggaaucag ccuggccaac 1020cugcuguugu caaagggcgc caagaauauc
cucgacgugg cccggcagcu gaacgacgcc 1080cac 10831081083RNAArtificial
SequencePBGD-CO41B 108augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcaccagaaa gagccagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agagucuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccgccugg cuucaccauc ggcgccaucu gcaagcggga
gaacccgcac 360gacgccgugg uguuccaccc caaguucgug ggcaagacuc
uggaaacccu gccugagaag 420uccguggucg gaacaagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccacac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau
ccuggccaca gccggccuuc agaggauggg cuggcacaau 600cggguaggcc
agauccugca ccccgaggag ugcauguacg cgguagguca gggcgcccug
660ggcguggagg ugcgggccaa ggaccaggac aucuuagauc ugguuggcgu
gcugcacgac 720cccgaaacac ugcugcggug caucgccgag cgggccuucc
ugcggcaccu cgagggcggc 780ugcagugugc ccguggccgu gcacaccgcc
augaaggacg gccagcugua ccugaccggc 840ggcgugugga gccuggacgg
cagcgacagc auccaggaga caaugcaggc caccauccac 900gugccagcuc
agcacgaaga cggaccagag gacgacccac aguuaguggg aaucaccgcc
960cggaacaucc cgcggggccc ucagcucgcg gcccagaacc ugggcaucag
ccuggccaac 1020cugcugcugu cuaagggcgc caagaacauc cuagacgugg
cccggcagcu gaacgacgcc 1080cac 10831091083RNAArtificial
SequencePBGD-CO42B 109augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcaccaggaa gucacagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agucccuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccgccagg cuucaccauc ggcgccaucu gcaagcggga
gaacccgcac 360gacgccgugg uguuccaccc caaguucgug ggcaagaccc
uugaaacccu gccagagaag 420ucuguggucg gcaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccgcac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggccacc gcuggccuac agcggauggg cuggcacaau
600agaguugguc agauccugca ccccgaggag ugcauguacg ccgucggcca
gggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac auacuagacc
ucgugggcgu gcugcacgac 720cccgaaaccu ugcugcggug caucgccgag
cgggccuucc ugcggcaccu ggaaggcggu 780ugcagcgugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaga caaugcaggc caccauccac
900gugccggccc aacacgagga cggaccugag gacgacccac agcuuguggg
aaucaccgcc 960cggaacaucc cacggggccc ucaacuggca gcccagaacu
uaggcauaag ccuggccaac 1020cugcugcugu ccaagggcgc caagaacauc
cucgaugugg cccggcagcu gaacgacgcc 1080cac 10831101083RNAArtificial
SequencePBGD-CO43B 110augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccgcaa gagucagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agagucuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccaccugg cuucaccauc ggcgccaucu gcaagcggga
gaacccacac 360gacgccgugg uguuccaccc caaguucgug ggcaagacac
uggaaacccu gccggagaag 420uccgugguag gcaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccgcac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggcaaca gccggcuuac agcguauggg cuggcacaac
600agggugggac agauccugca ccccgaggag ugcauguacg cugugggcca
gggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac aucuuagauc
ucgucggcgu gcugcacgac 720cccgaaaccu ugcugcggug caucgccgag
cgggccuucc ugcggcaucu ugagggcgga 780ugcuccgugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaga cuaugcaggc caccauccac
900gugccagccc agcacgaaga cggcccagag gacgacccac agcugguggg
aaucaccgcc 960cggaacaucc cgcggggccc ucaacuggcc gcacagaacc
uaggcaucag ccuggccaac 1020cugcugcuca gcaagggcgc caagaauauc
uuggacgugg cccggcagcu gaacgacgcc 1080cac 10831111083RNAArtificial
SequencePBGD-CO44B 111augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcaccagaaa gagccagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agucucuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccuccugg cuucaccauc ggcgccaucu gcaagcggga
gaacccacac 360gacgccgugg uguuccaccc caaguucgug ggcaagaccc
uugagacucu gccagagaag 420ucuguagugg gaaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccucac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggccacg gccggauuac agagaauggg cuggcacaac
600cgagugggac agauccugca ccccgaggag ugcauguaug ccguuggcca
aggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac auccucgauc
ucgugggcgu gcugcacgac 720cccgagacuu ugcugcggug caucgccgag
cgggccuucc ugcggcaccu agagggcggc 780ugcucggugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaga caaugcaggc caccauccac
900gugccagccc agcacgagga uggcccugaa gacgacccac agcugguggg
caucaccgcc 960cggaacaucc cgcggggccc acaauuggcc gcucagaacu
uaggcauuag ccuggccaac 1020cugcugcugu cuaagggcgc caagaacaua
cuggacgugg cccggcagcu gaacgacgcc 1080cac 10831121083RNAArtificial
SequencePBGD-CO45B 112augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccggaa gagccagcug gcccggaucc
agaccgacag cgugguggcc 120acccugaagg ccagcuaccc cggccugcag
uucgagauca ucgccaugag caccaccggc 180gacaagaucc uggacaccgc
ccugagcaag aucggcgaga agagccuguu caccaaggag 240cuggagcacg
cccuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300cccaccgugc ugccgcccgg cuucaccauc ggcgccaucu gcaagcggga
gaacccgcac 360gacgccgugg uguuccaccc caaguucgug ggcaagaccc
uggagacccu gcccgagaag 420agcguggugg gcaccagcag ccugcggcgg
gccgcccagc ugcagcggaa guucccucac 480cuggaguucc ggagcauccg
gggcaaccug aacacccggc ugcggaagcu ggacgagcag 540caggaguuca
gcgccaucau ccuggccacc gccggccugc agcggauggg cuggcacaac
600cgggugggcc agauccugca ccccgaggag ugcauguacg ccgugggcca
gggcgcccug 660ggcguggagg ugcgggccaa ggaccaggac auccuggacc
uggugggcgu gcugcacgac 720cccgagaccc ugcugcggug caucgccgag
cgggccuucc ugcggcaccu ggagggcggc 780ugcagcgugc ccguggccgu
gcacaccgcc augaaggacg gccagcugua ccugaccggc 840ggcgugugga
gccuggacgg cagcgacagc auccaggaga ccaugcaggc caccauccac
900gugcccgccc agcacgagga cggccccgag gacgacccuc agcugguggg
caucaccgcc 960cggaacaucc cacggggccc ucagcuggcc gcccagaacc
ugggcaucag ccuggccaac 1020cugcugcuga gcaagggcgc caagaacauc
cuggacgugg cccggcagcu gaacgacgcc 1080cac 10831131083RNAArtificial
SequencePBGD-CO46B 113augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccggaa gagccagcug gcgaggaucc
agacggacag cgugguggcg 120acgcugaagg cgagcuaccc ggggcugcag
uucgagauca ucgcgaugag cacgacgggc 180gacaagaucc uggacacggc
gcugagcaag aucggggaga agagccuguu cacgaaggag 240cuggagcacg
cgcuggagaa gaacgaggug gaccuggugg ugcacagccu gaaggaccug
300ccgacggugc ugccgccggg guucacgauc ggggcgaucu gcaagaggga
gaacccgcac 360gacgcggugg uguuccaccc gaaguucgug gggaagacgc
uggagacgcu gccggagaag 420agcguggugg ggacgagcag ccugaggagg
gcggcgcagc ugcagaggaa guucccgcac 480cuggaguuca ggagcaucag
ggguaaccug aacacgaggc ugaggaagcu ggacgagcag 540caggaguuca
gcgcgaucau ccuggcgacg gcggggcugc agaggauggg guggcacaac
600aggguggggc agauccugca cccggaggag ugcauguacg cgguggggca
gggcgcgcug 660ggcguggagg ugagggcgaa ggaccaggac auccuggacc
uggugggcgu gcugcacgac 720ccggagacgc ugcugaggug caucgcggag
agggcguucc ugaggcaccu ggagggcggg 780ugcagcgugc cgguggcggu
gcacacggcg augaaggacg ggcagcugua ccugacggga 840ggggugugga
gccuggacgg gagcgacagc auccaggaga cgaugcaggc gacgauccac
900gugccggcgc agcacgagga cgggccggag gacgacccgc agcugguggg
gaucacggcg 960aggaacaucc cgaggggucc gcagcuggcg gcgcagaacc
uggggaucag ccuggcgaac 1020cugcugcuga gcaagggagc gaagaacauc
cuggacgugg cgaggcagcu gaacgacgcg 1080cac 10831141083RNAArtificial
SequencePBGD-CO47B 114augagcggca acggcaacgc cgccgccacc gccgaggaga
acagccccaa gaugcgggug 60auccgggugg gcacccggaa gagccagcug gcccgcaucc
agaccgacuc cgucgucgcc 120acccucaagg ccuccuaccc cggccuccag
uucgagauca ucgccauguc caccaccggc 180gacaagaucc ucgacaccgc
ccucuccaag aucggcgaga agucccucuu caccaaggag 240cucgagcacg
cccucgagaa gaacgagguc gaccucgucg uccacucccu caaggaccuc
300cccaccgucc ucccacccgg cuucaccauc ggcgccaucu gcaagcgcga
gaacccucac 360gacgccgucg ucuuccaccc caaguucguc ggcaagaccc
ucgagacccu ccccgagaag 420uccgucgucg gcaccuccuc ccuccgccgc
gccgcccagc uccagcgcaa guucccacac 480cucgaguucc gcuccauccg
cggcaaccuc aacacccgcc uccgcaagcu cgacgagcag 540caggaguucu
ccgccaucau ccucgccacc gccggccucc agcgcauggg cuggcacaac
600cgcgucggcc agauccucca ccccgaggag ugcauguacg ccgucggcca
gggcgcccuc 660ggcgucgagg uccgcgccaa ggaccaggac auccucgacc
ucgucggcgu ccuccacgac 720cccgagaccc uccuccgcug caucgccgag
cgcgccuucc uccgccaccu cgagggcggc 780ugcuccgucc ccgucgccgu
ccacaccgcc augaaggacg gccagcucua ccucaccggc 840ggcgucuggu
cccucgacgg cuccgacucc auccaggaga ccaugcaggc caccauccac
900guccccgccc agcacgagga cggccccgag gacgacccuc agcucgucgg
caucaccgcc 960cgcaacaucc cgcgcggccc ucagcucgcc gcccagaacc
ucggcaucuc ccucgccaac 1020cuccuccucu ccaagggcgc caagaacauc
cucgacgucg cccgccagcu caacgacgcc 1080cac 10831151083RNAArtificial
SequenceCONSTRUCT #29 115auguccggaa acggaaacgc cgccgcuacc
gccgaggaaa auagcccgaa gaugagagug 60auccgggugg guaccaggaa gucccagcuc
gccaggaucc aaacggacuc ggugguggcc 120acccucaagg cuagcuaccc
gggccugcaa uucgagauca uugcuauguc caccaccggc 180gacaagaucc
uggauacggc ccuguccaag aucggcgaaa agagccucuu caccaaggag
240cuggaacacg cgcucgagaa gaacgaggug gaccuggucg uccacagccu
caaggaccug 300ccuacggugc ugccgccggg auucaccauc ggcgccaucu
guaagcggga gaauccgcac 360gacgccgugg uguuccaccc uaaguucgug
ggcaagaccc ucgagacacu gccggaaaag 420uccgucgugg gcaccuccuc
ccugagaagg gccgcucagc uccagagaaa guucccgcac 480cuggaauuca
ggagcauccg gggcaaccug aauacccggc uucgcaagcu ggacgagcag
540caggaguuca gcgccaucau ccuggccacc gccggccugc agcggauggg
cuggcacaac 600cgggugggcc agauccugca cccggaggag ugcauguaug
ccguggguca gggagcccug 660ggcguggagg uccgggccaa ggaccaggac
auccuggacc ucgugggcgu gcuccacgac 720ccugaaaccc uccugaggug
caucgccgag agggccuucc uccggcaccu ggagggcggc 780uguuccgucc
cuguggccgu gcauaccgcc augaaggacg gacagcugua ccugaccggc
840ggcguguggu cccuggacgg cuccgacagc auccaggaaa ccaugcaggc
cacuauccac 900gugccggccc agcacgaaga cggcccagag gaugacccgc
aacuggucgg cauuaccgcc 960aggaacauac caaggggccc gcagcuggcc
gcccagaacc ugggcaucuc ccuggccaac 1020cugcugcugu ccaagggagc
caagaacauu cuggacgugg ccaggcagcu caaugaugcc 1080cac
10831161083RNAArtificial SequenceCONSTRUCT #30 116augagcggca
acggcaacgc cgccgccacc gcagaggaga auagcccgaa gaugagggug 60auccgagugg
gcaccaggaa gucccagcuu gcgcgaauuc agaccgacag cgugguggcc
120acccucaagg ccuccuaccc gggacuccag uucgagauca ucgccaugag
caccacggga 180gacaagaucc uggacaccgc ccuguccaag aucggcgaaa
agagccucuu caccaaggag 240cuggagcacg cccuggagaa gaacgagguc
gaucuggugg ugcacagccu gaaggaccug 300ccgaccgucc ugccgccggg
auucaccauc ggugccaucu guaagcggga gaacccgcac 360gacgccgugg
uguuccaccc gaaguucguc ggcaagaccc uggaaacccu gccggagaag
420uccguggugg gcaccagcag ccugaggcgg gccgcccagc ugcagcggaa
guucccgcac 480cuggaauuca ggagcauccg gggcaaccug aacacccggc
ugcggaagcu ggacgagcag 540caggaguuca gcgccaucau ccuggccacc
gcaggccucc agcgcauggg auggcacaac 600aggguaggcc aaauccugca
cccggaggag uguauguacg ccgugggcca gggagcccug 660ggcguggagg
ugagagccaa ggaccaggac auccuagacc uggucggcgu gcugcacgac
720ccggagacac ugcugagaug caucgcggag agagccuucc ugcgacaccu
ggagggcggc 780ugcuccgugc cgguggccgu gcacaccgcc augaaggacg
gccagcugua ucugaccggc 840ggcgugugga gccuggacgg cagcgacucc
auccaagaaa ccaugcaggc uaccauccac 900gugccggccc agcacgagga
uggaccagag gacgauccuc aacugguggg caucacugcc 960aggaacaucc
caagaggccc gcagcuggcc gcccagaacc ugggcaucag ccuggccaac
1020cugcugcugu ccaagggcgc caagaacauu cucgaugugg ccaggcagcu
gaacgaugcc 1080cac 10831171083RNAArtificial SequenceCONSTRUCT #31
117augucgggaa acggcaacgc cgccgccacg gccgaggaga acagcccgaa
gaugagagug 60auuagggugg gcacccggaa gucccaacuc gcgcggaucc agaccgacuc
cgugguggcc 120acccucaagg ccagcuaccc gggccuccag uucgagauua
ucgccauguc caccacaggc 180gacaagaucc ucgacaccgc acucucgaag
aucggcgaga agucccuguu caccaaggaa 240cuggagcacg cccuggagaa
gaacgaggug gaccuggugg ugcacucccu gaaggaccug 300ccgaccgugc
ucccaccagg cuucaccauc ggcgcaaucu guaagcgcga gaauccgcac
360gacgccgugg uguuccaccc aaaguucgug ggcaagaccc ucgaaacccu
cccggaaaag 420agcguggugg guaccagcuc ccugcggaga gcugcccagc
ugcagagaaa guucccgcau 480cuggaauuca ggagcaucag gggaaaucug
aauaccagac ugcgcaagcu ggacgagcag 540caggaguuca gcgccaucau
ccuggccacc gccggccugc agcggauggg cuggcacaac 600agggugggcc
agauacugca uccggaggag uguauguacg ccgugggcca gggcgcccuc
660ggcguggagg ugagagccaa ggaccaagac auccuggacc uagugggcgu
gcugcaugac 720ccugaaaccc ugcucaggug caucgccgag agggccuucc
ugcggcaccu ggagggcggc 780ugcagcgugc cgguggccgu ccacaccgcc
augaaggacg gccagcugua ccugaccggc 840ggcgucugga gccuggacgg
auccgacagc auccaggaaa ccaugcaggc caccauccac 900gugccggccc
agcacgagga cggcccugag gacgacccuc agcugguggg caucaccgcu
960aggaacaucc caaggggccc gcagcuggcc gcccagaacc ucggcaucag
ccuggccaac 1020cugcugcugu ccaagggcgc caagaauauc cuggacgugg
ccaggcagcu gaacgacgcc 1080cac 10831181294RNAArtificial
SequenceCONSTRUCT #1 118gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug agcggcaacg 60gcaacgccgc agccaccgcc gaggaaaaca gccccaagau
gcgggugauc agagugggca 120cccggaagag ccagcuggcc cggauccaga
ccgacagcgu gguggccacc cugaaggccu 180ccuaccccgg ccugcaguuc
gagaucauug ccaugagcac caccggcgac aagauccugg 240acaccgcccu
gagcaagauc ggcgagaaga gccuguucac aaaagagcug gaacacgccc
300uggaaaagaa cgagguggac cugguggugc acagccugaa ggaccugccc
accgugcugc 360ccccuggcuu caccaucggc gccaucugca agagagagaa
cccccacgac gccguggugu 420uccacccuaa guucgugggc aagacacugg
aaacccugcc cgagaagucc guggugggca 480ccagcagccu gcggagagcc
gcccagcugc agcggaaguu cccccaccug gaauuucgga 540gcauccgggg
caaccugaac acccggcugc ggaagcugga cgagcagcag gaauuuuccg
600cuaucauccu ggccacagcc ggacugcagc ggaugggcug gcacaacaga
gugggccaga 660uccugcaccc cgaggaaugc auguacgccg ugggccaggg
agcccugggc guggaagugc 720gggccaagga ccaggacauc cuggaucugg
ugggcgugcu gcaugacccc gagacacugc 780ugcgguguau cgccgagcgg
gccuuccugc ggcaccugga aggcggcugc agcgugcccg 840uggccgugca
caccgccaug aaggacggac agcuguaccu gacaggcggc guguggagcc
900uggacggcag cgacagcauc caggagacca ugcaggccac cauccacgug
cccgcccagc 960acgaggacgg ccccgaggac gacccucagc uggucggcau
caccgcccgg aacaucccca 1020gaggccccca gcuggccgcc cagaaccugg
gcaucagccu ggccaaccug cugcugucca 1080agggcgccaa gaacauccug
gacguggccc ggcagcugaa cgacgcccac ugauaauagu 1140ccauaaagua
ggaaacacua cagcuggagc cucgguggcc augcuucuug ccccuugggc
1200cuccccccag ccccuccucc ccuuccugca cccguacccc ccgcauuauu
acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941191294RNAArtificial SequenceCONSTRUCT #2 119gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgcuaccgcc gaagagaaca gcccaaagau gcgcgugauc agggucggca
120cgcgcaaguc ccagcucgcc cggauccaaa ccgauagcgu gguggccacg
cucaaggcga 180gcuauccggg cuuacaguuc gagaucaucg ccaugagcac
caccggcgau aagauacugg 240acaccgcccu guccaagauc ggcgaaaaga
gccuguucac caaggaacug gagcacgcgc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccg accgugcugc 360cgccgggauu
caccaucggc gccaucugca agagggagaa uccgcacgau gccguggugu
420uccacccaaa guucgugggc aagaccuugg aaacccugcc agagaagucu
guggucggca 480ccuccagccu gcggcgagcc gcccagcugc agcgaaaguu
cccgcaccug gaguucaggu 540ccauccgcgg aaaucugaac accaggcugc
gcaagcucga cgagcagcag gaguucuccg 600ccaucauccu ggccaccgca
ggccuccaaa gaaugggcug gcauaaccga gucggccaga 660uccuccaccc
ggaggagugc auguacgcag ugggccaagg cgcccugggc gucgaggugc
720gugccaagga ccaggacauc cuggaccugg ugggcgugcu ccacgaucca
gagacacugc 780ugagaugcau cgcggagcgc gccuuccugc gccaucugga
gggaggcugc uccgucccgg 840uggccguaca uaccgccaug aaggacgguc
agcuguaccu caccggcggc guaugguccc 900ucgacgguag cgacagcaua
caggagacga ugcaggccac cauccacgug ccggcgcagc 960acgaggaugg
accagaggac gacccgcagc ugguggguau caccgccagg aauaucccgc
1020ggggaccuca gcuggccgcc cagaaccugg gcaucucccu cgccaaccuc
cugcugagca 1080agggcgccaa gaacauccug gacguggcca ggcagcucaa
cgaugcccau ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941201294RNAArtificial
SequenceCONSTRUCT #3 120gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggaaaaca gcccgaagau
gcgggugauc agggugggca 120ccaggaaguc ccagcucgcc cggauccaga
ccgacagcgu ggucgccacc uugaaggccu 180ccuacccggg ccuccaguuc
gagaucaucg ccauguccac aaccggcgac aagauccugg 240auaccgcccu
cagcaagauc ggcgagaagu cccuguucac caaggagcug gagcacgccc
300uggagaagaa ugagguggac cugguggugc acagccugaa ggaccugccu
accgugcugc 360caccaggcuu cacaaucggc gccaucugca agagagagaa
cccgcacgac gccguggugu 420uccauccgaa guucgugggc aagacccugg
aaacccugcc ggagaagucc guagugggaa 480ccucaagccu gaggcgcgcc
gcccagcucc agaggaaguu cccucaccug gaauuccggu 540ccaucagggg
caaccugaac acgcgccugc ggaagcucga cgagcagcag gaguucuccg
600ccaucauccu ggccacagcc ggccuucagc gcaugggcug gcacaacagg
gugggccaga 660uccugcaccc ggaagaaugc auguacgccg ugggccaagg
cgcccucggc guggaagugc 720gugccaagga ccaggacauc cuggaccugg
ugggcgugcu gcacgacccu gagacgcugc 780ucaggugcau cgccgaacgc
gcguuccugc ggcaccugga gggaggcugc agcgucccgg 840uggccgucca
caccgccaug aaggacggcc agcucuaccu gacuggcggc guguggagcc
900uggacggcag cgacagcauu caggaaacca ugcaggccac cauccacgug
ccugcccagc 960acgaggacgg cccggaggac gacccucaac uggugggcau
uacugcgcga aacaucccgc 1020gcggaccuca gcuggccgcc cagaaccugg
gcaucagccu ggccaaccug cuccugucca 1080agggcgccaa gaacauccuc
gacguggcca ggcagcugaa cgacgcgcac ugauaauagu 1140ccauaaagua
ggaaacacua cagcuggagc cucgguggcc augcuucuug ccccuugggc
1200cuccccccag ccccuccucc ccuuccugca cccguacccc ccgcauuauu
acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941211294RNAArtificial SequenceCONSTRUCT #4 121gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gaaacgccgc
cgcgaccgcg gaggagaacu cgccuaagau gagagugaua aggguaggca
120cccggaaguc ucaacucgcc aggauccaga ccgacagcgu gguggccacc
cucaaggcca 180gcuauccagg acuccaguuc gaaaucaucg ccauguccac
cacaggcgau aagauccugg 240acaccgcccu guccaagauc ggcgagaagu
cccucuucac caaggaacug gagcacgccc 300uggagaagaa cgaggucgau
cuggucgugc acagccugaa ggaucugccu accgugcucc 360cgccgggcuu
caccaucggc gccaucugca agagggagaa uccucacgac gccguggugu
420uccacccgaa guucgugggc aagacccugg agacacugcc agaaaagucg
guggugggca 480ccagcagccu gcggcgggcg gcccagcugc agcggaaguu
cccacaccug gaguucaggu 540ccauccgugg caaucugaac acccggcugc
guaagcugga cgagcagcag gaauucagcg 600cgaucauccu ggcaaccgcc
ggucugcaaa ggaugggcug gcacaacagg gugggccaga 660uccugcaccc
ugaggagugc auguacgccg ugggccaggg agcccugggc guggaagugc
720gggccaagga ccaggacauc cuggaccugg ugggugugcu ccacgacccu
gaaacccugc 780ugcggugcau cgccgaaagg gccuuccuga ggcaccucga
gggcggcugc agcgugccgg 840ucgccgugca caccgccaug aaggacggcc
agcuguaccu gaccggagga guguggagcc 900uggacggcuc cgacuccauc
caggagacua ugcaggccac cauucaugug ccggcccagc 960augaggacgg
uccggaggac gauccacagc uggucggcau caccgcgcgg aacaucccaa
1020gaggcccgca acuggccgcu cagaaccugg gcauaucccu ggccaaccug
cuccugagca 1080agggcgccaa gaacauccug gacguggcca ggcagcugaa
ugacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941221294RNAArtificial
SequenceCONSTRUCT #5 122gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug uccggcaacg 60gcaacgccgc cgcuaccgcc gaggagaacu ccccuaagau
gcgggucauc agggugggca 120cccgaaaguc ccaacuugcc cggauccaga
ccgacuccgu cguggccacc cucaaggcua 180gcuauccagg ccuccaguuc
gaaaucaucg ccaugagcac caccggcgac aagauucugg 240acaccgcccu
guccaagauc ggcgagaaga gucuguucac gaaggagcuc gagcacgccc
300uggaaaagaa cgagguggac cugguggugc auucccugaa ggaccugcca
accgugcugc 360cgccgggcuu cacuauagga gccaucugca agcgggagaa
cccgcacgac gcgguggugu 420uccauccgaa guucgugggc aagacucugg
aaacccugcc ggagaagucc guggugggaa 480cuagcucccu gcggcgggcc
gcccagcugc agaggaaguu cccgcaccug gaguucagga 540gcauacgcgg
caaccugaac acccgccugc guaagcucga cgagcagcag gaauucagug
600ccaucauccu ggccacggcg ggccugcagc ggaugggcug gcacaacagg
gugggccaga 660uccuccaccc ggaggaaugu auguacgccg ugggccaggg
cgcacugggc guggaggucc 720gcgccaagga ccaagacauc cuggaccugg
ucggcgugcu gcacgacccu gaaacccugc 780ugaggugcau ugccgagaga
gccuuccuga ggcaucugga gggcggcugc agcgugccug 840uggccgugca
cacagccaug aaggacgguc agcuguaccu gaccggcggc guguggagcc
900uggacggcag cgacuccauc caggagacaa ugcaggccac cauccacguc
ccggcccaac 960acgaggacgg accugaggac gauccucagc uggugggcau
caccgccagg aacaucccuc 1020ggggcccgca gcuggccgcc cagaaccugg
gcaucucccu cgccaaccug cugcugucca 1080agggcgccaa gaacauccuc
gacguggcca gacagcugaa cgacgcccac ugauaauagu 1140ccauaaagua
ggaaacacua cagcuggagc cucgguggcc augcuucuug ccccuugggc
1200cuccccccag ccccuccucc ccuuccugca cccguacccc ccgcauuauu
acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941231294RNAArtificial SequenceCONSTRUCT #6 123gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gcccgaagau gagggugaua agggugggca
120cacggaaguc ccagcucgcc cgcauccaaa ccgacuccgu gguggccacc
cucaaggcca 180gcuacccggg ccuccaauuc gagaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gucuaagaua ggcgaaaaga
gccuguucac caaggagcug gagcaugccc 300uggagaagaa cgagguggac
cugguggucc acagucucaa ggaccugcca accgugcugc 360cgccaggcuu
caccaucggc gccaucugca agcgugagaa cccgcacgau gcuguggugu
420uccacccuaa guucguggga aagacccugg agacgcugcc ggaaaagagc
guggucggca 480ccuccagccu gcggagggcc gcccaacucc agaggaaguu
cccgcaccug gaguucagga 540gcauccgcgg caaccugaac accaggcugc
gaaagcugga cgagcagcag gaauucucgg 600ccaucauccu cgccaccgcc
ggcuugcaaa gaaugggcug gcauaaucgc gugggccaga 660uccugcaccc
ugaggagugc auguacgccg ugggccaggg ugcucuggga guggaggugc
720gggccaagga ccaggauauc cuggaccugg ucggcgugcu ucaugacccg
gagacgcucc 780ugaggugcau cgccgagcgg gccuuccuga gacaccugga
gggcggcugc uccgugccag 840uggccgugca caccgccaug aaggacggac
agcuguaccu gaccggcggc guguggagcc 900uggacggaag cgacagcauc
caagaaacca ugcaggcgac cauucacguc ccugcccagc 960acgaggaugg
accagaggac gacccgcagc uggugggcau caccgcccgc aacaucccua
1020gaggcccaca gcuggccgcc cagaaucugg gcaucagccu ggccaaccug
cugcugucua 1080agggagccaa gaacauccug gacguggcca ggcagcugaa
cgacgcccau ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941241294RNAArtificial
SequenceCONSTRUCT #7 124gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug uccggcaacg 60gcaacgccgc agccaccgcc gaggagaauu ccccgaagau
gcgggugauc cgggugggca 120ccagaaagag ccagcucgcc cgcauccaaa
ccgacuccgu gguggccacc cucaaggccu 180ccuacccagg cuugcaguuc
gaaaucaucg ccaugagcac caccggcgac aagauccugg 240acaccgcccu
gagcaagauu ggcgagaagu cccuguucac caaggagcug gagcaugcuc
300uggagaagaa cgagguggac cucguggugc acucccugaa ggaccugccg
acugugcugc 360cgccuggcuu cacgaucggc gccauaugca agcgggaaaa
cccacacgac gccguggucu 420uccacccaaa guucgugggc aagacccugg
aaacccugcc ggaaaagagc guggucggca 480caagcucccu gaggagagcc
gcccaacugc aaaggaaguu cccucaccuc gaguucaggu 540ccauccgggg
caaccugaac accaggcuga gaaagcucga cgaacagcag gaguucagcg
600ccaucauccu ggccacggcc ggccugcaga ggaugggaug gcauaacagg
gugggccaga 660uccugcaccc ggaggagugc auguacgccg ugggccaggg
agcccucggc guggagguca 720gggccaagga ucaggauauc cuggaccugg
ugggcgugcu gcacgauccu gagacgcugc 780ugaggugcau cgccgagcgg
gccuuccugc ggcaccuaga gggcggaugc agcgugccgg 840ucgcggucca
caccgcgaug aaggacggcc agcuguaccu gaccggcggc gugugguccc
900uggacggcag cgauucaauc caggagacga ugcaggccac cauccacgug
ccagcccagc 960acgaggaugg cccggaggac gacccgcagc uggugggcau
uacagccagg aacaucccuc 1020ggggcccgca gcuggccgcc cagaaucugg
gcaucagccu ggcgaaccug cugcucagca 1080agggagcgaa gaacauccug
gacguggccc gccagcugaa cgaugcccac ugauaauagu 1140ccauaaagua
ggaaacacua cagcuggagc cucgguggcc augcuucuug ccccuugggc
1200cuccccccag ccccuccucc ccuuccugca cccguacccc ccgcauuauu
acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941251294RNAArtificial SequenceCONSTRUCT #8 125gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gcccaaagau gcgggugauc agggugggca
120cccgcaagag ccaacucgcc agaauccaga ccgacagcgu gguggccacc
uugaaggcca 180gcuacccggg ccuccaguuc gagaucaucg cuauguccac
caccggcgac aagauccugg 240acaccgcgcu guccaagauc ggcgaaaaga
gccuguucac caaggaacug gagcacgccc 300ucgagaagaa cgagguggac
cugguggugc acucccugaa ggaccugccg acgguccugc 360cgccgggcuu
caccaucggc gccaucugca agcgggaaaa cccgcacgac gcuguggugu
420uccacccaaa guucgugggc aagacccugg aaacccugcc agaaaagagc
guggugggca 480ccagcagccu caggagagcc gcccagcugc agaggaaguu
cccgcaccug gaguucagga 540gcaucagggg caaccugaac accaggcugc
guaagcugga cgagcagcag gaguucuccg 600ccaucauccu cgccacagcc
ggccuccaga ggauggguug gcacaacagg gugggccaga 660uccugcaccc
ggaagagugc auguacgcag ugggccaggg cgcccuuggc guggaagugc
720gagccaagga ucaggauauc cuggaccugg ugggcgugcu gcacgacccg
gaaacucugc 780ugcggugcau cgccgaaagg gccuuccugc gccaccucga
aggcggcugu agcgugccgg 840uggccgugca caccgccaug aaggacggcc
agcuguaccu gaccggcggc guguggagcc 900ucgacggcag cgacagcauc
caggagacaa ugcaggccac cauccacgug ccggcccagc 960augaggaugg
cccggaggac gacccucagc uggugggcau caccgcccgc aacaucccaa
1020gaggaccgca acuggccgcc cagaaccugg gcaucucccu ggccaaccug
cuccugagca 1080agggcgcgaa gaacauccuc gacgucgcac ggcagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941261294RNAArtificial
SequenceCONSTRUCT #9 126gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug agcggcaacg 60gcaacgccgc cgcgacggcc gaggaaaaua gcccgaagau
gcgggugauc agggugggca 120ccaggaaguc ccagcucgcc aggauccaga
ccgacagcgu gguggccacc cucaaggccu 180ccuacccggg ccuccaauuc
gagaucaucg ccauguccac caccggcgac aagauccucg 240acaccgcccu
gagcaagauc ggcgaaaagu cgcuguucac caaggagcug gagcacgccc
300ucgagaagaa cgagguggac cugguagugc acucccuaaa ggaccugccg
accgugcugc 360cgccgggcuu cacgaucggc gccaucugca agcgcgagaa
cccgcaugau gccgucguuu 420uccacccuaa guucgugggc aagacccugg
agacgcugcc ggagaagucg guggugggaa 480ccagcagccu gaggagggcc
gcacaacugc agaggaaguu cccgcaucug gaguuccgca 540gcauucgagg
caaccugaac acgcgccuga gaaagcucga ugaacagcag gaguucagcg
600ccaucauucu ggccacugcc ggacugcagc ggaugggcug gcacaacaga
gugggccaga 660uccugcaucc ggaagagugu auguacgccg ugggccaggg
ugcccugggc guggaggugc 720gggccaagga ccaggauaua cuggaucugg
ucggcgugcu ccacgaccca gaaacacucc 780ugaggugcau cgcugagaga
gccuuccucc ggcaccucga gggcggcugu uccgugccgg 840uggccgucca
uaccgccaug aaggacgguc agcuguaccu gaccggaggc guuugguccc
900uggacggcag cgacagcauc caggaaacca ugcaggccac cauccacgug
ccggcgcagc 960acgaggacgg cccggaagac gacccgcagc uggucggcau
cacggccaga aacaucccgc 1020ggggcccgca gcuggcggcc cagaaccugg
gaaucucccu ggccaaccug cugcugagca 1080agggcgcgaa gaacauccug
gacguggcca ggcagcugaa cgaugcccac ugauaauagu 1140ccauaaagua
ggaaacacua cagcuggagc cucgguggcc augcuucuug ccccuugggc
1200cuccccccag ccccuccucc ccuuccugca cccguacccc ccgcauuauu
acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941271294RNAArtificial SequenceCONSTRUCT #10 127gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcgguaacg 60gcaacgccgc
cgccaccgcc gaggagaacu ccccgaagau gcgcgugauu cgggucggca
120caagaaaguc ucaacucgcc cgaauccaaa cggacagcgu gguggccacc
cucaaggcga 180gcuacccggg ccuccaguuc gaaaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gucgaagauu ggcgaaaagu
cccuguucac caaggagcug gagcacgccc 300uggagaagaa cgaagucgac
cuggucgugc acagccugaa ggaccugccg accguucugc 360cgccgggcuu
caccaucgga gccaucugca agcgggagaa uccgcacgac gccguggucu
420uccacccaaa guucguggga aagacccucg agacacugcc ggagaagucc
guggugggaa 480ccuccucccu gcggagggcc gcccaacugc agcggaaguu
cccacaccug gaauuccggu 540ccaucagagg caaccucaac accaggcuga
ggaagcucga ugagcagcag gaguucagcg 600ccaucauccu ggccacagcc
ggacugcagc gcaugggcug gcauaacaga gugggccaga 660uccuccaccc
ggaggagugc auguacgccg ugggacaagg cgcgcugggc guggaaguuc
720gggccaagga ccaggauauc cuggaccugg ugggcgugcu ccacgaccca
gagacgcugc 780ugcggugcau cgccgagcgc gccuuccugc ggcaccucga
gggcggcugc agcgugccgg 840ucgcugugca cacagccaug aaggacggcc
agcuguaccu gaccggcggc guguggaguc 900uggacggcag cgacuccauc
caggagacua ugcaagccac cauccaugug ccggcccaac 960augaggacgg
cccggaggac gacccgcaac uggugggcau caccgcccgg aacaucccga
1020ggggcccgca gcuggccgcc cagaaccugg gcauuagccu ggccaaccug
cuccugagca 1080agggcgcuaa gaacauccug gacgucgcca gacagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941281294RNAArtificial
SequenceCONSTRUCT #11 128gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120cccguaagag ccagcuggcc
cggauccaga ccgacagcgu gguggccacc cugaaggcca 180gcuaccccgg
ccugcaguuc gagaucaucg ccaugagcac caccggcgac aagauccugg
240acaccgcccu gagcaagauc ggcgagaagu cccuguucac caaggagcug
gagcacgccc 300uggagaagaa cgagguggac cugguggugc acagccugaa
ggaccugccc accgugcugc 360cuccaggcuu caccaucggc gccaucugca
agcgggagaa cccccacgac gccguggugu 420uccaccccaa guucgugggc
aagacccugg aaacccugcc ugagaagucc gucguaggaa 480ccagcagccu
gcggcgggcc gcccagcugc agcggaaguu cccccaccug gaguuccgga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggcuaccgcc ggucugcaac gaaugggcug
gcacaauagg gugggccaga 660uccugcaccc cgaggagugc auguacgcgg
ugggacaggg cgcccugggc guggaggugc 720gggccaagga ccaggacauc
cuugaucugg ugggcgugcu gcacgacccc gagacgcugc 780ugcggugcau
cgccgagcgg gccuuccugc ggcauuugga gggcggaugc agcgugcccg
840uggccgugca caccgccaug aaggacggcc agcuguaccu gaccggcggc
guguggagcc 900uggacggcag cgacagcauc caggaaacca ugcaggccac
cauccacgug ccugcucagc 960acgaagacgg cccagaggac gacccccagc
ugguaggcau caccgcccgg aacauccccc 1020ggggcccuca gcucgccgca
cagaaccuug gaaucagccu ggccaaccug cuguugucaa 1080agggcgccaa
gaauauccuc gacguggccc ggcagcugaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941291294RNAArtificial SequenceCONSTRUCT #12 129gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120ccagaaagag ccagcuggcc cggauccaga ccgacagcgu gguggccacc
cugaaggcca 180gcuaccccgg ccugcaguuc gagaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gagcaagauc ggcgagaaga
gucuguucac caaggagcug gagcacgccc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccc accgugcugc 360cgccuggcuu
caccaucggc gccaucugca agcgggagaa cccccacgac gccguggugu
420uccaccccaa guucgugggc aagacucugg aaacccugcc ugagaagucc
guggucggaa 480caagcagccu gcggcgggcc gcccagcugc agcggaaguu
cccccaccug gaguuccgga 540gcauccgggg caaccugaac acccggcugc
ggaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggccacagcc
ggccuucaga ggaugggcug gcacaaucgg guaggccaga 660uccugcaccc
cgaggagugc auguacgcgg uaggucaggg cgcccugggc guggaggugc
720gggccaagga ccaggacauc uuagaucugg uuggcgugcu gcacgacccc
gaaacacugc 780ugcggugcau cgccgagcgg gccuuccugc ggcaccucga
gggcggcugc agugugcccg 840uggccgugca caccgccaug aaggacggcc
agcuguaccu gaccggcggc guguggagcc 900uggacggcag cgacagcauc
caggagacaa ugcaggccac cauccacgug ccagcucagc 960acgaagacgg
accagaggac gacccccagu uagugggaau caccgcccgg aacauccccc
1020ggggcccuca gcucgcggcc cagaaccugg gcaucagccu ggccaaccug
cugcugucua 1080agggcgccaa gaacauccua gacguggccc ggcagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941301294RNAArtificial
SequenceCONSTRUCT #13 130gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120ccaggaaguc acagcuggcc
cggauccaga ccgacagcgu gguggccacc cugaaggcca 180gcuaccccgg
ccugcaguuc gagaucaucg ccaugagcac caccggcgac aagauccugg
240acaccgcccu gagcaagauc ggcgagaagu cccuguucac caaggagcug
gagcacgccc 300uggagaagaa cgagguggac cugguggugc acagccugaa
ggaccugccc accgugcugc 360cgccaggcuu caccaucggc gccaucugca
agcgggagaa cccccacgac gccguggugu 420uccaccccaa guucgugggc
aagacccuug aaacccugcc agagaagucu guggucggca 480ccagcagccu
gcggcgggcc gcccagcugc agcggaaguu cccccaccug gaguuccgga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggccaccgcu ggccuacagc ggaugggcug
gcacaauaga guuggucaga 660uccugcaccc cgaggagugc auguacgccg
ucggccaggg cgcccugggc guggaggugc 720gggccaagga ccaggacaua
cuagaccucg ugggcgugcu gcacgacccc gaaaccuugc 780ugcggugcau
cgccgagcgg gccuuccugc ggcaccugga aggcgguugc agcgugcccg
840uggccgugca caccgccaug aaggacggcc agcuguaccu gaccggcggc
guguggagcc 900uggacggcag cgacagcauc caggagacaa ugcaggccac
cauccacgug ccggcccaac 960acgaggacgg accugaggac gacccccagc
uugugggaau caccgcccgg aacauccccc 1020ggggcccuca acuggcagcc
cagaacuuag gcauaagccu ggccaaccug cugcugucca 1080agggcgccaa
gaacauccuc gauguggccc ggcagcugaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941311294RNAArtificial SequenceCONSTRUCT #14 131gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120cccgcaagag ucagcuggcc cggauccaga ccgacagcgu gguggccacc
cugaaggcca 180gcuaccccgg ccugcaguuc gagaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gagcaagauc ggcgagaaga
gucuguucac caaggagcug gagcacgccc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccc accgugcugc 360caccuggcuu
caccaucggc gccaucugca agcgggagaa cccccacgac gccguggugu
420uccaccccaa guucgugggc aagacacugg aaacccugcc ggagaagucc
gugguaggca 480ccagcagccu gcggcgggcc gcccagcugc agcggaaguu
cccccaccug gaguuccgga 540gcauccgggg caaccugaac acccggcugc
ggaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggcaacagcc
ggcuuacagc guaugggcug gcacaacagg gugggacaga 660uccugcaccc
cgaggagugc auguacgcug ugggccaggg cgcccugggc guggaggugc
720gggccaagga ccaggacauc uuagaucucg ucggcgugcu gcacgacccc
gaaaccuugc 780ugcggugcau cgccgagcgg gccuuccugc ggcaucuuga
gggcggaugc uccgugcccg 840uggccgugca caccgccaug aaggacggcc
agcuguaccu gaccggcggc guguggagcc 900uggacggcag cgacagcauc
caggagacua ugcaggccac cauccacgug ccagcccagc 960acgaagacgg
cccagaggac gacccccagc uggugggaau caccgcccgg aacauccccc
1020ggggcccuca acuggccgca cagaaccuag gcaucagccu ggccaaccug
cugcucagca 1080agggcgccaa gaauaucuug gacguggccc ggcagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc
1200cuccccccag ccccuccucc ccuuccugca cccguacccc ccgcauuauu
acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941321294RNAArtificial SequenceCONSTRUCT #15 132gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug aguggcaacg 60gaaacgccgc
cgcuacagca gaggagaacu ccccgaagau gcgcgugauu aggguaggca
120ccagaaaguc ucagcuggcc agaauccaaa ccgauagcgu uguggccaca
uugaaggcua 180gcuaucccgg ccugcaguuc gagaucaucg ccaugagcac
caccggcgac aagauacucg 240acaccgcucu gaguaaaauc ggcgagaaga
gccuguuuac caaggagcug gagcacgccc 300uggaaaagaa cgaaguggac
cugguggugc auagucugaa agaccuuccc accguccuuc 360caccaggcuu
cacuaucggc gccaucugca agagggagaa uccucacgau gccgucgugu
420uucaucccaa guucgugggc aagaccuuag aaacccugcc agagaaaagc
gucguuggga 480ccuccucccu gcgacgggcc gcccagcugc agagaaaguu
cccccacuug gaauucagau 540ccaucagagg gaaucugaau acucgccuga
gaaagcugga cgagcagcag gaguuuagcg 600cuaucauccu ggccacggcu
gguuugcaga gaaugggcug gcacaaccgg gugggacaga 660uccugcaccc
cgaggagugc auguaugcag uaggccaggg ggcccugggg guggagguca
720gagccaaaga ucaggacauc cuggaccugg ucggcgugcu gcacgauccc
gaaacacugc 780ugcgguguau cgccgagagg gcuuuccucc ggcacuuaga
gggcggcugc uccguccccg 840uggccguuca cacugccaug aaagacgggc
agcuguaccu gacgggcggc gugugguccc 900uggacggcuc agacuccauu
caggagacca ugcaagcuac cauccacguc ccugcccaac 960acgaagaugg
ccccgaggac gacccccagc uggugggcau caccgccagg aauaucccaa
1020gaggccccca guuggccgcc cagaaccugg gcaucagucu ggccaaccug
cugcugagua 1080agggcgccaa aaacauccug gacguggcuc ggcagcugaa
ugacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941331294RNAArtificial
SequenceCONSTRUCT #16 133gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120cccggaagag ccagcuggcc
cggauccaga ccgacagcgu gguggccacc cugaaggcca 180gcuaccccgg
ccugcaguuc gagaucaucg ccaugagcac caccggcgac aagauccugg
240acaccgcccu gagcaagauc ggcgagaaga gccuguucac caaggagcug
gagcacgccc 300uggagaagaa cgagguggac cugguggugc acagccugaa
ggaccugccc accgugcugc 360cccccggcuu caccaucggc gccaucugca
agcgggagaa cccccacgac gccguggugu 420uccaccccaa guucgugggc
aagacccugg agacccugcc cgagaagagc guggugggca 480ccagcagccu
gcggcgggcc gcccagcugc agcggaaguu cccccaccug gaguuccgga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggccaccgcc ggccugcagc ggaugggcug
gcacaaccgg gugggccaga 660uccugcaccc cgaggagugc auguacgccg
ugggccaggg cgcccugggc guggaggugc 720gggccaagga ccaggacauc
cuggaccugg ugggcgugcu gcacgacccc gagacccugc 780ugcggugcau
cgccgagcgg gccuuccugc ggcaccugga gggcggcugc agcgugcccg
840uggccgugca caccgccaug aaggacggcc agcuguaccu gaccggcggc
guguggagcc 900uggacggcag cgacagcauc caggagacca ugcaggccac
cauccacgug cccgcccagc 960acgaggacgg ccccgaggac gacccccagc
uggugggcau caccgcccgg aacauccccc 1020ggggccccca gcuggccgcc
cagaaccugg gcaucagccu ggccaaccug cugcugagca 1080agggcgccaa
gaacauccug gacguggccc ggcagcugaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941341294RNAArtificial SequenceCONSTRUCT #16 134gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120cccggaagag ccagcuggcg aggauccaga cggacagcgu gguggcgacg
cugaaggcga 180gcuacccggg gcugcaguuc gagaucaucg cgaugagcac
gacgggggac aagauccugg 240acacggcgcu gagcaagauc ggggagaaga
gccuguucac gaaggagcug gagcacgcgc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccg acggugcugc 360cgccgggguu
cacgaucggg gcgaucugca agagggagaa cccgcacgac gcgguggugu
420uccacccgaa guucgugggg aagacgcugg agacgcugcc ggagaagagc
guggugggga 480cgagcagccu gaggagggcg gcgcagcugc agaggaaguu
cccgcaccug gaguucagga 540gcaucagggg gaaccugaac acgaggcuga
ggaagcugga cgagcagcag gaguucagcg 600cgaucauccu ggcgacggcg
gggcugcaga ggauggggug gcacaacagg guggggcaga 660uccugcaccc
ggaggagugc auguacgcgg uggggcaggg ggcgcugggg guggagguga
720gggcgaagga ccaggacauc cuggaccugg ugggggugcu gcacgacccg
gagacgcugc 780ugaggugcau cgcggagagg gcguuccuga ggcaccugga
gggggggugc agcgugccgg 840uggcggugca cacggcgaug aaggacgggc
agcuguaccu gacggggggg guguggagcc 900uggacgggag cgacagcauc
caggagacga ugcaggcgac gauccacgug ccggcgcagc 960acgaggacgg
gccggaggac gacccgcagc ugguggggau cacggcgagg aacaucccga
1020gggggccgca gcuggcggcg cagaaccugg ggaucagccu ggcgaaccug
cugcugagca 1080agggggcgaa gaacauccug gacguggcga ggcagcugaa
cgacgcgcac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941351294RNAArtificial
SequenceCONSTRUCT #17 135gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120cccggaagag ccagcuggcc
cgcauccaga ccgacuccgu cgucgccacc cucaaggccu 180ccuaccccgg
ccuccaguuc gagaucaucg ccauguccac caccggcgac aagauccucg
240acaccgcccu cuccaagauc ggcgagaagu cccucuucac caaggagcuc
gagcacgccc 300ucgagaagaa cgaggucgac cucgucgucc acucccucaa
ggaccucccc accguccucc 360cccccggcuu caccaucggc gccaucugca
agcgcgagaa cccccacgac gccgucgucu 420uccaccccaa guucgucggc
aagacccucg agacccuccc cgagaagucc gucgucggca 480ccuccucccu
ccgccgcgcc gcccagcucc agcgcaaguu cccccaccuc gaguuccgcu
540ccauccgcgg caaccucaac acccgccucc gcaagcucga cgagcagcag
gaguucuccg 600ccaucauccu cgccaccgcc ggccuccagc gcaugggcug
gcacaaccgc gucggccaga 660uccuccaccc cgaggagugc auguacgccg
ucggccaggg cgcccucggc gucgaggucc 720gcgccaagga ccaggacauc
cucgaccucg ucggcguccu ccacgacccc gagacccucc 780uccgcugcau
cgccgagcgc gccuuccucc gccaccucga gggcggcugc uccguccccg
840ucgccgucca caccgccaug aaggacggcc agcucuaccu caccggcggc
gucugguccc 900ucgacggcuc cgacuccauc caggagacca ugcaggccac
cauccacguc cccgcccagc 960acgaggacgg ccccgaggac gacccccagc
ucgucggcau caccgcccgc aacauccccc 1020gcggccccca gcucgccgcc
cagaaccucg gcaucucccu cgccaaccuc cuccucucca 1080agggcgccaa
gaacauccuc gacgucgccc gccagcucaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941361294RNAArtificial SequenceCONSTRUCT #18 136gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120cccguaagag ccagcuggcc cggauccaga ccgacagcgu gguggccacc
cugaaggcca 180gcuaccccgg ccugcaguuc gagaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gagcaagauc ggcgagaagu
cccuguucac caaggagcug gagcacgccc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccc accgugcugc 360cuccaggcuu
caccaucggc gccaucugca agcgggagaa cccucacgac gccguggugu
420uccaccccaa guucgugggc aagacccugg aaacccugcc ugagaagucc
gucguaggaa 480ccagcagccu gcggcgggcc gcccagcugc agcggaaguu
cccacaccug gaguuccgga 540gcauccgggg caaccugaac acccggcugc
ggaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggcuaccgcc
ggucugcaac gaaugggcug gcacaauagg gugggccaga 660uccugcaccc
cgaggagugc auguacgcgg ugggacaggg cgcccugggc guggaggugc
720gggccaagga ccaggacauc cuugaucugg ugggcgugcu gcacgacccc
gagacgcugc 780ugcggugcau cgccgagcgg gccuuccugc ggcauuugga
gggcggaugc agcgugcccg 840uggccgugca caccgccaug aaggacggcc
agcuguaccu gaccggcggc guguggagcc 900uggacggcag cgacagcauc
caggaaacca ugcaggccac cauccacgug ccugcucagc 960acgaagacgg
cccagaggac gacccucagc ugguaggcau caccgcccgg aacaucccuc
1020ggggcccuca gcucgccgca cagaaccuug gaaucagccu ggccaaccug
cuguugucaa 1080agggcgccaa gaauauccuc gacguggccc ggcagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941371294RNAArtificial
SequenceCONSTRUCT #19 137gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120ccagaaagag ccagcuggcc
cggauccaga ccgacagcgu gguggccacc cugaaggcca 180gcuaccccgg
ccugcaguuc gagaucaucg ccaugagcac caccggcgac aagauccugg
240acaccgcccu gagcaagauc ggcgagaaga gucuguucac caaggagcug
gagcacgccc 300uggagaagaa cgagguggac cugguggugc acagccugaa
ggaccugccc accgugcugc 360cgccuggcuu caccaucggc gccaucugca
agcgggagaa cccgcacgac gccguggugu 420uccaccccaa guucgugggc
aagacucugg aaacccugcc ugagaagucc guggucggaa 480caagcagccu
gcggcgggcc gcccagcugc agcggaaguu cccacaccug gaguuccgga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggccacagcc ggccuucaga ggaugggcug
gcacaaucgg guaggccaga 660uccugcaccc cgaggagugc auguacgcgg
uaggucaggg cgcccugggc guggaggugc 720gggccaagga ccaggacauc
uuagaucugg uuggcgugcu gcacgacccc gaaacacugc 780ugcggugcau
cgccgagcgg gccuuccugc ggcaccucga gggcggcugc agugugcccg
840uggccgugca caccgccaug aaggacggcc agcuguaccu gaccggcggc
guguggagcc 900uggacggcag cgacagcauc caggagacaa ugcaggccac
cauccacgug ccagcucagc 960acgaagacgg accagaggac gacccacagu
uagugggaau caccgcccgg aacaucccgc 1020ggggcccuca gcucgcggcc
cagaaccugg gcaucagccu ggccaaccug cugcugucua 1080agggcgccaa
gaacauccua gacguggccc ggcagcugaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941381294RNAArtificial SequenceCONSTRUCT #20 138gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120ccaggaaguc acagcuggcc cggauccaga ccgacagcgu gguggccacc
cugaaggcca 180gcuaccccgg ccugcaguuc gagaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gagcaagauc ggcgagaagu
cccuguucac caaggagcug gagcacgccc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccc accgugcugc 360cgccaggcuu
caccaucggc gccaucugca agcgggagaa cccgcacgac gccguggugu
420uccaccccaa guucgugggc aagacccuug aaacccugcc agagaagucu
guggucggca 480ccagcagccu gcggcgggcc gcccagcugc agcggaaguu
cccgcaccug gaguuccgga 540gcauccgggg caaccugaac acccggcugc
ggaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggccaccgcu
ggccuacagc ggaugggcug gcacaauaga guuggucaga 660uccugcaccc
cgaggagugc auguacgccg ucggccaggg cgcccugggc guggaggugc
720gggccaagga ccaggacaua cuagaccucg ugggcgugcu gcacgacccc
gaaaccuugc 780ugcggugcau cgccgagcgg gccuuccugc ggcaccugga
aggcgguugc agcgugcccg 840uggccgugca caccgccaug aaggacggcc
agcuguaccu gaccggcggc guguggagcc 900uggacggcag cgacagcauc
caggagacaa ugcaggccac cauccacgug ccggcccaac 960acgaggacgg
accugaggac gacccacagc uugugggaau caccgcccgg aacaucccac
1020ggggcccuca acuggcagcc cagaacuuag gcauaagccu ggccaaccug
cugcugucca 1080agggcgccaa gaacauccuc gauguggccc ggcagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941391294RNAArtificial
SequenceCONSTRUCT #21 139gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120cccgcaagag ucagcuggcc
cggauccaga ccgacagcgu gguggccacc cugaaggcca 180gcuaccccgg
ccugcaguuc gagaucaucg ccaugagcac caccggcgac aagauccugg
240acaccgcccu gagcaagauc ggcgagaaga gucuguucac caaggagcug
gagcacgccc 300uggagaagaa cgagguggac cugguggugc acagccugaa
ggaccugccc accgugcugc 360caccuggcuu caccaucggc gccaucugca
agcgggagaa cccacacgac gccguggugu 420uccaccccaa guucgugggc
aagacacugg aaacccugcc ggagaagucc gugguaggca 480ccagcagccu
gcggcgggcc gcccagcugc agcggaaguu cccgcaccug gaguuccgga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggcaacagcc ggcuuacagc guaugggcug
gcacaacagg gugggacaga 660uccugcaccc cgaggagugc auguacgcug
ugggccaggg cgcccugggc guggaggugc 720gggccaagga ccaggacauc
uuagaucucg ucggcgugcu gcacgacccc gaaaccuugc 780ugcggugcau
cgccgagcgg gccuuccugc ggcaucuuga gggcggaugc uccgugcccg
840uggccgugca caccgccaug aaggacggcc agcuguaccu gaccggcggc
guguggagcc 900uggacggcag cgacagcauc caggagacua ugcaggccac
cauccacgug ccagcccagc 960acgaagacgg cccagaggac gacccacagc
uggugggaau caccgcccgg aacaucccgc 1020ggggcccuca acuggccgca
cagaaccuag gcaucagccu ggccaaccug cugcucagca 1080agggcgccaa
gaauaucuug gacguggccc ggcagcugaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941401294RNAArtificial SequenceCONSTRUCT #22 140gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120ccagaaagag ccagcuggcc cggauccaga ccgacagcgu gguggccacc
cugaaggcca 180gcuaccccgg ccugcaguuc gagaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gagcaagauc ggcgagaagu
cucuguucac caaggagcug gagcacgccc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccc accgugcugc 360cuccuggcuu
caccaucggc gccaucugca agcgggagaa cccacacgac gccguggugu
420uccaccccaa guucgugggc aagacccuug agacucugcc agagaagucu
guagugggaa 480ccagcagccu gcggcgggcc gcccagcugc agcggaaguu
cccucaccug gaguuccgga 540gcauccgggg caaccugaac acccggcugc
ggaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggccacggcc
ggauuacaga gaaugggcug gcacaaccga gugggacaga 660uccugcaccc
cgaggagugc auguaugccg uuggccaagg cgcccugggc guggaggugc
720gggccaagga ccaggacauc cucgaucucg ugggcgugcu gcacgacccc
gagacuuugc 780ugcggugcau cgccgagcgg gccuuccugc ggcaccuaga
gggcggcugc ucggugcccg 840uggccgugca caccgccaug aaggacggcc
agcuguaccu gaccggcggc guguggagcc 900uggacggcag cgacagcauc
caggagacaa ugcaggccac cauccacgug ccagcccagc 960acgaggaugg
cccugaagac gacccacagc uggugggcau caccgcccgg aacaucccgc
1020ggggcccaca auuggccgcu cagaacuuag gcauuagccu ggccaaccug
cugcugucua 1080agggcgccaa gaacauacug gacguggccc ggcagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941411294RNAArtificial
SequenceCONSTRUCT #24 141gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120cccggaagag ccagcuggcc
cggauccaga ccgacagcgu gguggccacc cugaaggcca 180gcuaccccgg
ccugcaguuc gagaucaucg ccaugagcac caccggcgac aagauccugg
240acaccgcccu gagcaagauc ggcgagaaga gccuguucac caaggagcug
gagcacgccc 300uggagaagaa cgagguggac cugguggugc acagccugaa
ggaccugccc accgugcugc 360cgcccggcuu caccaucggc gccaucugca
agcgggagaa cccgcacgac gccguggugu 420uccaccccaa guucgugggc
aagacccugg agacccugcc cgagaagagc guggugggca 480ccagcagccu
gcggcgggcc gcccagcugc agcggaaguu cccucaccug gaguuccgga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggccaccgcc ggccugcagc ggaugggcug
gcacaaccgg gugggccaga 660uccugcaccc cgaggagugc auguacgccg
ugggccaggg cgcccugggc guggaggugc 720gggccaagga ccaggacauc
cuggaccugg ugggcgugcu gcacgacccc gagacccugc 780ugcggugcau
cgccgagcgg gccuuccugc ggcaccugga gggcggcugc agcgugcccg
840uggccgugca caccgccaug aaggacggcc agcuguaccu gaccggcggc
guguggagcc 900uggacggcag cgacagcauc caggagacca ugcaggccac
cauccacgug cccgcccagc 960acgaggacgg ccccgaggac gacccucagc
uggugggcau caccgcccgg aacaucccac 1020ggggcccuca gcuggccgcc
cagaaccugg gcaucagccu ggccaaccug cugcugagca 1080agggcgccaa
gaacauccug gacguggccc ggcagcugaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941421294RNAArtificial SequenceCONSTRUCT #23 142gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120cccggaagag ccagcuggcg aggauccaga cggacagcgu gguggcgacg
cugaaggcga 180gcuacccggg gcugcaguuc gagaucaucg cgaugagcac
gacgggcgac aagauccugg 240acacggcgcu gagcaagauc ggggagaaga
gccuguucac gaaggagcug gagcacgcgc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccg acggugcugc 360cgccgggguu
cacgaucggg gcgaucugca agagggagaa cccgcacgac gcgguggugu
420uccacccgaa guucgugggg aagacgcugg agacgcugcc ggagaagagc
guggugggga 480cgagcagccu gaggagggcg gcgcagcugc agaggaaguu
cccgcaccug gaguucagga 540gcaucagggg uaaccugaac acgaggcuga
ggaagcugga cgagcagcag gaguucagcg 600cgaucauccu ggcgacggcg
gggcugcaga ggauggggug gcacaacagg guggggcaga 660uccugcaccc
ggaggagugc auguacgcgg uggggcaggg cgcgcugggc guggagguga
720gggcgaagga ccaggacauc cuggaccugg ugggcgugcu gcacgacccg
gagacgcugc 780ugaggugcau cgcggagagg gcguuccuga ggcaccugga
gggcgggugc agcgugccgg 840uggcggugca cacggcgaug aaggacgggc
agcuguaccu gacgggaggg guguggagcc 900uggacgggag cgacagcauc
caggagacga ugcaggcgac gauccacgug ccggcgcagc 960acgaggacgg
gccggaggac gacccgcagc ugguggggau cacggcgagg aacaucccga
1020gggguccgca gcuggcggcg cagaaccugg ggaucagccu ggcgaaccug
cugcugagca 1080agggagcgaa gaacauccug gacguggcga ggcagcugaa
cgacgcgcac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc
12941431294RNAArtificial SequenceCONSTRUCT #24 143gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120cccggaagag ccagcuggcc cgcauccaga ccgacuccgu cgucgccacc
cucaaggccu 180ccuaccccgg ccuccaguuc gagaucaucg ccauguccac
caccggcgac aagauccucg 240acaccgcccu cuccaagauc ggcgagaagu
cccucuucac caaggagcuc gagcacgccc 300ucgagaagaa cgaggucgac
cucgucgucc acucccucaa ggaccucccc accguccucc 360cacccggcuu
caccaucggc gccaucugca agcgcgagaa cccucacgac gccgucgucu
420uccaccccaa guucgucggc aagacccucg agacccuccc cgagaagucc
gucgucggca 480ccuccucccu ccgccgcgcc gcccagcucc agcgcaaguu
cccacaccuc gaguuccgcu 540ccauccgcgg caaccucaac acccgccucc
gcaagcucga cgagcagcag gaguucuccg 600ccaucauccu cgccaccgcc
ggccuccagc gcaugggcug gcacaaccgc gucggccaga 660uccuccaccc
cgaggagugc auguacgccg ucggccaggg cgcccucggc gucgaggucc
720gcgccaagga ccaggacauc cucgaccucg ucggcguccu ccacgacccc
gagacccucc 780uccgcugcau cgccgagcgc gccuuccucc gccaccucga
gggcggcugc uccguccccg 840ucgccgucca caccgccaug aaggacggcc
agcucuaccu caccggcggc gucugguccc 900ucgacggcuc cgacuccauc
caggagacca ugcaggccac cauccacguc cccgcccagc 960acgaggacgg
ccccgaggac gacccucagc ucgucggcau caccgcccgc aacaucccgc
1020gcggcccuca gcucgccgcc cagaaccucg gcaucucccu cgccaaccuc
cuccucucca 1080agggcgccaa gaacauccuc gacgucgccc gccagcucaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941441294RNAArtificial
SequenceCONSTRUCT #27 144gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgcc gaggagaaca
gccccaagau gcgggugauc cgggugggca 120cccggaagag ccagcuggcc
cggauccaga ccgacagcgu gguggccacc cugaaggcca 180gcuaccccgg
ccugcaguuc gagaucaucg ccaugagcac caccggcgac aagauccugg
240acaccgcccu gagcaagauc ggcgagaaga gccuguucac caaggagcug
gagcacgccc 300uggagaagaa cgagguggac cugguggugc acagccugaa
ggaccugccc accgugcugc 360cgcccggcuu caccaucggc gccaucugca
agcgggagaa cccgcacgac gccguggugu 420uccaccccaa guucgugggc
aagacccugg agacccugcc cgagaagagc guggugggca 480ccagcagccu
gcggcgggcc gcccagcugc agcggaaguu cccucaccug gaguuccgga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggccaccgcc ggccugcagc ggaugggcug
gcacaaccgg gugggccaga 660uccugcaccc cgaggagugc auguacgccg
ugggccaggg cgcccugggc guggaggugc 720gggccaagga ccaggacauc
cuggaccugg ugggcgugcu gcacgacccc gagacccugc 780ugcggugcau
cgccgagcgg gccuuccugc ggcaccugga gggcggcugc agcgugcccg
840uggccgugca caccgccaug aaggacggcc agcuguaccu gaccggcggc
guguggagcc 900uggacggcag cgacagcauc caggagacca ugcaggccac
cauccacgug cccgcccagc 960acgaggacgg ccccgaggac gacccucagc
uggugggcau caccgcccgg aacaucccac 1020ggggcccuca gcuggccgcc
cagaaccugg gcaucagccu ggccaaccug cugcugagca 1080agggcgccaa
gaacauccug gacguggccc ggcagcugaa cgacgcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc uagcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941451272RNAArtificial SequenceCONSTRUCT #28 145gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc
cgccaccgcc gaggagaaca gccccaagau gcgggugauc cgggugggca
120cccggaagag ccagcuggcc cggauccaga ccgacagcgu gguggccacc
cugaaggcca 180gcuaccccgg ccugcaguuc gagaucaucg ccaugagcac
caccggcgac aagauccugg 240acaccgcccu gagcaagauc ggcgagaaga
gccuguucac caaggagcug gagcacgccc 300uggagaagaa cgagguggac
cugguggugc acagccugaa ggaccugccc accgugcugc 360cgcccggcuu
caccaucggc gccaucugca agcgggagaa cccgcacgac gccguggugu
420uccaccccaa guucgugggc aagacccugg agacccugcc cgagaagagc
guggugggca 480ccagcagccu gcggcgggcc gcccagcugc agcggaaguu
cccucaccug gaguuccgga 540gcauccgggg caaccugaac acccggcugc
ggaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggccaccgcc
ggccugcagc ggaugggcug gcacaaccgg gugggccaga 660uccugcaccc
cgaggagugc auguacgccg ugggccaggg cgcccugggc guggaggugc
720gggccaagga ccaggacauc cuggaccugg ugggcgugcu gcacgacccc
gagacccugc 780ugcggugcau cgccgagcgg gccuuccugc ggcaccugga
gggcggcugc agcgugcccg 840uggccgugca caccgccaug aaggacggcc
agcuguaccu gaccggcggc guguggagcc 900uggacggcag cgacagcauc
caggagacca ugcaggccac cauccacgug cccgcccagc 960acgaggacgg
ccccgaggac gacccucagc uggugggcau caccgcccgg aacaucccac
1020ggggcccuca gcuggccgcc cagaaccugg gcaucagccu ggccaaccug
cugcugagca 1080agggcgccaa gaacauccug gacguggccc ggcagcugaa
cgacgcccac ugauaauagg 1140cuggagccuc gguggccuag cuucuugccc
cuugggccuc cccccagccc cuccuccccu 1200uccugcaccc guacccccuc
cauaaaguag gaaacacuac aguggucuuu gaauaaaguc 1260ugagugggcg gc
12721461294RNAArtificial SequenceCONSTRUCT #25 146gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug uccggaaacg 60gaaacgccgc
cgcuaccgcc gaggaaaaua gcccgaagau gagagugauc cgggugggua
120ccaggaaguc ccagcucgcc aggauccaaa cggacucggu gguggccacc
cucaaggcua 180gcuacccggg ccugcaauuc gagaucauug cuauguccac
caccggcgac aagauccugg 240auacggcccu guccaagauc ggcgaaaaga
gccucuucac caaggagcug gaacacgcgc 300ucgagaagaa cgagguggac
cuggucgucc acagccucaa ggaccugccu acggugcugc 360cgccgggauu
caccaucggc gccaucugua agcgggagaa uccgcacgac gccguggugu
420uccacccuaa guucgugggc aagacccucg agacacugcc ggaaaagucc
gucgugggca 480ccuccucccu gagaagggcc gcucagcucc agagaaaguu
cccgcaccug gaauucagga 540gcauccgggg caaccugaau acccggcuuc
gcaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggccaccgcc
ggccugcagc ggaugggcug gcacaaccgg gugggccaga 660uccugcaccc
ggaggagugc auguaugccg ugggucaggg agcccugggc guggaggucc
720gggccaagga ccaggacauc cuggaccucg ugggcgugcu ccacgacccu
gaaacccucc 780ugaggugcau cgccgagagg gccuuccucc ggcaccugga
gggcggcugu uccgucccug 840uggccgugca uaccgccaug aaggacggac
agcuguaccu gaccggcggc gugugguccc 900uggacggcuc cgacagcauc
caggaaacca ugcaggccac uauccacgug ccggcccagc 960acgaagacgg
cccagaggau gacccgcaac uggucggcau uaccgccagg aacauaccaa
1020ggggcccgca gcuggccgcc cagaaccugg gcaucucccu ggccaaccug
cugcugucca 1080agggagccaa gaacauucug gacguggcca ggcagcucaa
ugaugcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 12941471294RNAArtificial
SequenceCONSTRUCT #26 147gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug agcggcaacg 60gcaacgccgc cgccaccgca gaggagaaua
gcccgaagau gagggugauc cgagugggca 120ccaggaaguc ccagcuugcg
cgaauucaga ccgacagcgu gguggccacc cucaaggccu 180ccuacccggg
acuccaguuc gagaucaucg ccaugagcac cacgggagac aagauccugg
240acaccgcccu guccaagauc ggcgaaaaga gccucuucac caaggagcug
gagcacgccc 300uggagaagaa cgaggucgau cugguggugc acagccugaa
ggaccugccg accguccugc 360cgccgggauu caccaucggu gccaucugua
agcgggagaa cccgcacgac gccguggugu 420uccacccgaa guucgucggc
aagacccugg aaacccugcc ggagaagucc guggugggca 480ccagcagccu
gaggcgggcc gcccagcugc agcggaaguu cccgcaccug gaauucagga
540gcauccgggg caaccugaac acccggcugc ggaagcugga cgagcagcag
gaguucagcg 600ccaucauccu ggccaccgca ggccuccagc gcaugggaug
gcacaacagg guaggccaaa 660uccugcaccc ggaggagugu auguacgccg
ugggccaggg agcccugggc guggagguga 720gagccaagga ccaggacauc
cuagaccugg ucggcgugcu gcacgacccg gagacacugc 780ugagaugcau
cgcggagaga gccuuccugc gacaccugga gggcggcugc uccgugccgg
840uggccgugca caccgccaug aaggacggcc agcuguaucu gaccggcggc
guguggagcc 900uggacggcag cgacuccauc caagaaacca ugcaggcuac
cauccacgug ccggcccagc 960acgaggaugg accagaggac gauccucaac
uggugggcau cacugccagg aacaucccaa 1020gaggcccgca gcuggccgcc
cagaaccugg gcaucagccu ggccaaccug cugcugucca 1080agggcgccaa
gaacauucuc gauguggcca ggcagcugaa cgaugcccac ugauaauagu
1140ccauaaagua ggaaacacua cagcuggagc cucgguggcc augcuucuug
ccccuugggc 1200cuccccccag ccccuccucc ccuuccugca cccguacccc
ccgcauuauu acucacggua 1260cgaguggucu uugaauaaag ucugaguggg cggc
12941481294RNAArtificial SequenceCONSTRUCT #27 148gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug ucgggaaacg 60gcaacgccgc
cgccacggcc gaggagaaca gcccgaagau gagagugauu agggugggca
120cccggaaguc ccaacucgcg cggauccaga ccgacuccgu gguggccacc
cucaaggcca 180gcuacccggg ccuccaguuc gagauuaucg ccauguccac
cacaggcgac aagauccucg 240acaccgcacu cucgaagauc ggcgagaagu
cccuguucac caaggaacug gagcacgccc 300uggagaagaa cgagguggac
cugguggugc acucccugaa ggaccugccg accgugcucc 360caccaggcuu
caccaucggc gcaaucugua agcgcgagaa uccgcacgac gccguggugu
420uccacccaaa guucgugggc aagacccucg aaacccuccc ggaaaagagc
guggugggua 480ccagcucccu gcggagagcu gcccagcugc agagaaaguu
cccgcaucug gaauucagga 540gcaucagggg aaaucugaau accagacugc
gcaagcugga cgagcagcag gaguucagcg 600ccaucauccu ggccaccgcc
ggccugcagc ggaugggcug gcacaacagg gugggccaga 660uacugcaucc
ggaggagugu auguacgccg ugggccaggg cgcccucggc guggagguga
720gagccaagga ccaagacauc cuggaccuag ugggcgugcu gcaugacccu
gaaacccugc 780ucaggugcau cgccgagagg gccuuccugc ggcaccugga
gggcggcugc agcgugccgg 840uggccgucca caccgccaug aaggacggcc
agcuguaccu gaccggcggc gucuggagcc 900uggacggauc cgacagcauc
caggaaacca ugcaggccac cauccacgug ccggcccagc 960acgaggacgg
cccugaggac gacccucagc uggugggcau caccgcuagg aacaucccaa
1020ggggcccgca gcuggccgcc cagaaccucg gcaucagccu ggccaaccug
cugcugucca 1080agggcgccaa gaauauccug gacguggcca ggcagcugaa
cgacgcccac ugauaauagu 1140ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug ccccuugggc 1200cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu acucacggua 1260cgaguggucu
uugaauaaag ucugaguggg cggc 1294149164RNAArtificial Sequence3'UTR
(miR142+miR126 variant 1) 149ugauaauagu ccauaaagua ggaaacacua
cagcuggagc cucgguggcc augcuucuug 60ccccuugggc cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu 120acucacggua cgaguggucu
uugaauaaag ucugaguggg cggc 164150164RNAArtificial Sequence3'UTR
(miR142+miR126 variant 2) 150ugauaauagu ccauaaagua ggaaacacua
cagcuggagc cucgguggcc uagcuucuug 60ccccuugggc cuccccccag ccccuccucc
ccuuccugca cccguacccc ccgcauuauu 120acucacggua cgaguggucu
uugaauaaag ucugaguggg cggc 164151142RNAArtificial Sequence3'UTR
(miR142) 151ugauaauagg cuggagccuc gguggccuag cuucuugccc cuugggccuc
cccccagccc 60cuccuccccu uccugcaccc guacccccuc cauaaaguag gaaacacuac
aguggucuuu 120gaauaaaguc ugagugggcg gc 142152361PRTArtificial
SequencePBGD GoF I291M/N340S 152Met Ser Gly Asn Gly Asn Ala Ala Ala
Thr Ala Glu Glu Asn Ser Pro1 5 10 15Lys Met Arg Val Ile Arg Val Gly
Thr Arg Lys Ser Gln Leu Ala Arg 20 25 30Ile Gln Thr Asp Ser Val Val
Ala Thr Leu Lys Ala Ser Tyr Pro Gly 35 40 45Leu Gln Phe Glu Ile Ile
Ala Met Ser Thr Thr Gly Asp Lys Ile Leu 50 55 60Asp Thr Ala Leu Ser
Lys Ile Gly Glu Lys Ser Leu Phe Thr Lys Glu65 70 75 80Leu Glu His
Ala Leu Glu Lys Asn Glu Val Asp Leu Val Val His Ser 85 90 95Leu Lys
Asp Leu Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala 100 105
110Ile Cys Lys Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys
115 120 125Phe Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val
Val Gly 130 135 140Thr Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg
Lys Phe Pro His145 150 155 160Leu Glu Phe Arg Ser Ile Arg Gly Asn
Leu Asn Thr Arg Leu Arg Lys 165 170 175Leu Asp Glu Gln Gln Glu Phe
Ser Ala Ile Ile Leu Ala Thr Ala Gly 180 185 190Leu Gln Arg Met Gly
Trp His Asn Arg Val Gly Gln Ile Leu His Pro 195 200 205Glu Glu Cys
Met Tyr Ala Val Gly Gln Gly Ala Leu Gly Val Glu Val 210 215 220Arg
Ala Lys Asp Gln Asp Ile Leu Asp Leu Val Gly Val Leu His Asp225 230
235 240Pro Glu Thr Leu Leu Arg Cys Ile Ala Glu Arg Ala Phe Leu Arg
His 245 250 255Leu Glu Gly Gly Cys Ser Val Pro Val Ala Val His Thr
Ala Met Lys 260 265 270Asp Gly Gln Leu Tyr Leu Thr Gly Gly Val Trp
Ser Leu Asp Gly Ser 275 280 285Asp Ser Met Gln Glu Thr Met Gln Ala
Thr Ile His Val Pro Ala Gln 290 295 300His Glu Asp Gly Pro Glu Asp
Asp Pro Gln Leu Val Gly Ile Thr Ala305 310 315 320Arg Asn Ile Pro
Arg Gly Pro Gln Leu Ala Ala Gln Asn Leu Gly Ile 325 330 335Ser Leu
Ala Ser Leu Leu Leu Ser Lys Gly Ala Lys Asn Ile Leu Asp 340 345
350Val Ala Arg Gln Leu Asn Asp Ala His 355 3601531083DNAArtificial
SequenceI291M / N340S 153atgagcggga acggcaacgc ggcagccacc
gctgaggaaa actctccaaa aatgagagtc 60attagggtgg gcaccagaaa gagtcaactc
gcaaggatcc agaccgactc tgtggtggcc 120actctaaaag cgagctaccc
cggactccag ttcgaaatca tcgcgatgag caccaccggc 180gataaaatcc
tagataccgc cttgagcaag atcggcgaga agagcctctt taccaaggag
240ctggagcacg ccctggagaa aaacgaggtg gatctggtgg tccatagcct
aaaggatctc 300cctacagtgc tgccccccgg gtttaccatc ggcgccattt
gtaagcggga gaacccgcac 360gacgcagtgg tcttccaccc caagtttgtg
ggcaagacac tggagaccct gccggaaaag 420tccgtggtgg gcacctccag
cctgagacga gccgcccagc tgcagcgcaa gtttccccac 480ctggagttta
ggagcatcag gggaaacctc aacaccagac tgagaaagct ggacgaacag
540caggagttca gcgccatcat cctggcgact gcaggactcc agaggatggg
ctggcataat 600agggtgggac aaattctgca ccccgaggaa tgcatgtatg
ctgtggggca gggcgcactc 660ggggtggagg tgcgggccaa ggatcaggac
atcctggacc tcgtgggcgt gcttcacgac 720ccggaaaccc tgctgaggtg
catagcagaa agagctttcc tgcggcacct ggagggggga 780tgcagcgtcc
ctgtggccgt gcatacagcc atgaaggacg gccagctgta cttgactggt
840ggcgtctggt cccttgatgg ctctgacagc atgcaggaaa caatgcaggc
cactatccac 900gtgcctgccc agcacgagga cggccccgag gacgaccctc
agctggtggg cattaccgcc 960agaaatatcc ctaggggacc ccagctggct
gcccaaaact tgggcatcag cctggccagc 1020ctgctgctga gcaaaggagc
taaaaacatc ctggatgtgg ccaggcagct gaatgacgcc 1080cac
1083154604PRTArtificial SequenceAPOA1-PBGD 154Met Asp Glu Pro Pro
Gln Ser Pro Trp Asp Arg Val Lys Asp Leu Ala1 5 10 15Thr Val Tyr Val
Asp Val Leu Lys Asp Ser Gly Arg Asp Tyr Val Ser 20 25 30Gln Phe Glu
Gly Ser Ala Leu Gly Lys Gln Leu Asn Leu Lys Leu Leu 35 40 45Asp Asn
Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln 50 55 60Leu
Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr65 70 75
80Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala
85 90 95Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu
Glu 100 105 110Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala
Glu Leu Gln 115 120 125Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln
Glu Lys Leu Ser Pro 130 135 140Leu Gly Glu Glu Met Arg Asp Arg Ala
Arg Ala His Val Asp Ala Leu145 150 155 160Arg Thr His Leu Ala Pro
Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala 165 170 175Ala Arg Leu Glu
Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu 180 185 190Tyr His
Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala 195 200
205Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu
210 215 220Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys225 230 235 240Leu Asn Thr Gln Ser Gly Asn Gly Asn Ala Ala
Ala Thr Ala Glu Glu 245 250 255Asn Ser Pro Lys Met Arg Val Ile Arg
Val Gly Thr Arg Lys Ser Gln 260 265 270Leu Ala Arg Ile Gln Thr Asp
Ser Val Val Ala Thr Leu Lys Ala Ser 275 280 285Tyr Pro Gly Leu Gln
Phe Glu Ile Ile Ala Met Ser Thr Thr Gly Asp 290 295 300Lys Ile Leu
Asp Thr Ala Leu Ser Lys Ile Gly Glu Lys Ser Leu Phe305 310 315
320Thr Lys Glu Leu Glu His Ala Leu Glu Lys Asn Glu Val Asp Leu Val
325 330 335Val His Ser Leu Lys Asp Leu Pro Thr Val Leu Pro Pro Gly
Phe Thr 340 345 350Ile Gly Ala Ile Cys Lys Arg Glu Asn Pro His Asp
Ala Val Val Phe 355 360 365His Pro Lys Phe Val Gly Lys Thr Leu Glu
Thr Leu Pro Glu Lys Ser 370 375 380Val Val Gly Thr Ser Ser Leu Arg
Arg Ala Ala Gln Leu Gln Arg Lys385 390 395 400Phe Pro His Leu Glu
Phe Arg Ser Ile Arg Gly Asn Leu Asn Thr Arg 405 410 415Leu Arg Lys
Leu Asp Glu Gln Gln Glu Phe Ser Ala Ile Ile Leu Ala 420 425 430Thr
Ala Gly Leu Gln Arg Met Gly Trp His Asn Arg Val Gly Gln Ile 435 440
445Leu
His Pro Glu Glu Cys Met Tyr Ala Val Gly Gln Gly Ala Leu Gly 450 455
460Val Glu Val Arg Ala Lys Asp Gln Asp Ile Leu Asp Leu Val Gly
Val465 470 475 480Leu His Asp Pro Glu Thr Leu Leu Arg Cys Ile Ala
Glu Arg Ala Phe 485 490 495Leu Arg His Leu Glu Gly Gly Cys Ser Val
Pro Val Ala Val His Thr 500 505 510Ala Met Lys Asp Gly Gln Leu Tyr
Leu Thr Gly Gly Val Trp Ser Leu 515 520 525Asp Gly Ser Asp Ser Met
Gln Glu Thr Met Gln Ala Thr Ile His Val 530 535 540Pro Ala Gln His
Glu Asp Gly Pro Glu Asp Asp Pro Gln Leu Val Gly545 550 555 560Ile
Thr Ala Arg Asn Ile Pro Arg Gly Pro Gln Leu Ala Ala Gln Asn 565 570
575Leu Gly Ile Ser Leu Ala Ser Leu Leu Leu Ser Lys Gly Ala Lys Asn
580 585 590Ile Leu Asp Val Ala Arg Gln Leu Asn Asp Ala His 595
6001551812DNAArtificial SequenceAPOA1-PBGD 155atggacgaac caccccagtc
accctgggat agagtcaaag acctggccac tgtgtacgtg 60gacgtgctga aagatagcgg
gcgggattac gtctcgcagt tcgagggctc cgccttaggc 120aaacagctga
acctcaaact gctggacaat tgggactccg tcactagcac attctccaag
180ctgagagagc agctgggacc tgtgactcag gagttctggg acaatctgga
gaaggaaact 240gagggcctga ggcaggagat gtccaaggat ctggaggagg
ttaaggcaaa ggtgcaaccc 300tacctcgacg acttccagaa aaaatggcaa
gaagagatgg agctataccg gcaaaaggtg 360gagcctctcc gggccgagct
ccaggagggc gctcggcaaa agctgcatga gctgcaggag 420aagctgagcc
ccctcggcga ggaaatgcgt gatagagcac gtgcccacgt ggacgccctg
480cggacacatc tggctcccta cagcgatgag cttaggcaga gactggccgc
cagactcgaa 540gctctgaagg agaatggcgg cgctcggctg gccgagtatc
atgccaaggc caccgagcat 600ttgtccactc tgagtgagaa ggctaagcct
gccctggagg acctgaggca gggcctgtta 660cccgtgctgg agagcttcaa
ggtgagcttt ctgagcgcct tggaggagta caccaaaaaa 720ctgaacaccc
agagcggcaa cggaaacgcc gccgctaccg ccgaggagaa cagccctaag
780atgagggtga tcagggtggg cacccgcaag tcccagcttg cacgaatcca
gaccgattca 840gtcgtggcga ccctgaaggc atcttacccc ggcctgcagt
tcgagattat cgccatgtct 900acaaccggcg acaagatcct cgataccgcc
ctgagcaaga tcggcgaaaa gagcctgttc 960accaaagagc tagagcacgc
cctcgagaaa aacgaggtgg accttgtggt gcatagcctc 1020aaggatcttc
ctaccgtgct gccccccggt ttcaccattg gggcaatctg caagagggag
1080aatccccacg atgccgtggt gttccatccc aaattcgtgg gcaaaacgtt
ggagaccctg 1140cccgagaaaa gtgtggtcgg cactagcagc ctgagaagag
cggcccaact tcagagaaag 1200tttcctcacc tggagttcag gagcatccgg
ggcaacctga acacccggct gcggaagctg 1260gacgagcagc aagagttcag
cgctatcatt ctagcaacag ccggcctgca gcgcatggga 1320tggcataaca
gggtgggaca gattctccac cccgaggaat gcatgtacgc cgttggccaa
1380ggcgcgctgg gggtggaggt cagagcaaag gaccaggaca ttcttgatct
ggtcggggtg 1440ctgcacgacc ccgagacact gctgagatgc atagctgagc
gggccttcct gcggcatctg 1500gagggcggct gctcagtccc cgtggccgtg
cacacagcca tgaaggacgg ccagctgtac 1560ctgaccggag gggtgtggag
cttggacggc agcgattcaa tgcaggagac aatgcaggca 1620accatccacg
ttccagctca gcacgaggat ggccccgaag atgaccctca gctggtgggg
1680attactgcta gaaacatccc ccgcggcccc cagctggccg cccaaaacct
gggaatctct 1740ctggcctccc tgctcctctc taaaggagcc aagaatattc
tggacgtggc acggcagctc 1800aacgatgcac ac 181215685RNAArtificial
SequencemiR-126 156cgcuggcgac gggacauuau uacuuuuggu acgcgcugug
acacuucaaa cucguaccgu 60gaguaauaau gcgccgucca cggca
8515722RNAArtificial SequencemiR 126-3p sequence 157ucguaccgug
aguaauaaug cg 2215822RNAArtificial SequencemiR-126-3p binding site
158cgcauuauua cucacgguac ga 2215921RNAArtificial SequencemiR 126-5p
sequence 159cauuauuacu uuugguacgc g 2116021RNAArtificial
SequencemiR-126-5p binding site 160cgcguaccaa aaguaauaau g
21161133RNAArtificial Sequence3'UTR with miR 142-3p binding site
161gcuggagccu cgguggccau gcuucuugcc ccuugggccu ccccccagcc
ccuccucccc 60uuccugcacc cguacccccu ccauaaagua ggaaacacua caguggucuu
ugaauaaagu 120cugagugggc ggc 133162141RNAArtificial Sequence3'UTR
with miR 126-3p binding site 162ugauaauagg cuggagccuc gguggccaug
cuucuugccc cuugggccuc cccccagccc 60cuccuccccu uccugcaccc guaccccccg
cauuauuacu cacgguacga guggucuuug 120aauaaagucu gagugggcgg c
141163119RNAArtificial Sequence(3' UTR, no miR binding sites
variant 2) 163ugauaauagg cuggagccuc gguggccuag cuucuugccc
cuugggccuc cccccagccc 60cuccuccccu uccugcaccc guacccccgu ggucuuugaa
uaaagucuga gugggcggc 119164141RNAArtificial Sequence(3' UTR with
miR 126-3p binding site variant 3) 164ugauaauagg cuggagccuc
gguggccuag cuucuugccc cuugggccuc cccccagccc 60cuccuccccu uccugcaccc
guaccccccg cauuauuacu cacgguacga guggucuuug 120aauaaagucu
gagugggcgg c 141165188RNAArtificial Sequence3' UTR with 3 miR
142-3p binding sites 165ugauaauagu ccauaaagua ggaaacacua cagcuggagc
cucgguggcc augcuucuug 60ccccuugggc cuccauaaag uaggaaacac uacauccccc
cagccccucc uccccuuccu 120gcacccguac ccccuccaua aaguaggaaa
cacuacagug gucuuugaau aaagucugag 180ugggcggc 188166140RNAArtificial
Sequence3'UTR with miR 142-5p binding site 166ugauaauagg cuggagccuc
gguggccaug cuucuugccc cuugggccuc cccccagccc 60cuccuccccu uccugcaccc
guacccccag uagugcuuuc uacuuuaugg uggucuuuga 120auaaagucug
agugggcggc 140167182RNAArtificial Sequence3'UTR with 3 miR 142-5p
binding sites 167ugauaauaga guagugcuuu cuacuuuaug gcuggagccu
cgguggccau gcuucuugcc 60ccuugggcca guagugcuuu cuacuuuaug uccccccagc
cccuccuccc cuuccugcac 120ccguaccccc aguagugcuu ucuacuuuau
gguggucuuu gaauaaaguc ugagugggcg 180gc 182168184RNAArtificial
Sequence3'UTR with 2 miR 142-5p binding sites and 1 miR 142-3p
binding site 168ugauaauaga guagugcuuu cuacuuuaug gcuggagccu
cgguggccau gcuucuugcc 60ccuugggccu ccauaaagua ggaaacacua caucccccca
gccccuccuc cccuuccugc 120acccguaccc ccaguagugc uuucuacuuu
augguggucu uugaauaaag ucugaguggg 180cggc 184169142RNAArtificial
Sequence3'UTR with miR 155-5p binding site 169ugauaauagg cuggagccuc
gguggccaug cuucuugccc cuugggccuc cccccagccc 60cuccuccccu uccugcaccc
guacccccac cccuaucaca auuagcauua aguggucuuu 120gaauaaaguc
ugagugggcg gc 142170188RNAArtificial Sequence3' UTR with 3 miR
155-5p binding sites 170ugauaauaga ccccuaucac aauuagcauu aagcuggagc
cucgguggcc augcuucuug 60ccccuugggc caccccuauc acaauuagca uuaauccccc
cagccccucc uccccuuccu 120gcacccguac ccccaccccu aucacaauua
gcauuaagug gucuuugaau aaagucugag 180ugggcggc 188171188RNAArtificial
Sequence3'UTR with 2 miR 155-5p binding sites and 1 miR 142-3p
binding site 171ugauaauaga ccccuaucac aauuagcauu aagcuggagc
cucgguggcc augcuucuug 60ccccuugggc cuccauaaag uaggaaacac uacauccccc
cagccccucc uccccuuccu 120gcacccguac ccccaccccu aucacaauua
gcauuaagug gucuuugaau aaagucugag 180ugggcggc 188172142RNAArtificial
Sequence3'UTR with miR 142-3p binding site, P3 insertion
172ugauaauagg cuggagccuc gguggccaug cuucuugccc cuugggccuc
cauaaaguag 60gaaacacuac auccccccag ccccuccucc ccuuccugca cccguacccc
cguggucuuu 120gaauaaaguc ugagugggcg gc 14217322RNAArtificial
SequencemiR 146-3p sequence 173ccucugaaau ucaguucuuc ag
2217422RNAArtificial SequencemiR 146-5p sequence 174ugagaacuga
auuccauggg uu 2217522RNAArtificial SequencemiR 155-3p sequence
175cuccuacaua uuagcauuaa ca 2217623RNAArtificial Sequence3' UTR
176uuaaugcuaa ucgugauagg ggu 2317722RNAArtificial SequencemiR 16-3p
sequence 177ccaguauuaa cugugcugcu ga 2217822RNAArtificial
SequencemiR 16-5p sequence 178uagcagcacg uaaauauugg cg
2217921RNAArtificial SequencemiR 21-3p sequence 179caacaccagu
cgaugggcug u 2118022RNAArtificial SequencemiR 21-5p sequence
180uagcuuauca gacugauguu ga 2218122RNAArtificial SequencemiR 223-3p
sequence 181ugucaguuug ucaaauaccc ca 2218222RNAArtificial
SequencemiR 223-5p sequence 182cguguauuug acaagcugag uu
2218322RNAArtificial SequencemiR 24-3p sequence 183uggcucaguu
cagcaggaac ag 2218422RNAArtificial SequencemiR 24-5p sequence
184ugccuacuga gcugauauca gu 2218521RNAArtificial SequencemiR 27-3p
sequence 185uucacagugg cuaaguuccg c 2118622RNAArtificial
SequencemiR 27-5p sequence 186agggcuuagc ugcuugugag ca
2218723RNAArtificial SequencemiR 155-5p sequence 187uuaaugcuaa
uugugauagg ggu 2318823RNAArtificial SequencemiR 155-5p binding site
188accccuauca caauuagcau uaa 2318970RNAArtificial Sequence5' UTR
with miR142-3p binding site at position p1 189gggaaauaag aguccauaaa
guaggaaaca cuacaagaaa agaagaguaa gaagaaauau 60aagagccacc
7019070RNAArtificial Sequence5' UTR with miR142-3p binding site at
position p2 190gggaaauaag agagaaaaga agaguaaucc auaaaguagg
aaacacuaca gaagaaauau 60aagagccacc 7019170RNAArtificial Sequence5'
UTR with miR142-3p binding site at position p3 191gggaaauaag
agagaaaaga agaguaagaa gaaauauaau ccauaaagua ggaaacacua 60cagagccacc
70192188RNAArtificial Sequence(3' UTR with 3 miR 142-3p binding
sites variant 2) 192ugauaauagu ccauaaagua ggaaacacua cagcuggagc
cucgguggcc uagcuucuug 60ccccuugggc cuccauaaag uaggaaacac uacauccccc
cagccccucc uccccuuccu 120gcacccguac ccccuccaua aaguaggaaa
cacuacagug gucuuugaau aaagucugag 180ugggcggc 188193142RNAArtificial
Sequence(3'UTR with miR 142-3p binding site, P1 insertion variant
2) 193ugauaauagu ccauaaagua ggaaacacua cagcuggagc cucgguggcc
uagcuucuug 60ccccuugggc cuccccccag ccccuccucc ccuuccugca cccguacccc
cguggucuuu 120gaauaaaguc ugagugggcg gc 142194142RNAArtificial
Sequence(3'UTR with miR 142-3p binding site, P2 insertion variant
2) 194ugauaauagg cuggagccuc gguggcucca uaaaguagga aacacuacac
uagcuucuug 60ccccuugggc cuccccccag ccccuccucc ccuuccugca cccguacccc
cguggucuuu 120gaauaaaguc ugagugggcg gc 142195181RNAArtificial
Sequence(3'UTR with 3 miR 142-5p binding sites) 195ugauaauaga
guagugcuuu cuacuuuaug gcuggagccu cgguggccau gcuucuugcc 60ccuugggcca
guagugcuuu cuacuuuaug uccccccagc cccucucccc uuccugcacc
120cguaccccca guagugcuuu cuacuuuaug guggucuuug aauaaagucu
gagugggcgg 180c 181196142RNAArtificial Sequence(3'UTR with miR
142-3p binding site, P3 insertion variant 2) 196ugauaauagg
cuggagccuc gguggccuag cuucuugccc cuugggccuc cauaaaguag 60gaaacacuac
auccccccag ccccuccucc ccuuccugca cccguacccc cguggucuuu
120gaauaaaguc ugagugggcg gc 142197142RNAArtificial Sequence(3'UTR
with miR 155-5p binding site variant 2) 197ugauaauagg cuggagccuc
gguggccuag cuucuugccc cuugggccuc cccccagccc 60cuccuccccu uccugcaccc
guacccccac cccuaucaca auuagcauua aguggucuuu 120gaauaaaguc
ugagugggcg gc 142198188RNAArtificial Sequence(3' UTR with 3 miR
155-5p binding sites variant 2) 198ugauaauaga ccccuaucac aauuagcauu
aagcuggagc cucgguggcc uagcuucuug 60ccccuugggc caccccuauc acaauuagca
uuaauccccc cagccccucc uccccuuccu 120gcacccguac ccccaccccu
aucacaauua gcauuaagug gucuuugaau aaagucugag 180ugggcggc
188199188RNAArtificial Sequence(3'UTR with 2 miR 155-5p binding
sites and 1 miR 142-3p binding site variant 2) 199ugauaauaga
ccccuaucac aauuagcauu aagcuggagc cucgguggcc uagcuucuug 60ccccuugggc
cuccauaaag uaggaaacac uacauccccc cagccccucc uccccuuccu
120gcacccguac ccccaccccu aucacaauua gcauuaagug gucuuugaau
aaagucugag 180ugggcggc 188
* * * * *
References