U.S. patent application number 12/915186 was filed with the patent office on 2012-05-03 for rnai modulation of tgf-beta and therapeutic uses thereof.
This patent application is currently assigned to ALNYLAM PHARMACEUTICALS, INC.. Invention is credited to Rainer Constien, Pamela Tan, Hans-Peter Vornlocher.
Application Number | 20120108646 12/915186 |
Document ID | / |
Family ID | 38522950 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120108646 |
Kind Code |
A1 |
Vornlocher; Hans-Peter ; et
al. |
May 3, 2012 |
RNAi Modulation Of TGF-BETA And Therapeutic Uses Thereof
Abstract
The present invention concerns methods of treatment using
transforming growth factor beta (TGF-beta) modulators. More
specifically, the invention concerns methods of treating disorders
associated with undesirable TGF-beta signaling, by administering
short interfering RNA which down-regulate the expression of
TGF-beta, and agents useful therein.
Inventors: |
Vornlocher; Hans-Peter;
(Bayreuth, DE) ; Tan; Pamela; (Kulmbach, DE)
; Constien; Rainer; (Kulmbach, DE) |
Assignee: |
ALNYLAM PHARMACEUTICALS,
INC.
Cambridge
MA
|
Family ID: |
38522950 |
Appl. No.: |
12/915186 |
Filed: |
October 29, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11724790 |
Mar 15, 2007 |
7846908 |
|
|
12915186 |
|
|
|
|
60782829 |
Mar 16, 2006 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/320.1; 435/325; 536/24.5 |
Current CPC
Class: |
C12N 2310/321 20130101;
A61P 1/16 20180101; C12N 15/1136 20130101; C12N 2310/321 20130101;
C12N 2310/14 20130101; A61P 9/00 20180101; A61P 43/00 20180101;
C12N 2310/3521 20130101 |
Class at
Publication: |
514/44.A ;
536/24.5; 435/320.1; 435/325 |
International
Class: |
C07H 21/02 20060101
C07H021/02; A61P 1/16 20060101 A61P001/16; A61K 31/7105 20060101
A61K031/7105; A61P 9/00 20060101 A61P009/00; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10 |
Claims
1. An isolated iRNA agent, comprising an antisense strand and a
sense strand substantially complementary to said antisense strand,
wherein the iRNA agent mediates the cleavage of a TGF-beta mRNA
within the target sequence of iRNA agent AL-DP-6857.
2. The iRNA agent of claim 1, wherein the target sequence is
nucleotides 1711 to 1733 of NM.sub.--000660.3.
3. The iRNA agent of claim 1, wherein the iRNA agent further
comprises a non-nucleotide moiety.
4. The iRNA of claim 1, wherein the iRNA agent is stabilized
against nucleolytic degradation.
5. The iRNA agent of claim 1, wherein the sense strand and/or the
antisense strand further comprises at least one 3'-overhang wherein
the 3'-overhang comprises from 1 to 6 nucleotides.
6. The iRNA agent of claim 1, further comprising a phosphorothioate
at the first internucleotide linkage at the 5' end of the sense
strand and/or the antisense strand.
7. The iRNA agent of claim 1, further comprising a phosphorothioate
at the first internucleotide linkage at the 3' end of the sense
strand and/or the antisense strand.
8. The iRNA agent of claim 1, further comprising a 2'-modified
nucleotide in the sense strand and/or the antisense strand.
9. The iRNA agent of claim 8, wherein the 2'-modified nucleotide
comprises a modification selected from the group consisting of:
2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl
(2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP),
2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and
2'-O--N-methylacetamido (2'-O-NMA).
10. The iRNA agent of claim 1, wherein the sense strand comprises
15 or more contiguous nucleotides of the nucleotide sequence of SEQ
ID NO:81.
11. The iRNA agent of claim 1, wherein the iRNA agent is
AL-DP-6857.
12. The iRNA agent of claim 1, wherein the antisense strand
comprises 15 or more contiguous nucleotides of the nucleotide
sequence of SEQ ID NO:82.
13. The iRNA agent of claim 1, wherein the sense strand comprises
SEQ ID NO:83.
14. The iRNA agent of claim 1, wherein the antisense strand
comprises SEQ ID NO:84.
15. The iRNA agent of claim 1, wherein the iRNA agent is
AL-DP-6270.
16. A vector encoding the iRNA agent of claim 1.
17. A cell comprising the iRNA agent of claim 1.
18. A method of making the iRNA agent of claim 1, the method
comprising the synthesis of the iRNA agent, wherein the sense and
antisense strands comprise at least one modification that
stabilizes the iRNA agent against nucleolytic degradation.
19. A pharmaceutical composition comprising the iRNA agent of claim
1 and a pharmaceutically acceptable carrier.
20. A method of treating a human diagnosed as having a disease or
disorder associated with undesired TGF-beta signaling, comprising
administering to a subject in need of such treatment a
therapeutically effective amount of the iRNA agent of claim 1.
21. The method of claim 20, wherein the human is diagnosed as
having idiopathic pulmonary fibrosis, diabetic nephropathy or
chronic liver disease.
22. A method of reducing the levels of a TGF-beta mRNA in a cell of
a subject, or of TGF-beta protein secreted by a cell of a subject,
comprising the step of administering the iRNA agent of claim 1 to
said subject.
23. The method of claim 22, wherein said iRNA agent mediates the
cleavage of a TGF-beta mRNA within the target sequence of iRNA
agent AL-DP-6857.
24. The method of claim 22, wherein said administration comprises
pulmonary administration.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/782,829, filed Mar. 16, 2006, and U.S. Ser. No.
11/724,790, filed Mar. 15, 2007, which are hereby incorporated by
reference in their entirety.
TECHNICAL FIELD
[0002] The present invention concerns methods of treatment using
transforming growth factor beta (TGF-beta) modulators. More
specifically, the invention concerns methods of treating disorders
associated with undesirable TGF-beta signaling, by administering
short interfering RNA which down-regulate the expression of
TGF-beta, and agents useful therein.
BACKGROUND
[0003] RNA interference or "RNAi" is a term initially coined by
Fire and co-workers to describe the observation that
double-stranded RNA (dsRNA) can block gene expression when it is
introduced into worms (Fire et al., Nature 391:806-811, 1998).
Short dsRNA directs gene-specific, post-transcriptional silencing
in many organisms, including vertebrates, and has provided a new
tool for studying gene function. RNAi has been suggested as a
method of developing a new class of therapeutic agents. However, to
date, these have remained mostly as suggestions with no demonstrate
proof that RNAi can be used therapeutically.
[0004] Transforming growth factor-beta (TGF-beta) isoforms (-1, -2,
& -3) are potent cytokines that act in autocrine and paracrine
fashion to effect a broad spectrum of biological processes,
including proliferation, differentiation, apoptosis, and
extracellular matrix production. TGF-beta exerts its biological
effects via binding to TGF-beta receptors (types I, II, & III).
The local over-expression of TGF-beta in the kidney, liver, or lung
mediates the pathophysiology observed in diabetic nephropathy,
chronic liver disease, and pulmonary fibrosis, respectively.
TGF-beta1 is up-regulated in patients with diabetic nephropathy
(for both type 1 or type 2 diabetes) and in animal models of the
disease. Antibodies to TGF-beta have been shown to prevent disease
in a mouse genetic model of type 2 diabetes (db/db mouse) and
antisense targeting a conserved sequence in TGF-beta RNA has been
shown to prevent disease in a streptozotocin-diabetic mouse
model.
[0005] About 100,000 diabetic patients per year are treated for
kidney disease in U.S. with health costs of $5.1 billion per year
in the U.S. Diabetic nephropathy is the leading cause of end-stage
renal disease in the industrialized world. Almost 40% of all new
patients with renal failure admitted to renal replacement programs
in the U.S have diabetic kidney disease. Albuminuria, proteinuria,
serum creatinine, as well as circulating TGF-beta1 plasma levels,
can be used as markers for efficacy determination in therapeutic
evaluation.
[0006] Liver disease affects all age groups and both genders. The
condition can be acute or chronic. The major causes of liver
diseases in the United States are viruses (hepatitis A, B, C) and
alcohol abuse. However, congenital, autoimmune and drug-induced
causes are also significant contributors to the origin of liver
diseases. Liver diseases disturb hepatic functions as a result of
destruction of functional liver tissue and development of fibrosis
(scarring), which blocks blood flow through the liver and causes
portal hypertension (pressure in the portal vein). Blood flow then
seeks an alternative route, leading to dilated swollen veins
(varices) in the esophagus that may hemorrhage. Liver disease and
portal hypertension also alter kidney function by causing retention
of salt and water (ascites), and can induce renal failure
(hepatorenal syndrome) and altered mental state (coma). The primary
cytokine involved in tissue scaring in chronic liver disease is
TGF-beta1. Recently it has been shown in a rat model of liver
fibrosis that blocking TGF-beta1 can lead to improvement in liver
histology. Further, in chronic Hepatitis C patients who have
responded to interferon alpha, decreased levels of TGF-beta1 are
associated with improvements in liver fibrosis.
[0007] TGF-beta1 has also been implicated in Idiopathic Pulmonary
Fibrosis. Similar to liver disease, the mechanisms of action of
TGF-beta1 in pulmonary fibrosis involves both induction and
inhibition of the degradation of extracellular matrix proteins,
leading to the formation of scar tissue. TGF-beta1 mediated cell
signaling, primarily at the local site of connective tissue, is
anabolic and leads to pulmonary fibrosis and angiogenesis, strongly
indicating that TGF-beta1 may be involved in the repair of tissue
injury caused by burns, trauma, or surgery. Pulmonary fibrosis,
also called Interstitial Lung Disease (ILD), is a broad category of
lung diseases that includes more than 130 disorders which are
characterized by scarring of the lungs. ILD accounts for 15% of the
cases seen by pulmonologists. Some of the interstitial lung
disorders include: Idiopathic pulmonary fibrosis, Hypersensitivity
pneumonitis, Sarcoidosis, Eosinophilic granuloma, Wegener's
granulomatosis, Idiopathic pulmonary hemosiderosis, and
Bronchiolitis obliterans. In ILD, scarring or fibrosis occurs as a
result of either an injury or an autoimmune process. Approximately
70% of ILD have no identifiable cause and are therefore termed
"idiopathic pulmonary fibrosis." Some of the known causes include
occupational and environmental exposure, dust (silica, hard metal
dusts), organic dust (bacteria, animal proteins), gases and fumes,
drugs, poisons, chemotherapy medications, radiation therapy,
infections, connective tissue disease, systemic lupus
erythematosus, and rheumatoid arthritis. In its severest form, ILD
can lead to death, which is often caused by respiratory failure due
to hypoxemic, right-heart failure, heart attack, stroke, blood clot
(embolism) in the lungs, or lung infection brought on by the
disease.
[0008] The use of small interfering nucleic acid molecules
targeting transforming growth factor beta (TGF-beta) genes provides
a class of novel therapeutic agents that can be used in the
treatment of various diseases and conditions associated with
undesired TGF-beta signaling, including diabetic nephropathy,
chronic liver disease, pulmonary fibrosis, hematopoietic
reconstitution, and any other inflammatory, respiratory,
autoimmune, and/or proliferative disease, condition, or trait that
responds to the level of TGF-beta in a cell, subject or organism,
and particularly idiopathic pulmonary fibrosis.
SUMMARY
[0009] The present invention is based on the in vitro demonstration
that TGF-beta expression can be inhibited by iRNA agents, and the
identification of potent iRNA agents from the TGF-beta gene that
can reduce RNA levels and protein levels of TGF-beta in cells, and
particularly in an organism. Based on these findings, the present
invention provides specific compositions and methods that are
useful in reducing TGF-beta mRNA levels and TGF-beta protein levels
in cells in vitro as well as in a subject in vivo, e.g., a mammal,
such as a human. These compositions are particularly useful in the
manufacturing of pharmaceutical compositions for the treatment of,
and ultimately in methods to treat, subjects having or at risk of
developing a disease or disorder associated with undesired TGF-beta
signaling such as, for example, idiopathic pulmonary fibrosis.
[0010] The present invention specifically provides methods of
reducing the levels of a TGF-beta mRNA in a cell of a subject, or
of TGF-beta protein secreted by a cell of a subject, comprising the
step of administering an iRNA agent to said subject, wherein the
iRNA agent comprises an antisense strand consisting of 15 to 30
nucleotides and having at least 15 or more contiguous nucleotides
complementary to a mammalian TGF-beta mRNA, and a sense strand
consisting of 15 to 30 nucleotides and having at least 15 or more
contiguous nucleotides complementary to said antisense strand. Said
iRNA agent may mediate the cleavage of a TGF-beta mRNA within the
target sequence of an iRNA agent selected from the group of:
AL-DP-6837 to AL-DP-6864 and AD-14419 to AD-14638. In one
embodiment, said iRNA agent comprises 15 or more contiguous
nucleotides from an iRNA agent selected from the group of:
AL-DP-6837 to AL-DP-6864, AL-DP-6140 to AL-DP-6151, AL-DP-6262 to
AL-DP-6277 and AD-14419 to AD-14638. In a further embodiment, said
iRNA agent is one of the iRNA agents selected from the group of:
AL-DP-6837 to AL-DP-6864, AL-DP-6140 to AL-DP-6151, AL-DP-6262 to
AL-DP-6277 and AD-14419 to AD-14638. In further embodiments, the
iRNA agents mediates the cleavage of a TGF-beta mRNA within the
target sequence of an iRNA agent, comprises 15 or more contiguous
nucleotides from an iRNA agent, or is an iRNA agent, selected from
one of the following groups: the 20%-group, the 30%-group, the
40%-group, the 50%-group, the 60%-group, the 70%-group, the
80%-group, or the 90%-group, as defined below. Said iRNA agent may
be administered to a subject, wherein administration may comprise
pulmonary administration. As a result of the inventive method, the
level of TGF-beta protein and/or TGF-beta mRNA may be reduced by at
least 20%.
[0011] In another aspect, the invention provides isolated iRNA
agents, comprising an antisense strand consisting of 15 to 30
nucleotides and having at least 15 or more contiguous nucleotides
complementary to a mammalian TGF-beta mRNA, and a sense strand
consisting of 15 to 30 nucleotides and having at least 15 or more
contiguous nucleotides complementary to said antisense strand. In
one embodiment, said iRNA agent mediates the cleavage of a TGF-beta
mRNA within the target sequence of an iRNA agent selected from one
of the following groups: the group of AL-DP-6837 to AL-DP-6864 and
AD-14419 to AD-14638, the 20%-group, the 30%-group, the 40%-group,
the 50%-group, the 60%-group, the 70%-group, the 80%-group, or the
90%-group, as defined below. In a further embodiment, said iRNA
agent comprises 15 or more contiguous nucleotides from an iRNA
agent selected from one of the following groups: the group of
AL-DP-6837 to AL-DP-6864, AL-DP-6140 to AL-DP-6151, AL-DP-6262 to
AL-DP-6277 and AD-14419 to AD-14638 the 20%-group, the 30%-group,
the 40%-group, the 50%-group, the 60%-group, the 70%-group, the
80%-group, or the 90%-group, as defined below. More specifically,
said iRNA agent may be an iRNA agent selected from one of the
following groups of: AL-DP-6837 to AL-DP-6864, AL-DP-6140 to
AL-DP-6151, AL-DP-6262 to AL-DP-6277 and AD-14419 to AD-14638; the
20%-group, the 30%-group, the 40%-group, the 50%-group, the
60%-group, the 70%-group, the 80%-group, or the 90%-group, as
defined below. The iRNA agent may further comprise a non-nucleotide
moiety. Furthermore, the sense and/or antisense strand may be
stabilized against nucleolytic degradation. The iRNA agent may
further comprise at least one 3'-overhang, wherein said 3'-overhang
comprises from 1 to 6 nucleotides. Also, the iRNA agent may
comprise a phosphorothioate at the first internucleotide linkage at
the 5' end of the antisense and/or sense sequence, and optionally a
further phosphorothioate at the first internucleotide linkage at
the 3' end of the antisense and/or sense sequence. The iRNA agent
may comprise a 2'-modified nucleotide, preferably selected from the
group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl,
2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and
2'-O--N-methylacetamido (2'-O-NMA).
[0012] In another aspect, the instant invention features a method
of reducing the levels of a TGF-beta mRNA in a cell, or of TGF-beta
protein secreted by a cell, comprising contacting the cell with an
iRNA agent of the invention.
[0013] In yet another aspect, the instant invention provides a
vector encoding an iRNA agent of the invention.
[0014] In yet another aspect, the instant invention provides a cell
comprising an iRNA agent or a vector of the invention.
[0015] In yet another aspect, the a method of making an iRNA agent
of the invention is provided, the method comprising the synthesis
of the iRNA agent, wherein the sense and antisense strands comprise
at least one modification that stabilizes the iRNA agent against
nucleolytic degradation.
[0016] In yet another aspect, the instant invention provides a
pharmaceutical composition comprising an iRNA agent of the
invention and a pharmaceutically acceptable carrier.
[0017] In yet another aspect, the instant invention provides a
method of treating a human diagnosed as having or at risk for
developing a disease or disorder associated with undesired TGF-beta
signaling, comprising administering to a subject in need of such
treatment a therapeutically effective amount of an iRNA agent of
the invention. In one embodiment, the human is diagnosed as having
or at risk for having idiopathic pulmonary fibrosis, diabetic
nephropathy or chronic liver disease.
[0018] Table 1 provides exemplary iRNA agents of the invention.
TABLE-US-00001 TABLE 1 Oligonucleotide sequences of siRNAs specific
for TGF-beta Position of target SEQ SEQ sequence in Duplex ID
antisense ID NM_000660.3 identifier sense strand sequence.sup.1 NO:
strand sequence.sup.1 NO: 1186-1208 AL-DP-6837
ggucacccgcgugcuaaugTT 1 cauuagcacgcgggugaccTT 2 1186-1208
AL-DP-6140 ggumcmacmcmcmgcmgumgcmumaaumgTT 3
cmauumagcmacgcgggugaccTT 4 1184-1206 AL-DP-6838
gaggucacccgcgugcuaaTT 5 uuagcacgcgggugaccucTT 6 1184-1206
AL-DP-6141 gaggumcmacmcmcmgcmgumgcmumaaTT 7 uumagcmacgcgggugaccucTT
8 1112-1134 AL-DP-6839 agcacccgcgaccggguggTT 9
ccacccggucgcgggugcuTT 10 1112-1134 AL-DP-6142
agcmacmcmcmgcmgacmcmgggumggTT 11 ccmacccggucgcgggugcuTT 12
1797-1819 AL-DP-6840 uccacgagcccaagggcuaTT 13 uagcccuugggcucguggaTT
14 1797-1819 AL-DP-6143 umcmcmacmgagcmcmcmaagggcmumaTT 15
umagcccuugggcucguggaTT 16 444-466 AL-DP-6841 auuccggaccagcccucggTT
17 ccgagggcugguccggaauTT 18 444-466 AL-DP-6144
aumumcmcmggacmcmagcmcmcmumcmggTT 19 ccgagggcugguccggaauTT 20
1185-1207 AL-DP-6842 aggucacccgcgugcuaauTT 21 auuagcacgcgggugaccuTT
22 1185-1207 AL-DP-6145 aggumcmacmcmcmgcmgumgcmumaaumTT 23
auumagcmacgcgggugaccuTT 24 445-467 AL-DP-6843 uuccggaccagcccucgggTT
25 cccgagggcugguccggaaTT 26 445-467 AL-DP-6146
umumcmcmggacmcmagcmcmcmumcmgggTT 27 cccgagggcugguccggaaTT 28
1104-1126 AL-DP-6844 uguacaacagcacccgcgaTT 29 ucgcgggugcuguuguacaTT
30 1104-1126 AL-DP-6147 umgumacmaacmagcmacmcmcmgcmgaTT 31
ucgcgggugcuguugumacmaTT 32 1189-1211 AL-DP-6845
cacccgcgugcuaauggugTT 33 caccauuagcacgcgggugTT 34 1189-1211
AL-DP-6148 cmacmcmcmgcmgcmgcmumaaumggumgTT 35
cmaccmauumagcmacgcgggugTT 36 446-468 AL-DP-6846
uccggaccagcccucgggaTT 37 ucccgagggcugguccggaTT 38 446-468
AL-DP-6149 umcmcmggacmcmagcmcmcmumcmgggaTT 39 ucccgagggcugguccggaTT
40 1787-1809 AL-DP-6847 uggaaguggauccacgagcTT 41
gcucguggauccacuuccaTT 42 1787-1809 AL-DP-6150
umggaagumggaumcmcmacmgagcmTT 43 gcucguggauccmacuuccmaTT 44
1806-1828 AL-DP-6848 ccaagggcuaccaugccaaTT 45 uuggcaugguagcccuuggTT
46 1806-1828 AL-DP-6151 cmcmaagggcmumacmcmaumgcmcmaaTT 47
uuggcmauggumagcccuuggTT 48 1187-1209 AL-DP-6849
gucacccgcgugcuaauggTT 49 ccauuagcacgcgggugacTT 50 1187-1209
AL-DP-6262 gumcmacmcmcmgcmgumgcmumaaumggTT 51
ccmauumagcmacgcgggugacTT 52 1106-1128 AL-DP-6850
uacaacagcacccgcgaccTT 53 ggucgcgggugcuguuguaTT 54 1106-1128
AL-DP-6263 umacmaacmagcmacmcmcmgcmgacmcmTT 55
ggucgcgggugcuguugumaTT 56 1804-1826 AL-DP-6851
gcccaagggcuaccaugccTT 57 ggcaugguagcccuugggcTT 58 1804-1826
AL-DP-6264 gcmcmcmaagggcmumacmcmaumgcmcmTT 59
ggcmauggumagcccuugggcTT 60 1790-1812 AL-DP-6852
aaguggauccacgagcccaTT 61 ugggcucguggauccacuuTT 62 1790-1812
AL-DP-6265 aagumggaumcmcmacmgagcmcmcmaTT 63 ugggcucguggauccmacuuTT
64 239-261 AL-DP-6853 gggaggagggacgagcuggTT 65
ccagcucgucccuccucccTT 66 239-261 AL-DP-6266
gggaggagggacmgagcmumggTT 67 ccmagcucgucccuccucccTT 68 1107-1129
AL-DP-6854 acaacagcacccgcgaccgTT 69 cggucgcgggugcuguuguTT 70
1107-1129 AL-DP-6267 acmaacmagcmacmcmcmgcmgacmcmgTT 71
cggucgcgggugcuguuguTT 72 1807-1829 AL-DP-6855 caagggcuaccaugccaacTT
73 guuggcaugguagcccuugTT 74 1807-1829 AL-DP-6268
cmaagggcmumacmcmaumgcmcmaacmTT 75 guuggcmauggumagcccuugTT 76
447-469 AL-DP-6356 ccggaccagcccucgggagTT 77 cucccgagggcugguccggTT
78 447-469 AL-DP-6269 cmcmggacmcmagcmcmcmumcmgggagTT 79
cucccgagggcugguccggTT 80 1711-1733 AL-DP-6857 caacuauugcuucagcuccTT
81 ggagcugaagcaauaguugTT 82 1711-1733 AL-DP-6270
cmaacmumaumumgcmumumcmagcmumcmcmTT 83 ggagcugaagcmaaumaguugTT 84
l748-1770 AL-DP-6858 gugcggcagcuguacauugTT 85 caauguacagcugccgcacTT
86 1748-1770 AL-DP-6271 gumgcmggcmagcmumgumacmaumumgTT 87
cmaaugumacmagcugccgcmacTT 88 1788-1810 AL-DP-6859
ggaaguggauccacgagccTT 89 ggcucguggauccacuuccTT 90 1788-1810
AL-DP-6272 ggaagumggaumcmcmacmgagcmcmTT 91 ggcucguggauccmacuuccTT
92 1789-1811 AL-DP-6860 gaaguggauccacgagcccTT 93
gggcucguggauccacuucTT 94 1789-1811 AL-DP-6273
gaagumggaumcmcmacmgagcmcmcmTT 95 gggcucguggauccmacuucTT 96
1798-1820 Al-DP-6861 ccacgagcccaagggcuacTT 97 guagcccuugggcucguggTT
98 1798-1820 AL-DP-6274 cmcmacmgagcmcmcmaagggcmumacmTT 99
gumagcccuugggcucguggTT 100 1802-1824 AL-DP-6862
gagcccaagggcuaccaugTT 101 caugguagcccuugggcucTT 102 1802-1824
AL-DP-6275 gagcmcmcmaagggcmumacmcmaumgTT 103
cmauggumagcccuugggcucTT 104 1803-1825 AL-DP-6863
agcccaagggcuaccaugcTT 105 gcaugguagcccuugggcuTT 106 1803-1825
AL-DP-6276 agcmcmcmaagggcmumacmcmaumgcmTT 107
gcmauggumagcccuugggcuTT 108 1926-1948 AL-DP-6864
cgugcugcgugccgcaggcTT 109 gccugcggcacgcagcacgTT 110 1926-1948
AL-DP-6277 cmgumgcmumgcmgumgcmcmgcmaggcmTT 111
gccugcggcmacgcmagcmacgTT 112 .sup.1a, g, c, u: ribonucleotide
5'-monophosphates (except where in 5'-most position, see below);
cm: 2'-O-methyl-cytidine-5'-monophosphate (except where in 5'-most
position, see below); um: 2'-O-methyl-uridine-5'-monophosphate
(except where in 5'-most position, see below); T: deoxythymidine
5'-monophosphate; T: deoxythymidine 5'-monothiophosphate; all
sequences given 5' .fwdarw. 3'; due to synthesis procedures, the
5'-most nucleic acid is a nucleoside, i.e. does not bear a
5'-monophosphate group
[0019] The iRNA agents of the invention can either contain only
naturally occurring ribonucleotide subunits, or can be synthesized
so as to contain one or more modifications to the base, the sugar
or the phosphate group of one or more of the ribonucleotide
5'-monophosphate subunits that is included in the agent. The iRNA
agent can be further modified so as to be attached to a ligand that
is selected to improve stability, distribution or cellular uptake
of the agent, e.g. cholesterol. The iRNA agents can further be in
isolated form or can be part of a pharmaceutical composition used
for the methods described herein, particularly as a pharmaceutical
composition formulated for delivery to the lung or formulated for
parenteral administration. The pharmaceutical compositions can
contain one or more iRNA agents, and in some embodiments, will
contain two or more iRNA agents, each one directed to a different
segment of a TGF-beta gene or a different TGF-beta gene.
[0020] In a preferred embodiment, an iRNA agent of the invention
reduces the level of TGF-beta protein secreted by, and/or TGF-beta
mRNA found inside, a cell by at least 20% or more. Thus, the iRNA
agent may preferably be chosen from the group of AD-14501,
AD-14503, AD-14507, AD-14554, AD-14594, AD-14597, AD-14633,
AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848, AL-DP-6862,
AD-14419, AD-14420, AD-14421, AD-14422, AD-14423, AD-14428,
AD-14430, AD-14434, AD-14438, AD-14449, AD-14453, AD-14459,
AD-14460, AD-14464, AD-14469, AD-14470, AD-14473, AD-14474,
AD-14476, AD-14486, AD-14490, AD-14495, AD-14496, AD-14497,
AD-14509, AD-14515, AD-14522, AD-14526, AD-14540, AD-14552,
AD-14555, AD-14557, AD-14565, AD-14567, AD-14568, AD-14582,
AD-14588, AD-14590, AD-14592, AD-14598, AD-14600, AD-14601,
AD-14603, AD-14604, AD-14608, AD-14610, AD-14612, AD-14614,
AD-14622, AD-14634, AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838,
AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837,
AD-14425, AD-14426, AD-14427, AD-14429, AD-14431, AD-14436,
AD-14437, AD-14441, AD-14448, AD-14458, AD-14463, AD-14467,
AD-14477, AD-14482, AD-14483, AD-14491, AD-14502, AD-14505,
AD-14508, AD-14512, AD-14513, AD-14519, AD-14524, AD-14537,
AD-14546, AD-14548, AD-14549, AD-14559, AD-14563, AD-14564,
AD-14569, AD-14578, AD-14579, AD-14587, AD-14595, AD-14599,
AD-14607, AD-14609, AD-14629, AD-14631, AD-14637, AL-DP-6840,
AL-DP-6860, AL-DP-6861, AL-DP-6849, AL-DP-6272, AL-DP-6271,
AL-DP-6847, AL-DP-6863, AD-14447, AD-14451, AD-14471, AD-14484,
AD-14487, AD-14498, AD-14523, AD-14527, AD-14529, AD-14539,
AD-14541, AD-14558, AD-14561, AD-14573, AD-14589, AD-14596,
AD-14605, AD-14615, AD-14626, AD-14630, AL-DP-6846, AL-DP-6853,
AL-DP-6856, AL-DP-6864, AL-DP-6844, AL-DP-6143, AD-14432, AD-14480,
AD-14485, AD-14488, AD-14499, AD-14504, AD-14516, AD-14518,
AD-14520, AD-14547, AD-14572, AD-14577, AD-14580, AD-14583,
AD-14584, AD-14618, AD-14621, AD-14625, AL-DP-6276, AD-14435,
AD-14445, AD-14450, AD-14456, AD-14466, AD-14475, AD-14489,
AD-14492, AD-14511, AD-14535, AD-14536, AD-14538, AD-14553,
AD-14560, AD-14562, AD-14576, AD-14581, AD-14586, AD-14627,
AL-DP-6150 AL-DP-6850, AD-14433, AD-14472, AD-14478, AD-14493,
AD-14510, AD-14544, AD-14566, AD-14570, AD-14575, AD-14593,
AD-14613, AL-DP-6269, AL-DP-6270, AL-DP-6147, AL-DP-6262,
AL-DP-6841, AL-DP-6274, AL-DP-6843, AL-DP-6145, AD-14424, AD-14442,
AD-14444, AD-14446, AD-14454, AD-14525, AD-14543, AD-14571,
AD-14606, AD-14611, AD-14616, AD-14617, AD-14619, and AD-14638 (the
"20% group").
[0021] More preferably, an iRNA agent of the invention reduces the
level of TGF-beta protein secreted by, and/or TGF-beta mRNA found
inside, a cell by at least 30% or more. Thus, the iRNA agent may
preferably be chosen from the group of AD-14501, AD-14503,
AD-14507, AD-14554, AD-14594, AD-14597, AD-14633, AL-DP-6858,
AL-DP-6857, AL-DP-6859, AL-DP-6848, AL-DP-6862, AD-14419, AD-14420,
AD-14421, AD-14422, AD-14423, AD-14428, AD-14430, AD-14434,
AD-14438, AD-14449, AD-14453, AD-14459, AD-14460, AD-14464,
AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,
AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,
AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557,
AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,
AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604,
AD-14608, AD-14610, AD-14612, AD-14614, AD-14622, AD-14634,
AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838, AL-DP-6855,
AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425, AD-14426,
AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,
AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482,
AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512,
AD-14513, AD-14519, AD-14524, AD-14537, AD-14546, AD-14548,
AD-14549, AD-14559, AD-14563, AD-14564, AD-14569, AD-14578,
AD-14579, AD-14587, AD-14595, AD-14599, AD-14607, AD-14609,
AD-14629, AD-14631, AD-14637, AL-DP-6840, AL-DP-6860, AL-DP-6861,
AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847, AL-DP-6863,
AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,
AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558,
AD-14561, AD-14573, AD-14589, AD-14596, AD-14605, AD-14615,
AD-14626, AD-14630, AL-DP-6846, AL-DP-6853, AL-DP-6856, AL-DP-6864,
AL-DP-6844, AL-DP-6143, AD-14432, AD-14480, AD-14485, AD-14488,
AD-14499, AD-14504, AD-14516, AD-14518, AD-14520, AD-14547,
AD-14572, AD-14577, AD-14580, AD-14583, AD-14584, AD-14618,
AD-14621, AD-14625, AL-DP-6276, AD-14435, AD-14445, AD-14450,
AD-14456, AD-14466, AD-14475, AD-14489, AD-14492, AD-14511,
AD-14535, AD-14536, AD-14538, AD-14553, AD-14560, AD-14562,
AD-14576, AD-14581, AD-14586, AD-14627, AL-DP-6150 AL-DP-6850,
AD-14433, AD-14472, AD-14478, AD-14493, AD-14510, AD-14544,
AD-14566, AD-14570, AD-14575, AD-14593, and AD-14613 (the "30%
group").
[0022] More preferably, an iRNA agent of the invention reduces the
level of TGF-beta protein secreted by, and/or TGF-beta mRNA found
inside, a cell by at least 40% or more. Thus, the iRNA agent may
preferably be chosen from the group of AD-14501, AD-14503,
AD-14507, AD-14554, AD-14594, AD-14597, AD-14633, AL-DP-6858,
AL-DP-6857, AL-DP-6859, AL-DP-6848, AL-DP-6862, AD-14419, AD-14420,
AD-14421, AD-14422, AD-14423, AD-14428, AD-14430, AD-14434,
AD-14438, AD-14449, AD-14453, AD-14459, AD-14460, AD-14464,
AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,
AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,
AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557,
AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,
AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604,
AD-14608, AD-14610, AD-14612, AD-14614, AD-14622, AD-14634,
AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838, AL-DP-6855,
AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425, AD-14426,
AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,
AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482,
AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512,
AD-14513, AD-14519, AD-14524, AD-14537, AD-14546, AD-14548,
AD-14549, AD-14559, AD-14563, AD-14564, AD-14569, AD-14578,
AD-14579, AD-14587, AD-14595, AD-14599, AD-14607, AD-14609,
AD-14629, AD-14631, AD-14637, AL-DP-6840, AL-DP-6860, AL-DP-6861,
AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847, AL-DP-6863,
AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,
AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558,
AD-14561, AD-14573, AD-14589, AD-14596, AD-14605, AD-14615,
AD-14626, AD-14630, AL-DP-6846, AL-DP-6853, AL-DP-6856, AL-DP-6864,
AL-DP-6844, AL-DP-6143, AD-14432, AD-14480, AD-14485, AD-14488,
AD-14499, AD-14504, AD-14516, AD-14518, AD-14520, AD-14547,
AD-14572, AD-14577, AD-14580, AD-14583, AD-14584, AD-14618,
AD-14621, AD-14625, AL-DP-6276, AD-14435, AD-14445, AD-14450,
AD-14456, AD-14466, AD-14475, AD-14489, AD-14492, AD-14511,
AD-14535, AD-14536, AD-14538, AD-14553, AD-14560, AD-14562,
AD-14576, AD-14581, AD-14586, and AD-14627 (the "40% group").
[0023] More preferably, an iRNA agent of the invention reduces the
level of TGF-beta protein secreted by, and/or TGF-beta mRNA found
inside, a cell by at least 50% or more. Thus, the iRNA agent may
preferably be chosen from the group of AD-14501, AD-14503,
AD-14507, AD-14554, AD-14594, AD-14597, AD-14633, AL-DP-6858,
AL-DP-6857, AL-DP-6859, AL-DP-6848, AL-DP-6862, AD-14419, AD-14420,
AD-14421, AD-14422, AD-14423, AD-14428, AD-14430, AD-14434,
AD-14438, AD-14449, AD-14453, AD-14459, AD-14460, AD-14464,
AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,
AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,
AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557,
AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,
AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604,
AD-14608, AD-14610, AD-14612, AD-14614, AD-14622, AD-14634,
AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838, AL-DP-6855,
AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425, AD-14426,
AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,
AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482,
AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512,
AD-14513, AD-14519, AD-14524, AD-14537, AD-14546, AD-14548,
AD-14549, AD-14559, AD-14563, AD-14564, AD-14569, AD-14578,
AD-14579, AD-14587, AD-14595, AD-14599, AD-14607, AD-14609,
AD-14629, AD-14631, AD-14637, AL-DP-6840, AL-DP-6860, AL-DP-6861,
AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847, AL-DP-6863,
AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,
AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558,
AD-14561, AD-14573, AD-14589, AD-14596, AD-14605, AD-14615,
AD-14626, AD-14630, AL-DP-6846, AL-DP-6853, AL-DP-6856, AL-DP-6864,
AL-DP-6844, AL-DP-6143, AD-14432, AD-14480, AD-14485, AD-14488,
AD-14499, AD-14504, AD-14516, AD-14518, AD-14520, AD-14547,
AD-14572, AD-14577, AD-14580, AD-14583, AD-14584, AD-14618,
AD-14621, and AD-14625 (the "50% group").
[0024] Yet more preferably, an iRNA agent of the invention reduces
the level of TGF-beta protein secreted by, and/or TGF-beta mRNA
found inside, a cell by at least 60% or more. Thus, the iRNA agent
may preferably be chosen from the group of AD-14501, AD-14503,
AD-14507, AD-14554, AD-14594, AD-14597, AD-14633, AL-DP-6858,
AL-DP-6857, AL-DP-6859, AL-DP-6848, AL-DP-6862, AD-14419, AD-14420,
AD-14421, AD-14422, AD-14423, AD-14428, AD-14430, AD-14434,
AD-14438, AD-14449, AD-14453, AD-14459, AD-14460, AD-14464,
AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,
AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,
AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557,
AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,
AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604,
AD-14608, AD-14610, AD-14612, AD-14614, AD-14622, AD-14634,
AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838, AL-DP-6855,
AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425, AD-14426,
AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,
AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482,
AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512,
AD-14513, AD-14519, AD-14524, AD-14537, AD-14546, AD-14548,
AD-14549, AD-14559, AD-14563, AD-14564, AD-14569, AD-14578,
AD-14579, AD-14587, AD-14595, AD-14599, AD-14607, AD-14609,
AD-14629, AD-14631, AD-14637, AL-DP-6840, AL-DP-6860, AL-DP-6861,
AL-DP-6849, AL-DP-6272, AL-DP-6271, AL-DP-6847, AL-DP-6863,
AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,
AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558,
AD-14561, AD-14573, AD-14589, AD-14596, AD-14605, AD-14615,
AD-14626, and AD-14630 (the "60% group").
[0025] Yet more preferably, an iRNA agent of the invention reduces
the level of TGF-beta protein secreted by, and/or TGF-beta mRNA
found inside, a cell by at least 70% or more. Thus, the iRNA agent
may preferably be chosen from the group of AD-14501, AD-14503,
AD-14507, AD-14554, AD-14594, AD-14597, AD-14633, AL-DP-6858,
AL-DP-6857, AL-DP-6859, AL-DP-6848, AL-DP-6862, AD-14419, AD-14420,
AD-14421, AD-14422, AD-14423, AD-14428, AD-14430, AD-14434,
AD-14438, AD-14449, AD-14453, AD-14459, AD-14460, AD-14464,
AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,
AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,
AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557,
AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,
AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604,
AD-14608, AD-14610, AD-14612, AD-14614, AD-14622, AD-14634,
AD-14635, AL-DP-6845, AL-DP-6852, AL-DP-6838, AL-DP-6855,
AL-DP-6264, AL-DP-6851, AL-DP-6842, AL-DP-6837, AD-14425, AD-14426,
AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,
AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482,
AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512,
AD-14513, AD-14519, AD-14524, AD-14537, AD-14546, AD-14548,
AD-14549, AD-14559, AD-14563, AD-14564, AD-14569, AD-14578,
AD-14579, AD-14587, AD-14595, AD-14599, AD-14607, AD-14609,
AD-14629, AD-14631, and AD-14637 (the "70% group").
[0026] Yet more preferably, an iRNA agent of the invention reduces
the level of TGF-beta protein secreted by, and/or TGF-beta mRNA
found inside, a cell by at least 80% or more. Thus, the iRNA agent
may preferably be chosen from the group of AD-14501, AD-14503,
AD-14507, AD-14554, AD-14594, AD-14597, AD-14633, AL-DP-6858,
AL-DP-6857, AL-DP-6859, AL-DP-6848, AL-DP-6862, AD-14419, AD-14420,
AD-14421, AD-14422, AD-14423, AD-14428, AD-14430, AD-14434,
AD-14438, AD-14449, AD-14453, AD-14459, AD-14460, AD-14464,
AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,
AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,
AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557,
AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,
AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604,
AD-14608, AD-14610, AD-14612, AD-14614, AD-14622, AD-14634, and
AD-14635 (the "80% group").
[0027] Yet more preferably, an iRNA agent of the invention reduces
the level of TGF-beta protein secreted by, and/or TGF-beta mRNA
found inside, a cell by at least 90% or more. Thus, the iRNA agent
may preferably be chosen from the group of AD-14501, AD-14503,
AD-14507, AD-14554, AD-14594, AD-14597, and AD-14633 (the "90%
group").
[0028] The present invention provides methods for reducing the
level of TGF-beta protein and TGF-beta mRNA in a cell. Such methods
optionally comprise the step of administering one of the iRNA
agents of the present invention to a subject as further described
below. The present methods utilize the cellular mechanisms involved
in RNA interference to selectively degrade the TGF-beta mRNA in a
cell and are comprised of the step of contacting a cell with one of
the TGF-beta iRNA agents of the present invention. Such methods can
be performed directly on a cell or can be performed on a mammalian
subject by administering to a subject one of the iRNA
agents/pharmaceutical compositions of the present invention.
Reduction of TGF-beta mRNA in a cell results in a reduction in the
amount of TGF-beta protein produced, and in an organism (as shown
in the Examples).
[0029] The methods and compositions of the invention, e.g., the
methods and iRNA agent compositions can be used with any dosage
and/or formulation described herein, as well as with any route of
administration described herein.
[0030] In another aspect, the invention features a method for
treating or preventing a disease or condition associated with
undesired TGF-beta signaling in a subject. The method can include
administering to the subject a composition of the invention under
conditions suitable for the treatment or prevention of the disease
or condition associated with undesired TGF-beta signaling in the
subject, alone or in conjunction with one or more other therapeutic
compounds.
[0031] In one embodiment, the iRNA agent is administered at or near
the site of undesired TGF-beta signaling, e.g., direct injection at
the site, or by a catheter or other placement device (e.g., an
implant including a porous, non-porous, or gelatinous material). In
another embodiment the iRNA agent is administered via pulmonary
delivery, e.g. by inhalation, which may be nasal and/or oral.
[0032] In one embodiment, an iRNA agent is administered repeatedly.
Administration of an iRNA agent can be carried out over a range of
time periods. It can be administered hourly, daily, once every few
days, weekly, or monthly. The timing of administration can vary
from patient to patient, depending upon such factors as the
severity of a patient's symptoms. For example, an effective dose of
an iRNA agent can be administered to a patient once a month for an
indefinite period of time, or until the patient no longer requires
therapy. In addition, sustained release compositions containing an
iRNA agent can be used to maintain a relatively constant dosage in
the area of the target TGF-beta nucleotide sequences.
[0033] In another embodiment, an iRNA agent is delivered at a
dosage on the order of about 0.00001 mg to about 3 mg per kg body
weight of the subject to be treated, or preferably about
0.0001-0.001 mg per kg body weight, about 0.03-3.0 mg per kg body
weight, about 0.1-3.0 mg per kg body weight or about 0.3-3.0 mg per
kg body weight.
[0034] In another embodiment, an iRNA agent is administered
prophylactically such as to prevent or slow the onset of a disease
or disorder or condition associated with undesired TGF-beta
signaling. For example, an iRNA can be administered to a patient
who is susceptible to or otherwise at risk for such a disease or
disorder.
[0035] In another aspect, a method of inhibiting TGF-beta
expression is provided. One such method includes administering an
effective amount of an iRNA agent including sense and antisense
sequences capable of forming an RNA duplex. The sense sequence of
the iRNA agent can include a nucleotide sequence substantially
identical to a target sequence of about 19 to 23 nucleotides of
TGF-beta mRNA, and the antisense sequence can include a nucleotide
sequence complementary to a target sequence of about 19-23
nucleotides of TGF-beta mRNA.
[0036] In one aspect, methods of treating any disease or disorder
associated with undesired TGF-beta signaling are provided.
[0037] In another aspect, a method of treating idiopathic pulmonary
fibrosis is provided. One such method includes administering a
therapeutically effective amount of an iRNA agent that includes
sense and antisense sequences capable of forming an RNA duplex. The
sense sequence can include a nucleotide sequence substantially
identical to a target sequence of about 19 to 23 nucleotides of
TGF-beta mRNA. The antisense sequence can include a nucleotide
sequence complementary to a target sequence of about 19 to 23
nucleotides of TGF-beta mRNA.
[0038] In one embodiment, a human has been diagnosed with a disease
or disorder associated with undesired TGF-beta signaling. In one
embodiment, said disease or disorder is Interstitial Lung Disease
(ILD). In a preferred embodiment, the human has been diagnosed with
one of: Idiopathic pulmonary fibrosis, Hypersensitivity
pneumonitis, Sarcoidosis, Eosinophilic granuloma, Wegener's
granulomatosis, Idiopathic pulmonary hemosiderosis, and
Bronchiolitis obliterans. In a particularly preferred embodiment,
the human has been diagnosed with idiopathic pulmonary
fibrosis.
[0039] In another aspect, the invention features a kit containing
an iRNA agent of the invention. The iRNA agent of the kit can be
chemically modified and can be useful for modulating the expression
of a TGF-beta target gene in a cell, tissue or organism. In one
embodiment, the kit contains more than one iRNA agent of the
invention.
[0040] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects, and
advantages of the invention will be apparent from this description
and from the claims. This application incorporates all cited
references, patents, and patent applications by references in their
entirety for all purposes.
DETAILED DESCRIPTION
[0041] For ease of exposition the term "nucleotide" or
"ribonucleotide" is sometimes used herein in reference to one or
more monomeric subunits of an RNA agent. It will be understood that
the usage of the term "ribonucleotide" or "nucleotide" herein can,
in the case of a modified RNA or nucleotide surrogate, also refer
to a modified nucleotide, or surrogate replacement moiety, as
further described below, at one or more positions.
[0042] An "RNA agent" as used herein, is an unmodified RNA,
modified RNA, or nucleoside surrogate, all of which are described
herein or are well known in the RNA synthetic art. While numerous
modified RNAs and nucleoside surrogates are described, preferred
examples include those which have greater resistance to nuclease
degradation than do unmodified RNAs. Examples include those that
have a 2' sugar modification, a modification in a single strand
overhang, such as a 3' single strand overhang, or, particularly if
single stranded, a 5'-modification which includes one or more
phosphate groups or one or more analogs of a phosphate group.
[0043] An "iRNA agent" (abbreviation for "interfering RNA agent")
as used herein, is an RNA agent, which can down-regulate the
expression of a target gene, e.g., a TGF-beta gene, and preferably
a human TGF-beta gene. While not wishing to be bound by theory, an
iRNA agent may act by one or more of a number of mechanisms,
including post-transcriptional cleavage of a target mRNA sometimes
referred to in the art as RNA interference or RNAi, or
pre-transcriptional or pre-translational mechanisms. An iRNA agent
can be a double stranded (ds) iRNA agent.
[0044] A "ds iRNA agent" (abbreviation for "double stranded iRNA
agent"), as used herein, is an iRNA agent which includes more than
one, and preferably two, strands in which interchain hybridization
can form a region of duplex structure. A "strand" herein refers to
a contiguous sequence of nucleotides (including non-naturally
occurring or modified nucleotides). The two or more strands may be,
or each form a part of, separate molecules, or they may be
covalently interconnected, e.g. by a linker, e.g. a polyethylene
glycol linker, to form but one molecule. At least one strand can
include a region which is sufficiently complementary to a target
RNA. Such strand is termed the "antisense strand". A second strand
comprised in the dsRNA agent that comprises a region complementary
to the antisense strand is termed the "sense strand". However, a ds
iRNA agent can also be formed from a single RNA molecule which is,
at least partly; self-complementary, forming, e.g., a hairpin or
panhandle structure, including a duplex region. In such case, the
term "strand" refers to one of the regions of the RNA molecule that
is complementary to another region of the same RNA molecule.
[0045] Although, in mammalian cells, long ds iRNA agents can induce
the interferon response which is frequently deleterious, short ds
iRNA agents do not trigger the interferon response, at least not to
an extent that is deleterious to the cell and/or host. The iRNA
agents of the present invention include molecules which are
sufficiently short that they do not trigger a deleterious
interferon response in mammalian cells. Thus, the administration of
a composition of an iRNA agent (e.g., formulated as described
herein) to a mammalian cell can be used to silence expression of an
TGF-beta gene while circumventing a deleterious interferon
response. Molecules that are short enough that they do not trigger
a deleterious interferon response are termed siRNA agents or siRNAs
herein. "siRNA agent" or "siRNA" as used herein, refers to an iRNA
agent, e.g., a ds iRNA agent, that is sufficiently short that it
does not induce a deleterious interferon response in a human cell,
e.g., it has a duplexed region of less than 60 but preferably less
than 50, 40, or 30 nucleotide pairs.
[0046] The isolated iRNA agents described herein, including ds iRNA
agents and siRNA agents, can mediate silencing of a gene, e.g., by
RNA degradation. For convenience, such RNA is also referred to
herein as the RNA to be silenced, or target RNA. Such a gene is
also referred to as a target gene. Preferably, the RNA to be
silenced is a gene product of a TGF-beta gene.
[0047] As used herein, the phrases "mediate RNAi", "silence a
TGF-beta gene", "reducing the level of TGF-beta mRNA" and/or
"reducing the level of TGF-beta protein" all refer to the at least
partial suppression of the expression of the TGF-beta gene, as
manifested, e.g., by a reduction of the amount of mRNA transcribed
from the TGF-beta gene which may be isolated from a first cell or
group of cells in which the TGF-beta gene is transcribed and which
has or have been treated such that the expression of the TGF-beta
gene is inhibited, as compared to a second cell or group of cells
substantially identical to the first cell or group of cells but
which has or have not been so treated (control cells).
Alternatively, the degree of inhibition may be given in terms of a
reduction of a parameter that is functionally linked to TGF-beta
gene transcription, e.g. the amount of protein encoded by the
TGF-beta gene which is secreted by a cell, or the number of cells
displaying a certain phenotype associated with TGF-beta signalling.
The degree of inhibition is usually expressed in terms of
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) 100 % ##EQU00001##
[0048] ("mRNA" may be replaced by another parameter used to assess
TGF-beta gene expression, where applicable)
[0049] For example, in certain instances, expression of the
TGF-beta gene is suppressed by at least about 20%, 25%, 35%, or 50%
by administration of the double-stranded oligonucleotide of the
invention. In a preferred embodiment, the TGF-beta gene is
suppressed by at least about 60%, 70%, or 80% by administration of
the double-stranded oligonucleotide of the invention. In a more
preferred embodiment, the TGF-beta gene is suppressed by at least
about 85%, 90%, or 95% by administration of the double-stranded
oligonucleotide of the invention. In a most preferred embodiment,
the TGF-beta gene is suppressed by at least about 98%, 99% or more
by administration of the double-stranded oligonucleotide of the
invention.
[0050] In principle, TGF-beta gene silencing may be determined in
any cell expressing the target, either constitutively or by genomic
engineering, and by any appropriate assay. However, when a
reference is needed in order to determine whether a given siRNA
inhibits the expression of the TGF-beta gene by a certain degree
and therefore is encompassed by the instant invention, the assay
provided in the Examples below shall serve as such reference.
[0051] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0052] This includes base-pairing of the oligonucleotide or
polynucleotide comprising the first nucleotide sequence to the
oligonucleotide or polynucleotide comprising the second nucleotide
sequence over the entire length of the first and second nucleotide
sequence. Such sequences can be referred to as "fully
complementary" with respect to each other herein. However, where a
first sequence is referred to as "substantially complementary" with
respect to a second sequence herein, the two sequences can be fully
complementary, or they may form one or more, but preferably not
more than 4, 3 or 2 mismatched base pairs upon hybridization, while
retaining the ability to hybridize under the conditions most
relevant to their ultimate application. However, where two
oligonucleotides are designed to form, upon hybridization, one or
more single stranded overhangs, such overhangs shall not be
regarded as mismatches with regard to the determination of
complementarity. For example, a dsRNA comprising one
oligonucleotide 21 nucleotides in length and another
oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 21 nucleotides that is
fully complementary to the shorter oligonucleotide, may yet be
referred to as "fully complementary" for the purposes of the
invention.
[0053] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, in as far
as the above requirements with respect to their ability to
hybridize are fulfilled.
[0054] The terms "complementary", "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of a dsRNA and a target
sequence, as will be understood from the context of their use.
[0055] As used herein, "essentially identical" when used referring
to a first nucleotide sequence in comparison to a second nucleotide
sequence means that the first nucleotide sequence is identical to
the second nucleotide sequence except for up to one, two or three
nucleotide substitutions (e.g. adenosine replaced by uracil).
[0056] As used herein, a "subject" refers to a mammalian organism
undergoing treatment for a disorder associated with undesired
TGF-beta signaling, such as undergoing treatment prophylactically
or therapeutically to prevent TGF-beta production. The subject can
be any mammal, such as a primate, cow, horse, mouse, rat, dog, pig,
goat. In the preferred embodiment, the subject is a human.
[0057] As used herein, "target sequence" refers to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of a TGF-beta gene, including mRNA that is
a product of RNA processing of a primary transcription product.
[0058] As used herein, treating a disorder associated with
undesired TGF-beta signaling refers to the amelioration of any
biological or pathological endpoints that 1) is mediated in part by
the unwanted expression or over expression of TGF-beta in the
subject and 2) whose outcome can be affected by reducing the level
of TGF-beta gene products present.
[0059] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" refer to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of pain or an overt symptom of pain. The specific
amount that is therapeutically effective can be readily determined
by ordinary medical practitioner, and may vary depending on factors
known in the art, such as, e.g. the type of pain, the patient's
history and age, the stage of pain, and the administration of other
anti-pain agents.
[0060] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a dsRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
RNA effective to produce the intended pharmacological, therapeutic
or preventive result. For example, if a given clinical treatment is
considered effective when there is at least a 25% reduction in a
measurable parameter associated with a disease or disorder, a
therapeutically effective amount of a drug for the treatment of
that disease or disorder is the amount necessary to effect at least
a 25% reduction in that parameter.
[0061] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The term
specifically excludes cell culture medium. For drugs administered
orally, pharmaceutically acceptable carriers include, but are not
limited to pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0062] As used herein, a "transformed cell" is a cell into which a
vector has been introduced from which a dsRNA molecule may be
expressed.
[0063] Design and Selection of iRNA Agents
[0064] The present invention is based on the demonstration of
target gene silencing of the TGF-beta gene in vitro following
administration of an iRNA agent results in reducing TGF-beta
signaling.
[0065] Based on these results, the invention specifically provides
an iRNA agent that can be used in reducing TGF-beta levels in a
cell or organism, particularly for use in reducing undesired
TGF-beta signaling, in isolated form and as a pharmaceutical
composition described below. Such agents will include a sense
strand having at least 15 or more contiguous nucleotides that are
complementary to a TGF-beta gene and an antisense strand having at
least 15 or more contiguous nucleotides that are complementary to
the sense strand sequence. Particularly useful are iRNA agents that
comprise a nucleotide sequence from the TGF-beta gene as provided
in Table 1 and Table 5.
[0066] Other candidate iRNA agents can be designed by performing,
for example, a gene walk analysis of the TGF-beta gene that will
serve as the iRNA target. Overlapping, adjacent, or closely spaced
candidate agents corresponding to all or some of the transcribed
region can be generated and tested. Each of the iRNA agents can be
tested and evaluated for the ability to down regulate the target
gene expression (see below, "Evaluation of Candidate iRNA
agents").
[0067] Preferably, the iRNA agents of the present invention are
based on and comprise at least 15 or more contiguous nucleotides
from one of the iRNA agents shown to be active in Table 1 and Table
5. In such agents, the agent can comprise the entire sequence
provided in the table or can comprise 15 or more contiguous
residues along with additional nucleotides from contiguous regions
of the target gene. Alternatively, an iRNA agent of the instant
invention may effect the cleavage of a TGF-beta mRNA within a
sequence on the TGF-beta mRNA that is the target sequence of one of
the iRNA agents provided in Table 1 and Table 5. The target
sequences of the iRNA agents in Table 5 is expressly provided
therein, the target sequence of the iRNA agents provided in Table 1
is equal to the sense strand sequence of these agents, minus the
5'-terminal deoxythymidines (and, where applicable, replacing
2'-.beta.-methyl ribonucleotides by ribonucleotides). The skilled
person is well aware of how an iRNA agent that cleaves a target
mRNA within a given mRNA sequence may be designed. Cleavage of a
target mRNA occurs between the two nucleotides hybridized to those
in positions 10 and 11, counting 5' to 3', of the iRNA agent's
antisense strand (Elbashir, S., et al., EMBO J. 2001, 23:6877).
Therefore, an iRNA agent may be designed to cleave at a given site
by including a sequence in its antisense strand sufficiently
complementary to the target mRNA to hybridize to the target mRNA
such that 10 nucleotides of the antisense strand are hybridized 3'
to the intended cleavage site on the mRNA, the remainder being
hybridized to a sequence 5' on the target mRNA.
[0068] An iRNA agent can be rationally designed based on sequence
information and desired characteristics and the information
provided in Table 1 and Table 5. For example, an iRNA agent can be
designed according to the relative melting temperature of the
candidate duplex. Generally, the duplex should have a lower melting
temperature at the 5' end of the antisense strand than at the 3'
end of the antisense strand.
[0069] The skilled person is well aware that dsRNAs comprising a
duplex structure of between 20 and 23, but specifically 21, base
pairs have been hailed as particularly effective in inducing RNA
interference (Elbashir et al., EMBO 2001, 20:6877-6888). However,
others have found that shorter or longer dsRNAs can be effective as
well. In the embodiments described above, by virtue of the nature
of the oligonucleotide sequences provided in Table 1 and Table 5,
the dsRNAs of the invention can comprise at least one strand of a
length of minimally 21 nt. It can be reasonably expected that
shorter dsRNAs comprising one of the sequences of Table 1 and Table
5 minus only a few nucleotides on one or both ends may be similarly
effective as compared to the dsRNAs described above. Hence, dsRNAs
comprising a partial sequence from one of the sequences of Table 1
and Table 5, and differing in their ability to inhibit the
expression of a TGF-beta gene in an assay as described herein below
by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA
comprising the full sequence, are contemplated by the
invention.
[0070] Accordingly, the present invention provides iRNA agents
comprising a sense strand and antisense strand each comprising a
sequence of at least 15, 16, 17, 18, 19, 20, 21 or 23 nucleotides
which is essentially identical to, as defined above, a portion of
the TGF-beta gene. Exemplified iRNA agents include those that
comprise 15 or more contiguous nucleotides from one of the agents
provided in Table 1 and Table 5.
[0071] The antisense strand of an iRNA agent should be equal to or
at least, 15, 16, 17, 18, 19, 25, 29, 40, or 50 nucleotides in
length. It should be equal to or less than 50, 40, or 30,
nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to
23, and 19 to 21 nucleotides in length. Exemplified iRNA agents
include those that comprise 15 or more nucleotides from one of the
agents in Table 1 and Table 5.
[0072] The sense strand of an iRNA agent should be equal to or at
least 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length.
It should be equal to or less than 50, 40, or 30 nucleotides in
length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to
21 nucleotides in length. Exemplified iRNA agents include those
that comprise 15 or more nucleotides from one of the agents Table 1
and Table 5.
[0073] The double stranded portion of an iRNA agent should be equal
to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40,
or 50 nucleotide pairs in length. It should be equal to or less
than 50, 40, or 30 nucleotides pairs in length. Preferred ranges
are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in
length.
[0074] The agents provided in Table 1 and Table 5 are 21
nucleotides in length for each strand. The iRNA agents contain a 19
nucleotide double stranded region with a 2 nucleotide overhang on
each of the 3' ends of the agent. These agents can be modified as
described herein to obtain equivalent agents comprising at least a
portion of these sequences (15 or more contiguous nucleotides) and
or modifications to the oligonucleotide bases and linkages.
[0075] Generally, the iRNA agents of the instant invention include
a region of sufficient complementarity to the TGF-beta gene, and
are of sufficient length in terms of nucleotides, that the iRNA
agent, or a fragment thereof, can mediate down regulation of the
specific TGF-beta gene. The antisense strands of the iRNA agents of
the present invention are preferably fully complementary to the
mRNA sequences of TGF-beta gene. However, it is not necessary that
there be perfect complementarity between the iRNA agent and the
target, but the correspondence must be sufficient to enable the
iRNA agent, or a cleavage product thereof, to direct sequence
specific silencing, e.g., by RNAi cleavage of an TGF-beta mRNA.
[0076] Therefore, the iRNA agents of the instant invention include
agents comprising a sense strand and antisense strand each
comprising a sequence of at least 16, 17 or 18 nucleotides which is
essentially identical, as defined below, to one of the sequences of
a TGF-beta gene such as those agent provided in Table 1 and Table
5, except that not more than 1, 2 or 3 nucleotides per strand,
respectively, have been substituted by other nucleotides (e.g.
adenosine replaced by uracil), while essentially retaining the
ability to inhibit TGF-beta expression in cultured human cells, as
defined below. These agents will therefore possess at least 15 or
more nucleotides identical to one of the sequences of a TGF-beta
gene but 1, 2 or 3 base mismatches with respect to either the
target TGF-beta mRNA sequence or between the sense and antisense
strand are introduced. Mismatches to the target TGF-beta mRNA
sequence, particularly in the antisense strand, are most tolerated
in the terminal regions and if present are preferably in a terminal
region or regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5'
and/or 3' terminus, most preferably within 6, 5, 4, or 3
nucleotides of the 5'-terminus of the sense strand or the
3'-terminus of the antisense strand. The sense strand need only be
sufficiently complementary with the antisense strand to maintain
the overall double stranded character of the molecule.
[0077] It is preferred that the sense and antisense strands be
chosen such that the iRNA agent includes a single strand or
unpaired region at one or both ends of the molecule, such as those
exemplified in Table 1 and Table 5. Thus, an iRNA agent contains
sense and antisense strands, preferably paired to contain an
overhang, e.g., one or two 5' or 3' overhangs but preferably a 3'
overhang of 2-3 nucleotides. Most embodiments will have a 3'
overhang. Preferred siRNA agents will have single-stranded
overhangs, preferably 3' overhangs, of 1 to 6, preferably 1 to 4,
or more preferably 2 or 3 nucleotides, in length, on one or both
ends of the iRNA agent. The overhangs can be the result of one
strand being longer than the other, or the result of two strands of
the same length being staggered. 5'-ends are preferably
phosphorylated.
[0078] Moreover, the present inventors have discovered that the
presence of only one nucleotide overhang strengthens the
interference activity of the dsRNA, without affecting its overall
stability. dsRNA having only one overhang has proven particularly
stable and effective in vivo, as well as in a variety of cells,
cell culture mediums, blood, and serum. Preferably, the
single-stranded overhang is located at the 3'-terminal end of the
antisense strand or, alternatively, at the 3'-terminal end of the
sense strand. The dsRNA may also have a blunt end, preferably
located at the 5'-end of the antisense strand. Such dsRNAs have
improved stability and inhibitory activity, thus allowing
administration at low dosages, i.e., less than 5 mg/kg body weight
of the recipient per day. Preferably, the antisense strand of the
dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is
blunt. In another embodiment, one or more of the nucleotides in the
overhang is replaced with a nucleoside thiophosphate.
[0079] Preferred lengths for the duplexed region is between 15 and
30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in
length, e.g., in the siRNA agent range discussed above. Embodiments
in which the two strands of the siRNA agent are linked, e.g.,
covalently linked are also included. Hairpin, or other single
strand structures which provide the required double stranded
region, and preferably a 3' overhang are also within the
invention.
[0080] Evaluation of Candidate iRNA Agents
[0081] A candidate iRNA agent can be evaluated for its ability to
down regulate target gene expression. For example, a candidate iRNA
agent can be provided, and contacted with a cell, e.g. a human cell
that expresses TGF-beta. Alternatively, the cell can be transfected
with a construct from which a target TGF-beta gene is expressed,
thus preventing the need for endogenous TGF-beta expression. The
level of target gene expression prior to and following contact with
the candidate iRNA agent can be compared, e.g. on an mRNA or
protein level. If it is determined that the amount of RNA or
protein expressed from the target gene is lower following contact
with the iRNA agent, then it can be concluded that the iRNA agent
down-regulates target gene expression. The level of target TGF-beta
mRNA or TGF-beta protein in the cell or tissue can be determined by
any method desired. For example, the level of target RNA can be
determined by Northern blot analysis, reverse transcription coupled
with polymerase chain reaction (RT-PCR), bDNA analysis, or RNAse
protection assay. The level of protein can be determined, for
example, by Western blot analysis or immunofluorescence.
[0082] Stability Testing, Modification, and Retesting of iRNA
Agents
[0083] A candidate iRNA agent can be evaluated with respect to
stability, e.g., its susceptibility to cleavage by an endonuclease
or exonuclease, such as when the iRNA agent is introduced into the
body of a subject. Methods can be employed to identify sites that
are susceptible to modification, particularly cleavage, e.g.,
cleavage by a component found in the body of a subject.
[0084] When sites susceptible to cleavage are identified, a further
iRNA agent can be designed and/or synthesized wherein the potential
cleavage site is made resistant to cleavage, e.g. by introduction
of a 2'-modification on the site of cleavage, e.g. a 2'-O-methyl
group. This further iRNA agent can be retested for stability, and
this process may be iterated until an iRNA agent is found
exhibiting the desired stability. Table 1 provides a variety of
sequence modifications as exemplars, which are nevertheless not to
be understood as limiting.
[0085] In Vivo Testing
[0086] An iRNA agent identified as being capable of inhibiting
TGF-beta gene expression can be tested for functionality in vivo in
an animal model (e.g., in a mammal, such as in mouse or rat) as
shown in the examples. For example, the iRNA agent can be
administered to an animal, and the iRNA agent evaluated with
respect to its biodistribution, stability, and its ability to
inhibit TGF-beta, e.g. lower TGF-beta protein or gene
expression.
[0087] The iRNA agent can be administered directly to the target
tissue, such as by injection, or the iRNA agent can be administered
to the animal model in the same manner that it would be
administered to a human, e.g. by inhalation, injection or
infusion.
[0088] The iRNA agent can also be evaluated for its intracellular
distribution. The evaluation can include determining whether the
iRNA agent was taken up into the cell. The evaluation can also
include determining the stability (e.g., the half-life) of the iRNA
agent. Evaluation of an iRNA agent in vivo can be facilitated by
use of an iRNA agent conjugated to a traceable marker (e.g., a
fluorescent marker such as fluorescein; a radioactive label, such
as .sup.35S, .sup.32P, .sup.33P or .sup.3H; gold particles; or
antigen particles for immunohistochemistry).
[0089] The iRNA agent can be evaluated with respect to its ability
to down regulate TGF-beta gene expression. Levels of TGF-beta gene
expression in vivo can be measured, for example, by in situ
hybridization, or by the isolation of RNA from tissue prior to and
following exposure to the iRNA agent. Where the animal needs to be
sacrificed in order to harvest the tissue, an untreated control
animal will serve for comparison. Target TGF-beta mRNA can be
detected by any desired method, including but not limited to
RT-PCR, Northern blot, branched-DNA assay, or RNAase protection
assay. Alternatively, or additionally, TGF-beta gene expression can
be monitored by performing Western blot analysis on tissue extracts
treated with the iRNA agent.
[0090] iRNA Chemistry
[0091] Described herein are isolated iRNA agents, e.g., ds RNA
agents, that mediate RNAi to inhibit expression of a TGF-beta
gene.
[0092] RNA agents discussed herein include otherwise unmodified RNA
as well as RNA which have been modified, e.g., to improve efficacy,
and polymers of nucleoside surrogates. Unmodified RNA refers to a
molecule in which the components of the nucleic acid, namely
sugars, bases, and phosphate moieties, are the same or essentially
the same as that which occur in nature, preferably as occur
naturally in the human body. The art has referred to rare or
unusual, but naturally occurring, RNAs as modified RNAs, see, e.g.,
Limbach et al., (1994) Nucleic Acids Res. 22: 2183-2196. Such rare
or unusual RNAs, often termed modified RNAs (apparently because
these are typically the result of a post-transcriptional
modification) are within the term unmodified RNA, as used herein.
Modified RNA as used herein refers to a molecule in which one or
more of the components of the nucleic acid, namely sugars, bases,
and phosphate moieties, are different from that which occurs in
nature, preferably different from that which occurs in the human
body. While they are referred to as modified "RNAs," they will of
course, because of the modification, include molecules which are
not RNAs. Nucleoside surrogates are molecules in which the
ribophosphate backbone is replaced with a non-ribophosphate
construct that allows the bases to the presented in the correct
spatial relationship such that hybridization is substantially
similar to what is seen with a ribophosphate backbone, e.g.,
non-charged mimics of the ribophosphate backbone. Examples of each
of the above are discussed herein.
[0093] Modifications described herein can be incorporated into any
double-stranded RNA and RNA-like molecule described herein, e.g.,
an iRNA agent. It may be desirable to modify one or both of the
antisense and sense strands of an iRNA agent. As nucleic acids are
polymers of subunits or monomers, many of the modifications
described below occur at a position which is repeated within a
nucleic acid, e.g., a modification of a base, or a phosphate
moiety, or the non-linking O of a phosphate moiety. In some cases
the modification will occur at all of the subject positions in the
nucleic acid but in many, and in fact in most, cases it will not.
By way of example, a modification may only occur at a 3' or 5'
terminal position, may only occur in a terminal region, e.g. at a
position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of a strand. A modification may occur in a double
strand region, a single strand region, or in both. E.g., a
phosphorothioate modification at a non-linking O position may only
occur at one or both termini, may only occur in a terminal regions,
e.g., at a position on a terminal nucleotide or in the last 2, 3,
4, 5, or 10 nucleotides of a strand, or may occur in double strand
and single strand regions, particularly at termini. Similarly, a
modification may occur on the sense strand, antisense strand, or
both. In some cases, the sense and antisense strand will have the
same modifications or the same class of modifications, but in other
cases the sense and antisense strand will have different
modifications, e.g., in some cases it may be desirable to modify
only one strand, e.g. the sense strand.
[0094] Two prime objectives for the introduction of modifications
into iRNA agents is their stabilization towards degradation in
biological environments and the improvement of pharmacological
properties, e.g. pharmacodynamic properties, which are further
discussed below. Other suitable modifications to a sugar, base, or
backbone of an iRNA agent are described in co-owned PCT Application
No. PCT/US2004/01193, filed Jan. 16, 2004. An iRNA agent can
include a non-naturally occurring base, such as the bases described
in co-owned PCT Application No. PCT/US2004/011822, filed Apr. 16,
2004. An iRNA agent can include a non-naturally occurring sugar,
such as a non-carbohydrate cyclic carrier molecule. Exemplary
features of non-naturally occurring sugars for use in iRNA agents
are described in co-owned PCT Application No. PCT/US2004/11829
filed Apr. 16, 2003.
[0095] An iRNA agent can include an internucleotide linkage (e.g.,
the chiral phosphorothioate linkage) useful for increasing nuclease
resistance. In addition, or in the alternative, an iRNA agent can
include a ribose mimic for increased nuclease resistance. Exemplary
internucleotide linkages and ribose mimics for increased nuclease
resistance are described in co-owned PCT Application No.
PCT/US2004/07070 filed on Mar. 8, 2004.
[0096] An iRNA agent can include ligand-conjugated monomer subunits
and monomers for oligonucleotide synthesis. Exemplary monomers are
described in co-owned U.S. application Ser. No. 10/916,185, filed
on Aug. 10, 2004.
[0097] An iRNA agent can have a ZXY structure, such as is described
in co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0098] An iRNA agent can be complexed with an amphipathic moiety.
Exemplary amphipathic moieties for use with iRNA agents are
described in co-owned PCT Application No. PCT/US2004/07070 filed on
Mar. 8, 2004.
[0099] In another embodiment, the iRNA agent can be complexed to a
delivery agent that features a modular complex. The complex can
include a carrier agent linked to one or more of (preferably two or
more, more preferably all three of): (a) a condensing agent (e.g.,
an agent capable of attracting, e.g., binding, a nucleic acid,
e.g., through ionic or electrostatic interactions); (b) a fusogenic
agent (e.g., an agent capable of fusing and/or being transported
through a cell membrane); and (c) a targeting group, 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.
iRNA agents complexed to a delivery agent are described in co-owned
PCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.
[0100] An iRNA agent can have non-canonical pairings, such as
between the sense and antisense sequences of the iRNA duplex.
Exemplary features of non-canonical iRNA agents are described in
co-owned PCT Application No. PCT/US2004/07070 filed on Mar. 8,
2004.
[0101] Enhanced Nuclease Resistance
[0102] An iRNA agent, e.g., an iRNA agent that targets TGF-beta,
can have enhanced resistance to nucleases.
[0103] For increased nuclease resistance and/or binding affinity to
the target, an iRNA agent, e.g., the sense and/or antisense strands
of the iRNA agent, can include, for example, 2'-modified ribose
units and/or phosphorothioate linkages. E.g., the 2' hydroxyl group
(OH) can be modified or replaced with a number of different "oxy"
or "deoxy" substituents.
[0104] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-AMINE and aminoalkoxy, O(CH.sub.2).sub.nAMINE, (e.g.,
AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl amino,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino). It is noteworthy that
oligonucleotides containing only the methoxyethyl group (MOE),
(OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative), exhibit nuclease
stabilities comparable to those modified with the robust
phosphorothioate modification.
[0105] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl
amino, heteroaryl amino, or diheteroaryl amino), --NHC(O)R(R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl
and alkynyl, which may be optionally substituted with e.g., an
amino functionality.
[0106] Preferred substituents are 2'-methoxyethyl,
2'-OCH3,2'-O-allyl, 2'-C-allyl, and 2'-fluoro.
[0107] One way to increase resistance is to identify cleavage sites
and modify such sites to inhibit cleavage, as described in co-owned
U.S. Application No. 60/559,917, filed on May 4, 2004. For example,
the dinucleotides 5'-ua-3',5'-ug-3',5'-ug-3',5'-uu-3', or 5'-cc-3'
can serve as cleavage sites. In certain embodiments, all the
pyrimidines of an iRNA agent carry a 2'-modification in either the
sense strand, the antisense strand, or both strands, and the iRNA
agent therefore has enhanced resistance to endonucleases. Enhanced
nuclease resistance can also be achieved by modifying the 5'
nucleotide, resulting, for example, in at least one
5'-uridine-adenine-3' (5'-ua-3') dinucleotide wherein the uridine
is a 2'-modified nucleotide; at least one 5'-cytidine-adenine-3'
(5'-ug-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified
nucleotide; at least one 5'-uridine-guanine-3' (5'-ug-3')
dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide;
at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide, wherein
the 5'-uridine is a 2'-modified nucleotide; or at least one
5'-cytidine-cytidine-3' (5'-cc-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, as described in co-owned
International Application No. PCT/US2005/018931, filed on May 27,
2005. The iRNA agent can include at least 2, at least 3, at least 4
or at least 5 of such dinucleotides. In a particularly preferred
embodiment, the 5' nucleotide in all occurrences of the sequence
motifs 5'-ua-3' and 5'-ug-3' in either the sense strand, the
antisense strand, or both strands is a modified nucleotide.
Preferably, the 5' nucleotide in all occurrences of the sequence
motifs 5'-ua-3',5'-ca-3' and 5'-ug-3' in either the sense strand,
the antisense strand, or both strands is a modified nucleotide.
More preferably, all pyrimidine nucleotides in the sense strand are
modified nucleotides, and the 5' nucleotide in all occurrences of
the sequence motifs 5'-ua-3' and 5'-ca-3' in the antisense strand
are modified nucleotides, or where the antisense strand does
comprise neither of a 5'-ua-3' and a 5'-ca-3' motif, in all
occurrences of the sequence motif 5'-ug-3'. Preferably, sites
particularly prone to nuclease degradation are first determined,
e.g. by mass spectrometry of degradation products, and only those
sites are sequentially modified until a desired level of stability
is attained.
[0108] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate), as further described below. The so-called
"chimeric" oligonucleotides are those that contain two or more
different modifications. Preferably, phosphate linker modifications
are located near the 3'- and/or 5'-end of either or both strands,
or at or near sites particularly prone to degradative attack.
[0109] The inclusion of furanose sugars in the oligonucleotide
backbone can also decrease endonucleolytic cleavage. An iRNA agent
can be further modified by including a 3' cationic group, or by
inverting the nucleoside at the 3'-terminus with a 3'-3' linkage.
In another alternative, the 3'-terminus can be blocked with an
aminoalkyl group, e.g., a 3' C5-aminoalkyl dT. Other 3' conjugates
can inhibit 3'-5' exonucleolytic cleavage. While not being bound by
theory, a 3' conjugate, such as naproxen or ibuprofen, may inhibit
exonucleolytic cleavage by sterically blocking the exonuclease from
binding to the 3'-end of oligonucleotide. Even small alkyl chains,
aryl groups, or heterocyclic conjugates or modified sugars
(D-ribose, deoxyribose, glucose etc.) can block
3'-5'-exonucleases.
[0110] Similarly, 5' conjugates can inhibit 5'-3' exonucleolytic
cleavage. While not being bound by theory, a 5' conjugate, such as
naproxen or ibuprofen, may inhibit exonucleolytic cleavage by
sterically blocking the exonuclease from binding to the 5'-end of
oligonucleotide. Even small alkyl chains, aryl groups, or
heterocyclic conjugates or modified sugars (D-ribose, deoxyribose,
glucose etc.) can block 3'-5'-exonucleases.
[0111] An iRNA agent can have increased resistance to nucleases
when a duplexed iRNA agent includes a single-stranded nucleotide
overhang on at least one end. In preferred embodiments, the
nucleotide overhang includes 1 to 4, preferably 2 to 3, unpaired
nucleotides. In a preferred embodiment, the unpaired nucleotide of
the single-stranded overhang that is directly adjacent to the
terminal nucleotide pair contains a purine base, and the terminal
nucleotide pair is a G-C pair, or at least two of the last four
complementary nucleotide pairs are G-C pairs. In further
embodiments, the nucleotide overhang may have 1 or 2 unpaired
nucleotides, and in an exemplary embodiment the nucleotide overhang
is 5'-GC-3'. In preferred embodiments, the nucleotide overhang is
on the 3'-end of the antisense strand. In one embodiment, the iRNA
agent includes the motif 5'-CGC-3' on the 3'-end of the antisense
strand, such that a 2-nt overhang 5'-GC-3' is formed.
[0112] Thus, an iRNA agent can include modifications so as to
inhibit degradation, e.g., by nucleases, e.g., endonucleases or
exonucleases, found in the body of a subject. These monomers are
referred to herein as NRMs, or Nuclease Resistance promoting
Monomers, the corresponding modifications as NRM modifications. In
many cases these modifications will modulate other properties of
the iRNA agent as well, e.g., the ability to interact with a
protein, e.g., a transport protein, e.g., serum albumin, or a
member of the RISC, or the ability of the first and second
sequences to form a duplex with one another or to form a duplex
with another sequence, e.g., a target molecule.
[0113] One or more different NRM modifications can be introduced
into an iRNA agent or into a sequence of an iRNA agent. An NRM
modification can be used more than once in a sequence or in an iRNA
agent.
[0114] NRM modifications include some which can be placed only at
the terminus and others which can go at any position. Some NRM
modifications that can inhibit hybridization are preferably used
only in terminal regions, and more preferably not at the cleavage
site or in the cleavage region of a sequence which targets a
subject sequence or gene, particularly on the antisense strand.
They can be used anywhere in a sense strand, provided that
sufficient hybridization between the two strands of the ds iRNA
agent is maintained. In some embodiments it is desirable to put the
NRM at the cleavage site or in the cleavage region of a sense
strand, as it can minimize off-target silencing.
[0115] In most cases, the NRM modifications will be distributed
differently depending on whether they are comprised on a sense or
antisense strand. If on an antisense strand, modifications which
interfere with or inhibit endonuclease cleavage should not be
inserted in the region which is subject to RISC mediated cleavage,
e.g., the cleavage site or the cleavage region (As described in
Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated
by reference). Cleavage of the target occurs about in the middle of
a 20 or 21 nt antisense strand, or about 10 or 11 nucleotides
upstream of the first nucleotide on the target mRNA which is
complementary to the antisense strand. As used herein cleavage site
refers to the nucleotides on either side of the site of cleavage,
on the target mRNA or on the iRNA agent strand which hybridizes to
it. Cleavage region means the nucleotides within 1, 2, or 3
nucleotides of the cleavage site, in either direction.
[0116] Such modifications can be introduced into the terminal
regions, e.g., at the terminal position or with 2, 3, 4, or 5
positions of the terminus, of a sequence which targets or a
sequence which does not target a sequence in the subject.
[0117] In a particular embodiment, the 5'-end of the antisense
strand and the 3'-end of the sense strand are chemically linked via
a hexaethylene glycol linker. In another embodiment, at least one
nucleotide of the dsRNA comprises a phosphorothioate or
phosphorodithioate groups. The chemical bond at the ends of the
dsRNA is preferably formed by triple-helix bonds.
[0118] In certain embodiments, a chemical bond may be formed by
means of one or several bonding groups, wherein such bonding groups
are preferably poly-(oxyphosphinicooxy-1,3-propandiol)- and/or
polyethylene glycol chains. In other embodiments, a chemical bond
may also be formed by means of purine analogs introduced into the
double-stranded structure instead of purines. In further
embodiments, a chemical bond may be formed by azabenzene units
introduced into the double-stranded structure. In still further
embodiments, a chemical bond may be formed by branched nucleotide
analogs instead of nucleotides introduced into the double-stranded
structure. In certain embodiments, a chemical bond may be induced
by ultraviolet light.
[0119] Teachings regarding the synthesis of particular modified
oligonucleotides may be found in the following U.S. patents: U.S.
Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated
oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for
the preparation of oligonucleotides having chiral phosphorus
linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to
oligonucleotides having modified backbones; U.S. Pat. No.
5,386,023, drawn to backbone-modified oligonucleotides and the
preparation thereof through reductive coupling; U.S. Pat. No.
5,457,191, drawn to modified nucleobases based on the 3-deazapurine
ring system and methods of synthesis thereof; U.S. Pat. No.
5,459,255, drawn to modified nucleobases based on N-2 substituted
purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing
oligonucleotides having chiral phosphorus linkages; U.S. Pat. No.
5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,
drawn to oligonucleotides having .beta.-lactam backbones; U.S. Pat.
No. 5,571,902, drawn to methods and materials for the synthesis of
oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides
having alkylthio groups, wherein such groups may be used as linkers
to other moieties attached at any of a variety of positions of the
nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to
oligonucleotides having phosphorothioate linkages of high chiral
purity; U.S. Pat. No. 5,506,351, drawn to processes for the
preparation of 2'-O-alkyl guanosine and related compounds,
including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469,
drawn to oligonucleotides having N-2 substituted purines; U.S. Pat.
No. 5,587,470, drawn to oligonucleotides having 3-deazapurines;
U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to
conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos.
5,602,240, and 5,610,289, drawn to backbone-modified
oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255,
drawn to, inter alia, methods of synthesizing
2'-fluoro-oligonucleotides.
[0120] In some preferred embodiments, functionalized nucleoside
sequences of the invention possessing an amino group at the
5'-terminus are prepared using a DNA synthesizer, and then reacted
with an active ester derivative of a selected ligand. Active ester
derivatives are well known to those skilled in the art.
Representative active esters include N-hydroxsuccinimide esters,
tetrafluorophenolic esters, pentafluorophenolic esters and
pentachlorophenolic esters. The reaction of the amino group and the
active ester produces an oligonucleotide in which the selected
ligand is attached to the 5'-position through a linking group. The
amino group at the 5'-terminus can be prepared utilizing a
5'-Amino-Modifier C6 reagent. In a preferred embodiment, ligand
molecules may be conjugated to oligonucleotides at the 5'-position
by the use of a ligand-nucleoside phosphoramidite wherein the
ligand is linked to the 5'-hydroxy group directly or indirectly via
a linker. Such ligand-nucleoside phosphoramidites are typically
used at the end of an automated synthesis procedure to provide a
ligand-conjugated oligonucleotide bearing the ligand at the
5'-terminus
[0121] In many cases, protecting groups are used during the
preparation of the compounds of the invention. As used herein, the
term "protected" means that the indicated moiety has a protecting
group appended thereon. In some preferred embodiments of the
invention, compounds contain one or more protecting groups. A wide
variety of protecting groups can be employed in the methods of the
invention. In general, protecting groups render chemical
functionalities inert to specific reaction conditions, and can be
appended to and removed from such functionalities in a molecule
without substantially damaging the remainder of the molecule.
[0122] Representative hydroxyl protecting groups, for example, are
disclosed by Beaucage et al. (Tetrahedron, 1992, 48:2223-2311).
Further hydroxyl protecting groups, as well as other representative
protecting groups, are disclosed in Greene and Wuts, Protective
Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley &
Sons, New York, 1991, and Oligonucleotides And Analogues A
Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991.
[0123] Examples of hydroxyl protecting groups include, but are not
limited to, t-butyl, t-butoxymethyl, methoxymethyl,
tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl,
2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl,
2,6-dichlorobenzyl, diphenylmethyl, p,p'-dinitrobenzhydryl,
p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,
benzoylformate, acetate, chloroacetate, trichloroacetate,
trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate,
9-fluorenylmethyl carbonate, mesylate and tosylate.
[0124] Amino-protecting groups stable to acid treatment are
selectively removed with base treatment, and are used to make
reactive amino groups selectively available for substitution.
Examples of such groups are the Fmoc (E. Atherton and R. C.
Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds.,
Academic Press, Orlando, 1987, volume 9, p. 1) and various
substituted sulfonylethyl carbamates exemplified by the Nsc group
(Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart and
Tesser, Rec. Tray. Chim. Pays-Bas, 1987, 107:621).
[0125] Additional amino-protecting groups include, but are not
limited to, carbamate protecting groups, such as
2-trimethylsilylethoxycarbonyl (Teoc),
1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl
(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl
(Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such
as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;
sulfonamide protecting groups, such as 2-nitrobenzenesulfonyl; and
imine and cyclic imide protecting groups, such as phthalimido and
dithiasuccinoyl. Equivalents of these amino-protecting groups are
also encompassed by the compounds and methods of the invention.
[0126] Many solid supports are commercially available and one of
ordinary skill in the art can readily select a solid support to be
used in the solid-phase synthesis steps. In certain embodiments, a
universal support is used. A universal support allows for
preparation of oligonucleotides having unusual or modified
nucleotides located at the 3'-terminus of the oligonucleotide.
Universal Support 500 and Universal Support II are universal
supports that are commercially available from Glen Research, 22825
Davis Drive, Sterling, Va. For further details about universal
supports see Scott et al., Innovations and Perspectives in
solid-phase Synthesis, 3rd International Symposium, 1994, Ed. Roger
Epton, Mayflower Worldwide, 115-124]; Azhayev, A. V. Tetrahedron
1999, 55, 787-800; and Azhayev and Antopolsky Tetrahedron 2001, 57,
4977-4986. In addition, it has been reported that the
oligonucleotide can be cleaved from the universal support under
milder reaction conditions when oligonucleotide is bonded to the
solid support via a syn-1,2-acetoxyphosphate group which more
readily undergoes basic hydrolysis. See Guzaev, A. I.; Manoharan,
M. J. Am. Chem. Soc. 2003, 125, 2380.
[0127] Ligand Conjugates
[0128] The properties of an iRNA agent, including its
pharmacological properties, can be influenced and tailored, for
example, by the introduction of ligands, e.g. tethered ligands.
[0129] A wide variety of entities, e.g., ligands, can be tethered
to an iRNA agent, e.g., to the carrier of a ligand-conjugated
monomer subunit. Examples are described below in the context of a
ligand-conjugated monomer subunit but that is only preferred,
entities can be coupled at other points to an iRNA agent.
[0130] Preferred moieties are ligands, which are coupled,
preferably covalently, either directly or indirectly via an
intervening tether, to the carrier. In preferred embodiments, the
ligand is attached to the carrier via an intervening tether. The
ligand or tethered ligand may be present on the ligand-conjugated
monomer when the ligand-conjugated monomer is incorporated into the
growing strand. In some embodiments, the ligand may be incorporated
into a "precursor" ligand-conjugated monomer subunit after a
"precursor" ligand-conjugated monomer subunit has been incorporated
into the growing strand. For example, a monomer having, e.g., an
amino-terminated tether, e.g., TAP-(CH.sub.2).sub.nNH.sub.2 may be
incorporated into a growing sense or antisense strand. In a
subsequent operation, i.e., after incorporation of the precursor
monomer subunit into the strand, a ligand having an electrophilic
group, e.g., a pentafluorophenyl ester or aldehyde group, can
subsequently be attached to the precursor ligand-conjugated monomer
by coupling the electrophilic group of the ligand with the terminal
nucleophilic group of the precursor ligand-conjugated monomer
subunit tether.
[0131] In preferred embodiments, a ligand alters the distribution,
targeting or lifetime of an iRNA agent into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g., molecule, cell or
cell type, compartment, e.g., a cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a
species absent such a ligand.
[0132] Preferred ligands can improve transport, hybridization, and
specificity properties and may also improve nuclease resistance of
the resultant natural or modified oligoribonucleotide, or a
polymeric molecule comprising any combination of monomers described
herein and/or natural or modified ribonucleotides.
[0133] Ligands in general can include therapeutic modifiers, e.g.,
for enhancing uptake; diagnostic compounds or reporter groups e.g.,
for monitoring distribution; cross-linking agents;
nuclease-resistance conferring moieties; and natural or unusual
nucleobases. General examples include lipophilic molecules, lipids,
lectins, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes
(e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol
derivatized lithocholic acid), vitamins, carbohydrates (e.g., a
dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or
hyaluronic acid), proteins, protein binding agents, integrin
targeting molecules, polycationics, peptides, polyamines, and
peptide mimics
[0134] The ligand may be a naturally occurring or recombinant or
synthetic molecule, such as a synthetic polymer, e.g., a synthetic
polyamino acid. 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-ethylacrylic 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
moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt
of a polyamine, or an alpha helical peptide.
[0135] Ligands can also include targeting groups, e.g., a cell or
tissue targeting agent, e.g., a thyrotropin, melanotropin,
surfactant protein A, Mucin carbohydrate, a glycosylated
polyaminoacid, transferrin, bisphosphonate, polyglutamate,
polyaspartate, or an RGD peptide or RGD peptide mimetic.
[0136] Ligands can be proteins, e.g., glycoproteins, lipoproteins,
e.g. low density lipoprotein (LDL), or albumins, e.g. human serum
albumin (HSA), 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. Ligands may also include hormones and hormone
receptors. They can also include non-peptidic species, such as
cofactors, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose,
or multivalent fucose. The ligand can be, for example, a
lipopolysaccharide, an activator of p38 MAP kinase, or an activator
of NF-KB.
[0137] The ligand can be a substance, e.g, a drug, which can
increase the uptake of the iRNA agent into the cell, for example,
by disrupting the cell's cytoskeleton, e.g., by disrupting the
cell's microtubules, microfilaments, and/or intermediate filaments.
The drug can be, for example, taxon, vincristine, vinblastine,
cytochalasin, nocodazole, jasplakinolide, latrunculin A,
phalloidin, swinholide A, indanocine, or myoservin.
[0138] In one aspect, the ligand is a lipid or lipid-based
molecule. Such a lipid or lipid-based molecule preferably binds a
serum protein, e.g., human serum albumin (HSA). An HSA binding
ligand allows for distribution of the conjugate to a target tissue,
e.g., liver tissue, including parenchymal cells of the liver. Other
molecules that can bind HSA can also be used as ligands. For
example, neproxen or aspirin can be used. A lipid or lipid-based
ligand can (a) increase resistance to degradation of the conjugate,
(b) increase targeting or transport into a target cell or cell
membrane, and/or (c) can be used to adjust binding to a serum
protein, e.g., HSA.
[0139] A lipid based ligand can be used to modulate, e.g., control
the binding of the conjugate to a target tissue. For example, a
lipid or lipid-based ligand that binds to HSA more strongly will be
less likely to be targeted to the kidney and therefore less likely
to be cleared from the body. A lipid or lipid-based ligand that
binds to HSA less strongly can be used to target the conjugate to
the kidney.
[0140] In a preferred embodiment, the lipid based ligand binds HSA.
Preferably, it binds HSA with a sufficient affinity such that the
conjugate will be preferably distributed to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that
the HSA-ligand binding cannot be reversed.
[0141] In another aspect, the ligand is a moiety, e.g., a vitamin
or nutrient, which is taken up by a target cell, e.g., a
proliferating cell. These are particularly useful for treating
disorders characterized by unwanted cell proliferation, e.g., of
the malignant or non-malignant type, e.g., cancer cells. Exemplary
vitamins include vitamin A, E, and K. Other exemplary vitamins
include the B vitamins, e.g., folic acid, B12, riboflavin, biotin,
pyridoxal or other vitamins or nutrients taken up by cancer
cells.
[0142] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, the agent
is amphipathic. An exemplary agent is a peptide such as tat or
antennapedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, inverters, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an alpha-helical agent, which preferably has a
lipophilic and a lipophobic phase.
[0143] Backbone Modifications
[0144] Specific examples of preferred modified oligonucleotides
envisioned for use in the ligand-conjugated oligonucleotides of the
invention include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. As defined here,
oligonucleotides having modified backbones or internucleoside
linkages include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the
backbone. For the purposes of the invention, modified
oligonucleotides that do not have a phosphorus atom in their
intersugar backbone can also be considered to be
oligonucleosides.
[0145] Specific oligonucleotide chemical modifications are
described below. It is not necessary for all positions in a given
compound to be uniformly modified. Conversely, more than one
modifications may be incorporated in a single dsRNA compound or
even in a single nucleotide thereof.
[0146] Preferred modified internucleoside linkages or backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free-acid forms are also
included.
[0147] Representative United States Patents relating to the
preparation of the above phosphorus-atom-containing linkages
include, but are not limited to, U.S. Pat. Nos. 3,687,808;
4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;
5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;
5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050;
and 5,697,248, each of which is herein incorporated by
reference.
[0148] Preferred modified internucleoside linkages or backbones
that do not include a phosphorus atom therein (i.e.,
oligonucleosides) have backbones that are formed by short chain
alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl
or cycloalkyl intersugar linkages, or one or more short chain
heteroatomic or heterocyclic intersugar linkages. These include
those having morpholino linkages (formed in part from the sugar
portion of a nucleoside); siloxane backbones; sulfide, sulfoxide
and sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
component parts.
[0149] Representative United States patents relating to the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0150] 5'-Terminal Modifications
[0151] In preferred embodiments, iRNA agents are 5' phosphorylated
or include a phosphoryl analog at the 5' prime terminus, e.g. 5'
prime terminus of the antisense strand. 5'-phosphate modifications
of the antisense strand include those which are compatible with
RISC mediated gene silencing. Suitable modifications include:
5'-monophosphate ((HO)2(O)P--O-5'); 5'-diphosphate
((HO2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap
structure. Other suitable 5'-phosphate modifications will be known
to the skilled person.
[0152] The sense strand can be modified in order to inactivate the
sense strand and prevent formation of an active RISC, thereby
potentially reducing off-target effects. This can be accomplished
by a modification which prevents 5'-phosphorylation of the sense
strand, e.g., by modification with a 5'-O-methyl ribonucleotide
(see Nykanen et al., (2001) ATP requirements and small interfering
RNA structure in the RNA interference pathway. Cell 107, 309-321.)
Other modifications which prevent phosphorylation can also be used,
e.g., simply substituting the 5'-OH by H rather than O-Me.
Alternatively, a large bulky group may be added to the 5'-phosphate
turning it into a phosphodiester linkage.
[0153] Disorders and Diseases Associated with Undesired TGF-Beta
Signaling
[0154] Diseases that can be treated in accordance with the present
invention include, without limitation, kidney disorders associated
with undesired TGF-beta signaling and excessive fibrosis and/or
sclerosis, such as glomerulonephritis (GN) of all etiologies, e.g.,
mesangial proliferative GN, immune GN, and crescentic GN; diabetic
nephropathy; renal interstitial fibrosis and all causes of renal
interstitial fibrosis, including hypertension; renal fibrosis
resulting from complications of drug exposure, including
cyclosporin treatment of transplant recipients, e.g. cyclosporin
treatment; HIV-associated nephropathy, transplant necropathy. The
invention further includes the treatment of hepatic diseases
associated with excessive scarring and progressive sclerosis,
including cirrhosis due to all etiologies, disorders of the biliary
tree, and hepatic dysfunction attributable to infections such as
infection with hepatitis virus or parasites; pulmonary fibrosis and
symptoms associates with pulmonary fibrosis with consequential loss
of gas exchange or ability to efficiently move air into and out of
the lungs, including adult respiratory distress syndrome (ARDS),
chronic obstructive pulmonary disease (COPD); idiopathic pulmonary
fibrosis (IPF), acute lung injury (ALI), or pulmonary fibrosis due
to infectious or toxic agents such as smoke, chemicals, allergens,
or autoimmune diseases, such as systemic lupus erythematosus and
scleroderma, chemical contact, or allergies. Fibroproliferative
diseases targeted by the agents, pharmaceutical compositions, and
treatment methods herein further include cardiovascular diseases,
such as congestive heart failure, dilated cardiomyopathy,
myocarditis, or vascular stenosis associated with atherosclerosis,
angioplasty treatment, or surgical incisions or mechanical trauma.
The invention also includes the treatment of all collagen vascular
disorders of a chronic or persistent nature including progressive
systemic sclerosis, polymyositis, scleroderma, dermatomyositis,
fascists, or Raynaud's syndrome, or arthritic conditions such as
rheumatoid arthritis; eye diseases associated with
fibroproliferative states, including proliferative
vitreoretinopathy of any etiology or fibrosis associated with
ocular surgery such as treatment of glaucoma, retinal reattachment,
cataract extraction, or drainage procedures of any kind; excessive
or hypertrophic scar formation in the dermis occurring during wound
healing resulting from trauma or surgical wounds.
[0155] While the above list is not meant in any way limiting, the
treatment of all the above disorders and diseases are contemplated
by the invention.
[0156] Delivery of iRNA Agents to Tissues and Cells
[0157] Formulation
[0158] For ease of exposition, the formulations, compositions, and
methods in this section are discussed largely with regard to
unmodified iRNA agents. It should be understood, however, that
these formulations, compositions, and methods can be practiced with
other iRNA agents, e.g., modified iRNA agents, and such practice is
within the invention.
[0159] The term "therapeutically effective amount" is the amount
present in the composition that is needed to provide the desired
level of drug in the subject to be treated to give the anticipated
physiological response.
[0160] The term "physiologically effective amount" is that amount
delivered to a subject to give the desired palliative or curative
effect.
[0161] The term "co-administration" refers to administering to a
subject two or more agents, and in particular two or more iRNA
agents. The agents can be contained in a single pharmaceutical
composition and be administered at the same time, or the agents can
be contained in separate formulation and administered serially to a
subject. So long as the two agents can be detected in the subject
at the same time, the two agents are said to be
co-administered.
[0162] In the present methods, the iRNA agent can be administered
to the subject either as naked iRNA agent, in conjunction with a
delivery reagent, or as a recombinant plasmid or viral vector which
expresses the iRNA agent. Preferably, the iRNA agent is
administered as naked iRNA.
[0163] The iRNA agents described herein can be formulated for
administration to a subject, preferably for administration locally
to a site of fibrosis or sclerosis, or parenterally, e.g. via
injection.
[0164] A formulated iRNA agent composition can assume a variety of
states. In some examples, the composition is at least partially
crystalline, uniformly crystalline, and/or anhydrous (e.g., less
than 80, 50, 30, 20, or 10% water). In other examples, the
composition is amorphous, such as a wax. In another example, the
iRNA agent is in an aqueous phase, e.g., in a solution that
includes water, this form being the preferred form for
administration via inhalation. The aqueous phase may contain other
solvents or excipients, e.g. ethanol.
[0165] The aqueous phase or the crystalline compositions can be
incorporated into a delivery vehicle, e.g., a liposome
(particularly for the aqueous phase), or a particle (e.g., a
microparticle as can be appropriate for a crystalline composition).
Generally, the iRNA agent composition is formulated in a manner
that is compatible with the intended method of administration.
[0166] Formulations for direct injection and parenteral
administration are well known in the art. Such formulation may
include sterile aqueous solutions which may also contain buffers,
diluents and other suitable additives. For intravenous use, the
total concentration of solutes should be controlled to render the
preparation isotonic.
[0167] The nucleic acid molecules of the present invention can also
be co-administered to a subject in combination with other
therapeutic compounds to increase the overall therapeutic effect.
The use of multiple compounds to treat an indication can increase
the beneficial effects while reducing the presence of side
effects.
[0168] An iRNA agent preparation can be formulated in combination
with another agent, e.g., another therapeutic agent or an agent
that stabilizes an iRNA agent, e.g., a protein that complexes with
the iRNA agent to form an iRNP. Still other agents include
chelators, e.g., EDTA (e.g., to remove divalent cations such as
Mg.sup.2+), salts, RNAse inhibitors (e.g., a broad specificity
RNAse inhibitor such as RNAsin) and so forth.
[0169] In one embodiment, the iRNA agent preparation includes
another iRNA agent, e.g., a second iRNA agent that can mediate RNAi
with respect to a second gene. Still other preparations can include
at least three, five, ten, twenty, fifty, or a hundred or more
different iRNA species. In some embodiments, the agents are
directed to the same gene but different target sequences. In
another embodiment, each iRNA agent is directed to a different
gene, e.g. TGF-beta.
[0170] An iRNA agent can be incorporated into pharmaceutical
compositions suitable for administration. For example, compositions
can include one or more iRNA agents and a pharmaceutically
acceptable carrier.
[0171] The iRNA agents featured by the invention are preferably
formulated as pharmaceutical compositions prior to administering to
a subject, according to techniques known in the art. Pharmaceutical
compositions of the present invention are characterized as being at
least sterile and pyrogen-free. As used herein, "pharmaceutical
formulations" include formulations for human and veterinary use.
Methods for preparing pharmaceutical compositions of the invention
are within the skill in the art, for example as described in
Remington's Pharmaceutical Science, 18th ed., Mack Publishing
Company, Easton, Pa. (1990), and The Science and Practice of
Pharmacy, 2003, Gennaro et al., the entire disclosures of which are
herein incorporated by reference.
[0172] The present pharmaceutical formulations comprise an iRNA
agent of the invention (e.g., 0.1 to 90% by weight), or a
physiologically acceptable salt thereof, mixed with a
physiologically acceptable carrier medium. Preferred
physiologically acceptable carrier media are water, buffered water,
normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the
like.
[0173] Pharmaceutical compositions of the invention can also
comprise conventional pharmaceutical excipients and/or additives.
Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (as for example calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example, calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate). Pharmaceutical compositions of the
invention can be packaged for use in liquid form, or can be
lyophilized.
[0174] For solid compositions, conventional non-toxic solid
carriers can be used; for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like.
[0175] For example, a solid pharmaceutical composition for oral
administration can comprise any of the carriers and excipients
listed above and 10-95%, preferably 25%-75%, of one or more iRNA
agents of the invention.
[0176] By "pharmaceutically acceptable formulation" is meant a
composition or formulation that allows for the effective
distribution of the nucleic acid molecules of the instant invention
in the physical location most suitable for their desired activity.
Non-limiting examples of agents suitable for formulation with the
nucleic acid molecules of the instant invention include:
P-glycoprotein inhibitors (such as PluronicP85), which can enhance
entry of drugs into the CNS (Jolliet-Riant and Tillement, Fundam.
Clin. Pharmacol. 13:16, 1999); biodegradable polymers, such as poly
(DL-lactide-coglycolide) microspheres for sustained release
delivery. Other non-limiting examples of delivery strategies for
the nucleic acid molecules of the instant invention include
material described in Boado et al., J. Pharm. Sci. 87:1308, 1998;
Tyler et al., FEBS Lett. 421:280, 1999; Pardridge et al., PNAS USA.
92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73, 1995;
Aldrian-Herrada et al., Nucleic Acids Res. 26:4910, 1998; and Tyler
et al., PNAS USA 96:7053, 1999.
[0177] The invention also features the use of the composition
comprising surface-modified liposomes containing poly (ethylene
glycol) lipids (PEG-modified, or long-circulating liposomes or
stealth liposomes). These formulations offer a method for
increasing the accumulation of drugs in target tissues. This class
of drug carriers resists opsonization and elimination by the
mononuclear phagocytic system (MPS or RES), thereby enabling longer
blood circulation times and enhanced tissue exposure for the
encapsulated drug (Lasic et al., Chem. Rev. 95:2601, 1995; Ishiwata
et al., Chem. Phare. Bull. 43:1005, 1995).
[0178] Such liposomes have been shown to accumulate selectively in
tumors, presumably by extravasation and capture in the
neovascularized target tissues (Lasic et al., Science 267:1275,
1995; Oku et al., Biochim. Biophys. Acta 1238:86, 1995). The
long-circulating liposomes enhance the pharmacokinetics and
pharmacodynamics of DNA and RNA, particularly compared to
conventional cationic liposomes which are known to accumulate in
tissues of the MPS (Liu et al., J. Biol. Chem. 42:24864, 1995; Choi
et al., International PCT Publication No. WO 96/10391; Ansell et
al., International PCT Publication No. WO 96/10390; Holland et al.,
International PCT Publication No. WO 96/10392). Long-circulating
liposomes are also likely to protect drugs from nuclease
degradation to a greater extent compared to cationic liposomes,
based on their ability to avoid accumulation in metabolically
aggressive MPS tissues such as the liver and spleen.
[0179] Alternatively, certain iRNA agents of the instant invention
can be expressed within cells from eukaryotic promoters (e.g.,
Izant and Weintraub, Science 229:345, 1985; McGarry and Lindquist,
Proc. Natl. Acad. Sci. USA 83:399, 1986; Scanlon et al., Proc.
Natl. Acad. Sci. USA 88:10591, 1991; Kashani-Sabet et al.,
Antisense Res. Dev. 2:3, 1992; Dropulic et al., J. Virol. 66:1432,
1992; Weerasinghe et al., J. Virol. 65:5531, 1991; Ojwanget al.,
Proc. Natl. Acad. Sci. USA 89:10802, 1992; Chen et al., Nucleic
Acids Res. 20:4581, 1992; Sarver et al., Science 247:1222, 1990;
Thompson et al., Nucleic Acids Res. 23:2259, 1995; Good et al.,
Gene Therapy 4:45, 1997). Those skilled in the art realize that any
nucleic acid can be expressed in eukaryotic cells from the
appropriate DNA/RNA vector. The activity of such nucleic acids can
be augmented by their release from the primary transcript by a
enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and
Sullivan et al., PCT WO 94/02595; Ohkawa et al., Nucleic Acids
Symp. Ser. 27:156, 1992; Taira et al., Nucleic Acids Res. 19:5125,
1991; Ventura et al., Nucleic Acids Res. 21:3249, 1993; Chowrira et
al., J. Biol. Chem. 269:25856, 1994).
[0180] In another aspect of the invention, RNA molecules of the
present invention can be expressed from transcription units (see
for example Couture et al., Trends in Genetics 12:510, 1996)
inserted into DNA or RNA vectors. The recombinant vectors can be
DNA plasmids or viral vectors. iRNA agent-expressing viral vectors
can be constructed based on, but not limited to, adeno-associated
virus, retrovirus, adenovirus, or alphavirus. In another
embodiment, pol III based constructs are used to express nucleic
acid molecules of the invention (see for example Thompson, U.S.
Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors
capable of expressing the iRNA agents can be delivered as described
above, and persist in target cells. Alternatively, viral vectors
can be used that provide for transient expression of nucleic acid
molecules. Such vectors can be repeatedly administered as
necessary. Once expressed, the iRNA agent interacts with the target
mRNA and generates an RNAi response. Delivery of iRNA
agent-expressing vectors can be systemic, such as by intravenous or
intra-muscular administration, by administration to target cells
ex-planted from a subject followed by reintroduction into the
subject, or by any other means that would allow for introduction
into the desired target cell (for a review see Couture et al.,
Trends in Genetics 12:510, 1996).
[0181] The present invention also includes compositions prepared
for storage or administration that include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier. The term "pharmaceutically acceptable carrier"
means that the carrier can be taken into the subject with no
significant adverse toxicological effects on the subject.
Acceptable carriers for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.
1985), hereby incorporated by reference herein. The term is
intended to include any and all solvents, dispersion media,
coatings, preservatives, e.g. antibacterial and antifungal agents,
isotonic and absorption delaying agents, stabilizers, antioxidants,
suspending agents, dyes, flavoring agents, and the like, compatible
with pharmaceutical administration. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0182] The types of pharmaceutical excipients that are useful as
carrier include stabilizers such as human serum albumin (HSA),
bulking agents such as carbohydrates, amino acids and polypeptides;
pH adjusters or buffers; salts such as sodium chloride; and the
like. These carriers may be in a crystalline or amorphous form or
may be a mixture of the two.
[0183] Preservatives include, for example, without limitation,
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic
acid.
[0184] Bulking agents that are particularly valuable include
compatible carbohydrates, polypeptides, amino acids or combinations
thereof. Suitable carbohydrates include monosaccharides such as
galactose, D-mannose, sorbose, and the like; disaccharides, such as
lactose, trehalose, and the like; cyclodextrins, such as
2-hydroxypropyl-beta-cyclodextrin; and polysaccharides, such as
raffinose, maltodextrins, dextrans, and the like; alditols, such as
mannitol, xylitol, and the like. A preferred group of carbohydrates
includes lactose, trehalose, raffinose maltodextrins, and mannitol.
Suitable polypeptides include aspartame. Amino acids include
alanine and glycine, with glycine being preferred.
[0185] Suitable pH adjusters or buffers include organic salts
prepared from organic acids and bases, such as sodium citrate,
sodium ascorbate, and the like; sodium citrate is preferred.
[0186] Treatment Methods and Delivery
[0187] Administration of the iRNA Agents A patient who has been
diagnosed with a disorder characterized by undesired TGF-beta
signaling can be treated by administration of an iRNA agent
described herein to block the negative effects of TGF-beta, thereby
alleviating the symptoms associated with undesired TGF-beta
signaling. For example, the iRNA agent can alleviate symptoms
associated with a disease of the lung, such as a fibrotic disorder.
In other examples, the iRNA agent can be administered to treat a
patient who has a fibrotic or sclerotic disease of the kidney or
liver; a fibroproliferative cardiovascular disease; a collagen
vascular disorder; or any other fibroproliferative disorder
associated with undesired TGF-beta signaling.
[0188] A composition that includes an iRNA agent of the present
invention, e.g., an iRNA agent that targets TGF-beta, can be
delivered to a subject by a variety of routes, depending upon
whether local or systemic treatment is desired and upon the area to
be treated. In general, the delivery of the iRNA agents of the
present invention is done to achieve delivery into the subject to
the site of undesired TGF-beta signaling. The preferred means of
administering the iRNA agents of the present invention is through
either a local administration to the site of fibrosis or sclerosis,
such as the lung, e.g. by inhalation, or systemically through
enteral or parenteral administration.
[0189] Suitable enteral administration routes include oral
delivery.
[0190] Suitable parenteral administration routes include
intravascular administration (e.g., intravenous bolus injection,
intravenous infusion, intra-arterial bolus injection,
intra-arterial infusion and catheter instillation into the
vasculature); peri- and intra-tissue injection; subcutaneous
injection, deposition including subcutaneous infusion (such as by
osmotic pumps), or transdermal application; intraperitoneal or
intramuscular injection; intrathecal or intraventricular
administration; intraocular, intraotic, intranasal, or
intrapulmonary administration; or direct application to the area at
or near the site of undesired TGF-beta signaling, for example by a
catheter or other placement device (e.g., a pellet or implant
comprising a porous, non-porous, or gelatinous material). It is
preferred that injections or infusions of the iRNA agent be given
at or near the site of undesired TGF-beta signaling.
[0191] The anti-TGF-beta iRNA agents can be administered
systemically, e.g., orally or by intramuscular injection or by
intravenous injection, in admixture with a pharmaceutically
acceptable carrier adapted for the route of administration. Methods
for the delivery of nucleic acid molecules are described in Akhtar
et al., Trends in Cell Bio. 2:139, 1992; Delivery Strategies for
Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995; Maurer et
al., Mol. Membr. Biol., 16:129, 1999; Hofland and Huang, Handb.
Exp. Pharmacol. 137:165, 1999; and Lee et al., ACS Symp. Ser.
752:184, 2000, all of which are incorporated herein by reference.
Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT
WO 94/02595 further describe the general methods for delivery of
nucleic acid molecules. Nucleic acid molecules can be administered
to cells by a variety of methods known to those of skill in the
art, including, but not restricted to, encapsulation in liposomes,
by iontophoresis, or by incorporation into other vehicles, such as
hydrogels, cyclodextrins (see for example Gonzalez et al.,
Bioconjugate Chem. 10:1068, 1999), biodegradable nanocapsules, and
bioadhesive microspheres, or by proteinaceous vectors (O'Hare and
Normand, International PCT Publication No. WO 00/53722).
[0192] Another aspect of the invention provides for the delivery of
IRNA agents to the respiratory tract. The respiratory tract
includes the upper airways, including the oropharynx and larynx,
followed by the lower airways, which include the trachea followed
by bifurcations into the bronchi and bronchioli. The upper and
lower airways are called the conductive airways. The terminal
bronchioli then divide into respiratory bronchioli which then lead
to the ultimate respiratory zone, the alveoli, or deep lung. The
deep lung, or alveoli, are the primary target of inhaled
therapeutic aerosols for systemic delivery of iRNA agents.
[0193] Pulmonary delivery compositions can be delivered by
inhalation by the patient of a dispersion so that the composition,
preferably the iRNA agent, within the dispersion can reach the lung
where it can, for example, be readily absorbed through the alveolar
region directly into blood circulation. Pulmonary delivery can be
effective both for systemic delivery and for localized delivery to
treat diseases of the lungs.
[0194] Pulmonary delivery can be achieved by different approaches,
including the use of nebulized, aerosolized, micellular and dry
powder-based formulations; administration by inhalation may be oral
and/or nasal. Delivery can be achieved with liquid nebulizers,
aerosol-based inhalers, and dry powder dispersion devices.
Metered-dose devices are preferred. One of the benefits of using an
atomizer or inhaler is that the potential for contamination is
minimized because the devices are self contained. Dry powder
dispersion devices, for example, deliver drugs that may be readily
formulated as dry powders. An iRNA composition may be stably stored
as lyophilized or spray-dried powders by itself or in combination
with suitable powder carriers. The delivery of a composition for
inhalation can be mediated by a dosing timing element which can
include a timer, a dose counter, time measuring device, or a time
indicator which when incorporated into the device enables dose
tracking, compliance monitoring, and/or dose triggering to a
patient during administration of the aerosol medicament.
[0195] Examples of pharmaceutical devices for aerosol delivery
include metered dose inhalers (MDIs), dry powder inhalers (DPIs),
and air-jet nebulizers. Exemplary delivery systems by inhalation
which can be readily adapted for delivery of the subject iRNA
agents are described in, for example, U.S. Pat. Nos. 5,756,353;
5,858,784; and PCT applications WO98/31346; WO98/10796; WO00/27359;
WO01/54664; WO02/060412. Other aerosol formulations that may be
used for delivering the iRNA agents are described in U.S. Pat. Nos.
6,294,153; 6,344,194; 6,071,497, and PCT applications WO02/066078;
WO02/053190; WO01/60420; WO00/66206. Further, methods for
delivering iRNA agents can be adapted from those used in delivering
other oligonucleotides (e.g., an antisense oligonucleotide) by
inhalation, such as described in Templin et al., Antisense Nucleic
Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al., Expert Opin
Biol Ther, 2001, 1:979-83; Sandrasagra et al., Antisense Nucleic
Acid Drug Dev, 2002, 12:177-81.
[0196] The delivery of the inventive agents may also involve the
administration of so called "pro-drugs", i.e. formulations or
chemical modifications of a therapeutic substance that require some
form of processing or transport by systems innate to the subject
organism to release the therapeutic substance, preferably at the
site where its action is desired. For example, the human lungs can
remove or rapidly degrade hydrolytically cleavable deposited
aerosols over periods ranging from minutes to hours. In the upper
airways, ciliated epithelia contribute to the "mucociliary
excalator" by which particles are swept from the airways toward the
mouth. Pavia, D., "Lung Mucociliary Clearance," in Aerosols and the
Lung: Clinical and Experimental Aspects, Clarke, S. W. and Pavia,
D., Eds., Butterworths, London, 1984. In the deep lungs, alveolar
macrophages are capable of phagocytosing particles soon after their
deposition. Warheit et al. Microscopy Res. Tech., 26: 412-422
(1993); and Brain, J. D., "Physiology and Pathophysiology of
Pulmonary Macrophages," in The Reticuloendothelial System, S. M.
Reichard and J. Filkins, Eds., Plenum, New. York., pp. 315-327,
1985.
[0197] In preferred embodiments, particularly where systemic dosing
with the iRNA agent is desired, the aerosoled iRNA agents are
formulated as microparticles. Microparticles having a diameter of
between 0.5 and ten microns can penetrate the lungs, passing
through most of the natural barriers. A diameter of less than ten
microns is required to bypass the throat; a diameter of 0.5 microns
or greater is required to avoid being exhaled.
[0198] The iRNA agent of the invention can be delivered using an
implant. Such implants can be biodegradable and/or biocompatible
implants, or may be non-biodegradable implants. The implants may be
permeable or impermeable to the active agent.
[0199] The iRNA agent of the invention can also be administered
topically, e.g. to the skin, for example by patch or by direct
application to the dermis, or by iontophoresis. Ointments, sprays,
or droppable liquids can be delivered by delivery systems known in
the art such as applicators or droppers.
[0200] The iRNA agent of the invention may be provided in sustained
release compositions, such as those described in, for example, U.S.
Pat. Nos. 5,672,659 and 5,595,760. The use of immediate or
sustained release compositions depends on the nature of the
condition being treated. If the condition consists of an acute or
over-acute disorder, treatment with an immediate release form will
be preferred over a prolonged release composition. Alternatively,
for certain preventative or long-term treatments, a sustained
release composition may be appropriate.
[0201] In addition to treating pre-existing disorders or diseases
associated with undesired TGF-beta signaling, iRNA agents of the
invention can be administered prophylactically in order to prevent
or slow the onset of these and related disorders or diseases. In
prophylactic applications, an iRNA of the invention is administered
to a patient susceptible to or otherwise at risk of a particular
disorder or disease associated with undesired TGF-beta
signaling.
[0202] The iRNA agent of the invention can be administered in a
single dose or in multiple doses. Where the administration of the
iRNA agent of the invention is by infusion, the infusion can be a
single sustained dose or can be delivered by multiple infusions.
Injection of the agent directly into the tissue is at or near the
site of undesired TGF-beta signaling is preferred. Multiple
injections of the agent into the tissue at or near the site of
undesired TGF-beta signaling are also preferred.
[0203] Dosage. The dosage can be an amount effective to treat or
prevent a disorder or disease associated with undesired TGF-beta
signaling.
[0204] One skilled in the art can readily determine an appropriate
dosage regimen for administering the iRNA agent of the invention to
a given subject. For example, the iRNA agent can be administered to
the subject once, e.g., as a single injection or deposition at or
near the site of undesired TGF-beta signaling. Alternatively, the
unit dose is administered less frequently than once a day, e.g.,
less than every 2, 4, 8 or 30 days. In another embodiment, the unit
dose is not administered with a frequency (e.g., not a regular
frequency). Because iRNA agent mediated silencing can persist for
several days after administering the iRNA agent composition, in
many instances, it is possible to administer the composition with a
frequency of less than once per day, or, for some instances, only
once for the entire therapeutic regimen, or only upon the
reoccurrence of a symptom or disease state. Alternatively, the iRNA
agent can be administered once or twice daily to a subject for a
period of from about three to about twenty-eight days, more
preferably from about seven to about ten days. In a preferred
dosage regimen, the iRNA agent is injected at or near a site of
unwanted TGF-beta expression (such as near a site of fibrosis or
sclerosis) once a day for seven days. Where a dosage regimen
comprises multiple administrations, it is understood that the
effective amount of iRNA agent administered to the subject can
comprise the total amount of iRNA agent administered over the
entire dosage regimen.
[0205] In one embodiment, a subject is administered an initial
dose, and one or more maintenance doses of an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, (e.g., a precursor,
e.g., a larger iRNA agent which can be processed into an siRNA
agent, or a DNA which encodes an iRNA agent, e.g., a
double-stranded iRNA agent, or siRNA agent, or precursor thereof).
The maintenance doses are preferably administered no more than once
every 5, 10, or 30 days. Further, the treatment regimen may last
for a period of time which will vary depending upon the nature of
the particular disease, its severity and the overall condition of
the patient. In preferred embodiments the dosage may be delivered
no more than once per day, e.g., no more than once per 24, 36, 48,
or more hours, e.g., no more than once every 5 or 8 days. Following
treatment, the patient can be monitored for changes in his
condition and for alleviation of the symptoms of the disease state.
The dosage of the compound may either be increased in the event the
patient does not respond significantly to current dosage levels, or
the dose may be decreased if an alleviation of the symptoms of the
disease state is observed, if the disease state has been ablated,
or if undesired side-effects are observed.
[0206] The effective dose can be administered in a single dose or
in two or more doses, as desired or considered appropriate under
the specific circumstances. If desired to facilitate repeated or
frequent infusions, implantation of a delivery device, e.g., a
pump, semi-permanent stent (e.g., intravenous, intraperitoneal,
intracisternal or intracapsular), or reservoir may be
advisable.
[0207] The concentration of the iRNA agent composition is an amount
sufficient to be effective in treating or preventing a disorder or
to regulate a physiological condition in humans. The concentration
or amount of iRNA agent administered will depend on the parameters
determined for the agent and the method of administration, e.g.
administration to the lung. For example, inhalable formulations may
require lower concentrations of some ingredients in order to avoid
irritation or discomfort compared to oral formulations. It is
sometimes desirable to dilute an oral formulation up to 10-100
times in order to provide a suitable inhalable formulation.
[0208] Certain factors may influence the dosage required to
effectively treat a subject, including but not limited to the
severity and responsiveness of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. It will also be appreciated that the effective
dosage of an iRNA agent such as an siRNA agent used for treatment
may increase or decrease over the course of a particular treatment.
Changes in dosage may result and become apparent from the results
of diagnostic assays. For example, the subject can be monitored
after administering an iRNA agent composition. Based on information
from the monitoring, an additional amount of the iRNA agent
composition can be administered.
[0209] Moreover, depending on the above factors, the course of
treatment may last from several days to several months, or until a
cure is effected or a diminution of disease state is achieved.
Optimal dosing schedules can be calculated from measurements of
drug accumulation in the body of the patient. Persons of ordinary
skill can easily determine optimum dosages, dosing methodologies
and repetition rates. Optimum dosages may vary depending on the
relative potency of individual compounds, and can generally be
estimated based on EC50s found to be effective in in vitro and in
vivo animal models.
[0210] Dosage levels on the order of about 1 .mu.g/kg to 100 mg/kg
of body weight per administration are useful in the treatment of
the disorders or diseases associated with undesired TGF-beta
signaling. The preferred dosage range is about 0.00001 mg to about
3 mg per kg body weight of the subject to be treated, or preferably
about 0.0001-0.001 mg per kg body weight, about 0.03-3.0 mg per kg
body weight, about 0.1-3.0 mg per kg body weight or about 0.3-3.0
mg per kg body weight.
[0211] An iRNA agent can be administered at a unit dose less than
about 75 mg per kg of bodyweight, or less than about 70, 60, 50,
40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or
0.0005 mg per kg of bodyweight, and less than 200 nmol of iRNA
agent (e.g., about 4.4.times.10.sup.16 copies) per kg of
bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5,
0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmol of
iRNA agent per kg of bodyweight. The unit dose, for example, can be
administered by injection (e.g., intravenous or intramuscular,
intrathecally, or directly into an organ), an inhaled dose, or a
topical application.
[0212] Delivery of an iRNA agent directly to an organ (e.g.,
directly to the liver or lung) can be at a dosage on the order of
about 0.00001 mg to about 3 mg per organ, or preferably about
0.0001-0.001 mg per organ, about 0.03-3.0 mg per organ, about
0.1-3.0 mg per kg body weight or about 0.3-3.0 mg per organ.
[0213] Where an initial dose/maintenance dose regimen is followed,
the maintenance dose or doses are generally lower than the initial
dose, e.g., one-half less of the initial dose. A maintenance
regimen can include treating the subject with a dose or doses
ranging from 0.01 .mu.g to 75 mg/kg of body weight per day, e.g.,
70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,
0.001, or 0.0005 mg per kg of bodyweight per day.
[0214] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Selection for the Targeting of TGF-Beta by Short Interfering
siRNAs
[0215] siRNA design was carried out to identify siRNAs targeting
human and mouse TGF-beta 1 genes. The siRNA in silicon selection
resulted in 28 siRNAs satisfying our selection criteria.
[0216] Human (NM.sub.--000660.3; May 4, 2005) and mouse
(NM.sub.--011577.1; Dec. 17, 2003) mRNA sequences to TGF-beta were
downloaded from NCBI resource
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide.
[0217] To setup an environment for sequence analysis, BioEdit
Sequence Alignment Editor software (Version 7.0.4.1; Hall, T. A.,
Nucl. Acids. Symp. Ser., 1999, 41:95-98) was downloaded from
http://www.mbio.ncsu.edu/BioEdit/bioedit.html and installed on a
windows 2000 computer.
[0218] The siRNA selection process was run as follows: ClustalW
multiple alignment (Thompson J D, et al., Nucleic Acids Res. 1994,
22:4673-80) function of the software was used to generate a global
alignment of human and mouse mRNA sequences. Conserved regions were
identified by embedded sequence analysis function of the software.
For this, conserved regions were defined as sequence stretches with
a minimum length of 19 bases for all aligned sequences containing
no internal gaps. In order to have a reference sequence available
for positions of conserved stretches and finally siRNA target
region coordination, a mapping file with positions of consensus
sequence relative to human mRNA sequence was generated by the
software and analyzed by a perl script.
[0219] The siRNA design web interface at Whitehead Institute for
Biomedical Research (http://jura.wi.mit.edu/siRNAext/) was used to
identify all candidate siRNAs targeting the conserved regions as
well as their predicted off-target hits to human and mouse
sequences. For this purpose, candidate siRNAs proposed by the tool
were subjected to the software embedded off-target analysis by
running the NCBI blast algorithm against the NCBI human and mouse
RefSeq database.
[0220] Blast results were downloaded and analyzed by a perl script
in order to extract an off-target score for each candidate siRNA
that was used for siRNA ranking. Final goal for ranking and
selection was to prefer siRNAs with high specificity. We defined an
off-target score to express off-target potential of each siRNA
based on the following assumptions:
[0221] 1) high specificity can be predicted by weak identity to
off-target hits
[0222] 2) positions 2 to 9 (counting 5' to 3') of a strand (seed
region) may contribute more to off-target potential than rest of
sequence (non-seed region)
[0223] Thus, the identity score of the antisense strand calculated
by blast was considered as well as the positions of occurring
mismatches. The off-target score was defined as the highest score
out of all scores for an siRNA calculated for each off-target hit
obtained by blast.
[0224] The off-target score was calculated as follows: identity
score--0.2 * mismatches in seed region.
[0225] siRNAs were sorted according to off-target score
(ascending). The top 28 siRNAs according to the above criteria were
selected and synthesized.
[0226] Designing siRNAs Against TGF-Beta mRNA
[0227] siRNA against TGF-beta mRNA were synthesized chemically
using procedures established in the art. Two different designs of
siRNAs were synthesized and tested for each base sequence obtained
in the selection step described above, for a total of 56 siRNAs.
The first group consisted of unmodified ribonucleotides, except
that the unpaired 3'-overhangs on both strands consisted of
2'-deoxy thymidines. The second group further included a
phosphorothioate linkage between the two unpaired 2'-deoxy
thymidines at the end of each strand, 2'O-methyl modifications at
every pyrimidine base in the sense strand, and 2'O-methyl
modifications at every uridine occurring in a sequence context of
5'-ua-3' and every cytidine occurring in a sequence context of
5'-ca-3'. Modifications in the oligonucleotide strands were
accomplished by using the appropriate modified monomer
phosphoramidite. The siRNA sequences of those siRNAs synthesized
are listed in Table 1, together with the position of the target
sequence for each siRNA in NM.sub.--000660.3.
Example 2
TGF-Beta siRNA in Vitro Screening Protocol
[0228] Human lung epithelial A549 carcinoma cells were transfected
with unstabilized and phosphorothioate/O-methyl exo-/endonuclease
stabilized siRNAs to select a set of most active siRNAs for further
development as potential drug candidates in preclinical and
clinical studies.
[0229] For transfections, A549 cells (ATCC, Manassas, USA, ATCC #:
CCL-185) were seeded at 1.5.times.10.sup.4 cells/well on 96-well
cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany)
in 100 .mu.l growth medium (RPMI 1640, 10% fetal calf serum, 100
u/ml penicillin 100 .mu.g/ml streptomycin, 2 mM L-glutamine;
Biochrom AG, Berlin, Germany) Transfection of each siRNA was
performed 24 h post-seeding at 100 nM and 10 nM final concentration
of siRNA duplex in quadruplets using Lipofectamine 2000 (Invitrogen
GmbH, Karlsruhe, Germany). For each well, 0.5 .mu.l Lipofectamine
2000 were mixed with 12.5 .mu.l Opti-MEM (Invitrogen) and incubated
for 5 min at room temperature. To achieve a final siRNA
concentration of 100 nM in the incubation mixture, 2.5 .mu.l of a 5
.mu.M solution of siRNA in annealing buffer per well were mixed
with 10 .mu.l Opti-MEM, per well. To achieve a final siRNA
concentration of 10 nM, 2.5 .mu.l of a 0.5 .mu.M solution of siRNA
in annealing buffer were mixed with 10 .mu.l Opti-MEM, per well.
The siRNA solution was combined with the diluted Lipofectamine 2000
solution, gently mixed and incubated for 20 minutes at room
temperature to allow complex formation. In the meantime, growth
medium was removed from cells and replaced by 100 .mu.l/well of
fresh medium. 25 .mu.l of the siRNA-Lipofectamine-2000-complex
solution were applied to the cells to a final volume of 125 .mu.l
per well and cells were incubated for 24 h at 37.degree. C. and 5%
CO.sub.2 in a humidified incubator (Heraeus GmbH, Hanau).
[0230] Following incubation, TGF-beta mRNA levels were determined
by QuantiGene bDNA-kit (Genospectra, Fremont, USA). The incubation
mixture was replaced by 100 .mu.l fresh growth medium and cells
were lysed by applying 50 .mu.l of lysis mixture from the
QuantiGene bDNA-kit and incubation for 30 min at 53.degree. C. Cell
lysates were incubated in parallel with bDNA probes specific to
hGAPDH (10 .mu.l lysate) and hTGF-beta (40 .mu.l lysate) on
QuantiGene Capture Plates and processed according to the
manufacturer's protocol for the QuantiGene bDNA kit. The nucleotide
sequences of the bDNA probes used for hTGF-beta and hGAPDH are
given in Table 2 and Table 3 below, respectively. Finally,
chemoluminescence was measured in a Victor2-Light (Perkin Elmer,
Wiesbaden, Germany) as relative light units (RLU) and values
obtained with hGAPDH and hTGF-beta probesets were normalized to the
respective GAPDH values for each well. TGF-beta/GAPDH ratios
obtained with siRNAs directed against TGF-beta finally were related
to the value obtained with the same concentration (100 nM or 10 nM)
of an unspecific siRNA (directed against ApoB) which was set to
100%.
[0231] Effective siRNAs from the screen were further characterized
by dose response curves (DRC). Transfections for dose response
curves were performed at the following concentrations: 100 nM, 25
nM, 6.3 nM, 1.6 nM, 0.4 nM, 0.1 nM, 25 pM according to the above
protocol. The % TGF-beta mRNA knockdown again was related to a 100%
value obtained from a control siRNA transfection. The dsRNA
concentrations at which 50% of their maximal activity was achieved
(Inhibitory concentration 50%, IC50) were calculated by curve
fitting with the computer software Xlfit, (ID Business Solutions
Ltd., Guildford, Surrey, UK) using the following parameters: Dose
Response One Site, 4 Parameter Logistic Model,
fit=(A+((B-A)/(1+(((10 C)/x) D)))), inv=((10 C)/((((B-A)/(y-A))-1)
(1/D))), res=(y-fit).
[0232] bDNA probes used in bDNA assay for TGF-.beta. (generated
with Bayer QuantiGene Probeset Software):
TABLE-US-00002 TABLE 2 TGF-.beta.-specific probes detecting
hsTGF-.beta.1 (Genbank accession no. NM_000660.3) SEQ ID FPL Name
Function Sequence NO hsTGFb 001 CE
GGGCTCCGGTTCTGCACTCTTTTTTCTCTTGGAAAGAAAGT 113 hsTGFb 002 CE
CGCGGGTGACCTCCTTGGTTTTTCTCTTGGAAAGAAAGT 114 hsTGFb 003 CE
GCAGCTCTGCCCGGGAGATTTTTCTCTTGGAAAGAAAGT 115 hsTGFb 004 CE
TTTTAACTTGAGCCTCAGCAGACTTTTTCTCTTGGAAAGAAAGT 116 hsTGFb 005 CE
GGTTGCTGAGGTATCGCCAGTTTTTCTCTTGGAAAGAAAGT 117 hsTGFb 006 CE
GCTGGGTGCCAGCAGCCTTTTTCTCTTGGAAAGAAAGT 118 hsTGFb 007 LE
CGTAGTAGTCGGCCTCAGGCTCTTTTTAGGCATAGGACCCGTGTCT 119 hsTGFb 008 LE
TGTGGGTTTCCACCATTAGCATTTTTAGGCATAGGACCCGTGTCT 120 hsTGFb 009 LE
GCTTCTCGGAGCTCTGATGTGTTTTTTAGGCATAGGACCCGTGTCT 121 hsTGFb 010 LE
GCAACACGGGTTCAGGTACCTTTTTAGGCATAGGACCCGTGTCT 122 hsTGFb 011 LE
GAATTGTTGCTGTATTTCTGGTACATTTTTAGGCATAGGACCCGTGTCT 123 hsTGFb 012 BL
TGCTTGAACTTGTCATAGATTTCGT 124 hsTGFb 013 BL
TGAAGAACATATATATGCTGTGTGTACTC 125 hsTGFb 014 BL GCTCCACGTGCTGCTCCAC
126
TABLE-US-00003 TABLE 3 GAPDH-specific probes detecting hsGAPDH
(Genbank accession no. NM_002046.2): SEQ ID FPL Name Function
Sequence NO hGAP001 CE GAATTTGCCATGGGTGGAATTTTTTCTCTTGGAAAGAAAGT
127 hGAP002 CE GGAGGGATCTCGCTCCTGGATTTTTCTCTTGGAAAGAAAGT 128
hGAP003 CE CCCCAGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT 129 hGAP004 CE
GCTCCCCCCTGCAAATGAGTTTTTCTCTTGGAAAGAAAGT 130 hGAP005 LE
AGCCTTGACGGTGCCATGTTTTTAGGCATAGGACCCGTGTCT 131 hGAP006 LE
GATGACAAGCTTCCCGTTCTCTTTTTAGGCATAGGACCCGTGTCT 132 hGAP007 LE
AGATGGTGATGGGATTTCCATTTTTTTAGGCATAGGACCCGTGTCT 133 hGAP008 LE
GCATCGCCCCACTTGATTTTTTTTTAGGCATAGGACCCGTGTCT 134 hGAP009 LE
CACGACGTACTCAGCGCCATTTTTAGGCATAGGACCCGTGTCT 135 hGAP010 LE
GGCAGAGATGATGACCCTTTTGTTTTTAGGCATAGGACCCGTGTCT 136 hGAP011 BL
GGTGAAGACGCCAGTGGACTC 137
[0233] Table 4 shows the relative activity of the dsRNA duplexes of
Table 1 towards reducing TGF-beta mRNA in A549 cells after
incubation with the respective dsRNA at 100 nM or 10 nM
concentration, expressed as % of the TGF-beta mRNA concentration
found in A549 cells that were similarly treated except for
incubation with the same concentration of a dsRNA specific for
ApoB. In addition, IC50 are given for select duplexes.
TABLE-US-00004 TABLE 4 Activity of siRNAs in lowering TGF-beta mRNA
in A549 cells compared to A549 cells treated with a control siRNA
specific for ApoB remaining TGF-beta RNA in % of control Duplex at
100 nM at 10 nM IC(50) identifier duplex conc. duplex conc. [nM]
AL-DP-6837 29 .+-. 5% 47 .+-. 19% AL-DP-6140 84 .+-. 7% 88 .+-. 9%
AL-DP-6838 22 .+-. 12% 34 .+-. 8% 0, 67 AL-DP-6141 81 .+-. 16% 118
.+-. 13% AL-DP-6839 99 .+-. 10% 105 .+-. 5% AL-DP-6142 87 .+-. 3%
115 .+-. 18% AL-DP-6840 31 .+-. 6% 37 .+-. 1% AL-DP-6143 48 .+-. 3%
86 .+-. 7% 23 AL-DP-6841 75 .+-. 13% 65 .+-. 29% AL-DP-6144 80 .+-.
7% 94 .+-. 12% AL-DP-6842 28 .+-. 10% 27 .+-. 4% 0, 12 AL-DP-6145
78 .+-. 9% 100 .+-. 22% AL-DP-6843 77 .+-. 9% 69 .+-. 13%
AL-DP-6146 81 .+-. 3% 102 .+-. 4% AL-DP-6844 48 .+-. 7% 45 .+-. 20%
AL-DP-6147 71 .+-. 18% 99 .+-. 12% AL-DP-6845 20 .+-. 17% 29 .+-.
5% 0, 16 AL-DP-6148 87 .+-. 20% 98 .+-. 11% AL-DP-6846 40 .+-. 14%
49 .+-. 29% AL-DP-6149 90 .+-. 13% 96 .+-. 13% AL-DP-6847 36 .+-.
22% 46 .+-. 27% AL-DP-6150 66 .+-. 22% 75 .+-. 9% AL-DP-6848 14
.+-. 5% 20 .+-. 6% 0, 31 AL-DP-6151 95 .+-. 5% 122 .+-. 20%
AL-DP-6849 33 .+-. 12% 31 .+-. 9% AL-DP-6262 75 .+-. 5% 113 .+-. 4%
AL-DP-6850 68 .+-. 30% 74 .+-. 6% AL-DP-6263 102 .+-. 27% 142 .+-.
19% AL-DP-6851 26 .+-. 11% 52 .+-. 11% AL-DP-6264 23 .+-. 3% 59
.+-. 9% 4, 4 AL-DP-6852 21 .+-. 9% 32 .+-. 18% AL-DP-6265 86 .+-.
15% 79 .+-. 16% AL-DP-6853 41 .+-. 12% 52 .+-. 14% AL-DP-6266 83
.+-. 3% 97 .+-. 32% AL-DP-6854 92 .+-. 12% 118 .+-. 33% AL-DP-6267
79 .+-. 12% 83 .+-. 11% AL-DP-6855 22 .+-. 2% 28 .+-. 6% AL-DP-6268
88 .+-. 15% 107 .+-. 46% AL-DP-6856 43 .+-. 4% 54 .+-. 3%
AL-DP-6269 70 .+-. 11% 92 .+-. 24% AL-DP-6857 13 .+-. 5% 35 .+-.
24% 0, 04 AL-DP-6270 71 .+-. 15% 89 .+-. 30% AL-DP-6858 11 .+-. 3%
28 .+-. 17% 0, 13 AL-DP-6271 34 .+-. 12% 51 .+-. 15% 0, 68
AL-DP-6859 14 .+-. 2% 26 .+-. 11% 0, 15 AL-DP-6272 33 .+-. 5% 56
.+-. 6% 2, 2 AL-DP-6860 31 .+-. 1% 45 .+-. 11% AL-DP-6273 118 .+-.
29% 161 .+-. 17% AL-DP-6861 32 .+-. 7% 65 .+-. 37% AL-DP-6274 76
.+-. 4% 111 .+-. 11% AL-DP-6862 16 .+-. 3% 27 .+-. 14% 0, 19
AL-DP-6275 93 .+-. 41% 96 .+-. 20% AL-DP-6863 36 .+-. 17% 37 .+-.
15% AL-DP-6276 56 .+-. 12% 74 .+-. 32% AL-DP-6864 46 .+-. 26% 62
.+-. 19% AL-DP-6277 110 .+-. 18% 83 .+-. 40%
[0234] From Table 4, it can be seen that, at 100 nM concentration,
AL-DP-6858, AL-DP-6857, AL-DP-6859, AL-DP-6848, and AL-DP-6862
reduced TGF-beta mRNA in A549 cells by more than 80%. Additionally,
under these circumstances, AL-DP-6845, AL-DP-6852, AL-DP-6838,
AL-DP-6855, AL-DP-6264, AL-DP-6851, AL-DP-6842, and AL-DP-6837
reduced TGF-beta mRNA in A549 cells by more than 70%. Additionally,
AL-DP-6840, AL-DP-6860, AL-DP-6861, AL-DP-6849, AL-DP-6272,
AL-DP-6271, AL-DP-6847, AL-DP-6863 reduced TGF-beta mRNA in A549
cells by more than 60%. Additionally, AL-DP-6846, AL-DP-6853,
AL-DP-6856, AL-DP-6864, AL-DP-6844, AL-DP-6143 reduced TGF-beta
mRNA in A549 cells by more than 50%. Additionally, AL-DP-6276
reduced TGF-beta mRNA in A549 cells by more than 40%. Additionally,
AL-DP-6150 and AL-DP-6850 reduced TGF-beta mRNA in A549 cells by
more than 30%. Finally, AL-DP-6269, AL-DP-6270, AL-DP-6147,
AL-DP-6262, AL-DP-6841, AL-DP-6274, AL-DP-6843, AL-DP-6145, and
AL-DP-6267 reduced TGF-beta mRNA in A549 cells by more than
20%.
Example 3
Screening of Additional siRNA for Activity in Inhibiting TGF-Beta
Expression
[0235] In order to identify as many RNAi agents with suitable
activity as possible, the above screen was extended to further
target sequences. For the selection of an expanded screening set we
re-calculated the predicted specificity based on the newly
available human RefSeq database (Human mRNA sequences in RefSeq
release version 21 (downloaded Jan. 12, 2007)) and selected only
those 220 non-poly-G siRNAs (no occurrence of 3 or more consecutive
guanines) targeting 19mer target sequences in NM.sub.--000660.3
with off-target scores of 3 or more for the antisense strand. These
agents were then screened for their activity in lowering the amount
of TGF-beta mRNA present in A549 cells treated with the agents as
described above, except that transfection with the agents was
performed directly after seeding, rather than 24 h after seeding,
and that the concentration of the agents in the transfection
mixture was 30 nM.
[0236] As may be concluded from the results shown in Table 5,
AD-14501, AD-14503, AD-14507, AD-14554, AD-14594, AD-14597, and
AD-14633 reduced TGF-beta mRNA in A549 cells by more than 90%.
Additionally, under these circumstances, AD-14419, AD-14420,
AD-14421, AD-14422, AD-14423, AD-14428, AD-14430, AD-14434,
AD-14438, AD-14449, AD-14453, AD-14459, AD-14460, AD-14464,
AD-14469, AD-14470, AD-14473, AD-14474, AD-14476, AD-14486,
AD-14490, AD-14495, AD-14496, AD-14497, AD-14509, AD-14515,
AD-14522, AD-14526, AD-14540, AD-14552, AD-14555, AD-14557,
AD-14565, AD-14567, AD-14568, AD-14582, AD-14588, AD-14590,
AD-14592, AD-14598, AD-14600, AD-14601, AD-14603, AD-14604,
AD-14608, AD-14610, AD-14612, AD-14614, AD-14622, AD-14634, and
AD-14635 reduced TGF-beta mRNA in A549 cells by more than 80%.
Additionally, under these circumstances, AD-14425, AD-14426,
AD-14427, AD-14429, AD-14431, AD-14436, AD-14437, AD-14441,
AD-14448, AD-14458, AD-14463, AD-14467, AD-14477, AD-14482,
AD-14483, AD-14491, AD-14502, AD-14505, AD-14508, AD-14512,
AD-14513, AD-14519, AD-14524, AD-14537, AD-14546, AD-14548,
AD-14549, AD-14559, AD-14563, AD-14564, AD-14569, AD-14578,
AD-14579, AD-14587, AD-14595, AD-14599, AD-14607, AD-14609,
AD-14629, AD-14631, and AD-14637 reduced TGF-beta mRNA in A549
cells by more than 70%. Additionally, under these circumstances,
AD-14447, AD-14451, AD-14471, AD-14484, AD-14487, AD-14498,
AD-14523, AD-14527, AD-14529, AD-14539, AD-14541, AD-14558,
AD-14561, AD-14573, AD-14589, AD-14596, AD-14605, AD-14615,
AD-14626, and AD-14630 reduced TGF-beta mRNA in A549 cells by more
than 60%. Additionally, under these circumstances, AD-14432,
AD-14480, AD-14485, AD-14488, AD-14499, AD-14504, AD-14516,
AD-14518, AD-14520, AD-14547, AD-14572, AD-14577, AD-14580,
AD-14583, AD-14584, AD-14618, AD-14621, and AD-14625 reduced
TGF-beta mRNA in A549 cells by more than 50%. Additionally, under
these circumstances, AD-14435, AD-14445, AD-14450, AD-14456,
AD-14466, AD-14475, AD-14489, AD-14492, AD-14511, AD-14535,
AD-14536, AD-14538, AD-14553, AD-14560, AD-14562, AD-14576,
AD-14581, AD-14586, and AD-14627 reduced TGF-beta mRNA in A549
cells by more than 40%. Additionally, under these circumstances,
AD-14433, AD-14472, AD-14478, AD-14493, AD-14510, AD-14544,
AD-14566, AD-14570, AD-14575, AD-14593, and AD-14613 reduced
TGF-beta mRNA in A549 cells by more than 30%. Additionally, under
these circumstances, AD-14424, AD-14442, AD-14444, AD-14446,
AD-14454, AD-14525, AD-14543, AD-14571, AD-14606, AD-14611,
AD-14616, AD-14617, AD-14619, and AD-14638 reduced TGF-beta mRNA in
A549 cells by more than 20%.
TABLE-US-00005 TABLE 5 Additional RNAi agents targeting hTGF-beta
Remaining TGF-beta Position of mRNA in % target SEQ SEQ of controls
Duplex 19mer target sequence in Sense ID Antisense ID .+-. stand.
identifer sequence NM_000660.3 strand sequence NO: strand sequence
NO: dev. AD-14419 aauuccuggcgauaccuca 1396-1414
aauuccuggcgauaccucaTT 138 ugagguaucgccaggaauuTT 139 16 .+-. 1
AD-14420 ucgcgcccaucuagguuau 654-672 ucgcgcccaucuagguuauTT 140
auaaccuagaugggcgcgaTT 141 19 .+-. 2 AD-14421 ggucacccgcgugcuaaug
1188-1206 ggucacccgcgugcuaaugTT 142 cauuagcacgcgggugaccTT 143 19
.+-. 2 AD-14422 aggucacccgcgugcuaau 1187-1205 aggucacccgccugcuaauTT
144 auuagcacgcgggugaccuTT 145 10 .+-. 1 AD-14423
cacccgcgugcuaauggug 1191-1209 cacccgcgugcuaauggugTT 146
caccauuagcacgcgggugTT 147 18 .+-. 1 AD-14424 uacaaccagcauaacccgg
1894-1912 uacaaccagcauaacccggTT 148 ccggguuaugcugguuguaTT 149 79
.+-. 5 AD-14425 aucgcgcccaucuagguua 653-671 aucgcgcccaucuagguuaTT
150 uaaccuagaugggcgcgauTT 151 24 .+-. 1 AD-14426
guaccagaucgcgcccauc 646-664 guaccagaucgcgcccaucTT 152
gaugggcgcgaucugguacTT 153 21 .+-. 1 AD-14427 agacggaucucucuccgac
589-607 agacggaucucucuccgacTT 154 gucggagagagauccgucuTT 155 26 .+-.
4 AD-14428 cgcgcccaucuagguuauu 655-673 cgcgcccaucuagguuauuTT 156
aauaaccuagaugggcgcgTT 157 19 .+-. 1 AD-14429 cccgcgcauccuagacccu
558-576 cccgcgcauccuagacccuTT 158 agggucuaggaugcgcgggTT 159 24 .+-.
3 AD-14430 gcgcccaucuagguuauuu 656-674 gcgcccaucuagguuauuuTT 160
aaauaaccuagaugggcgcTT 161 18 .+-. 1 AD-14431 uccgugggauacugagaca
674-692 uccgugggauacugagacaTT 162 ugucucaguaucccacggaTT 163 25 .+-.
3 AD-14432 cuuucgccuuagcgcccac 1515-1533 cuuucgccuuagcgcccacTT 164
gugggcgcuaaggcgaaagTT 165 41 .+-. 2 AD-14433 acccgcgugcuaauggugg
1192-1210 acccgcgugcuaaugguggTT 166 ccaccauuagcacgcggguTT 167 60
.+-. 4 AD-14434 gcaacaauuccuggcgaua 1391-1409 gcaacaauuccuggcgauaTT
168 uaucgccaggaauuguugcTT 169 10 .+-. 0 AD-14435
aacggguucacuaccggcc 1576-1594 aacggguucacuaccggccTT 170
ggccgguagugaacccguuTT 171 57 .+-. 4 AD-14436 cggguucacuaccggccgc
1578-1596 cggguucacuaccggccgcTT l72 gcggccgguagugaacccgTT 173 28
.+-. 3 AD-14437 cuguauuuaaggacacccg 2117-2135 cuguauuuaaggacacccgTT
174 cggguguccuuaaauacagTT 175 21 .+-. 1 AD-14438
agguuauuuccgugggaua 666-684 agguuauuuccgugggauaTT 176
uaucccacggaaauaaccuTT 177 19 .+-. 1 AD-14439 cccucgggagucgccgacc
458-476 cccucgggagucgccgaccTT 178 ggucggcgacucccgagggTT 179 90 .+-.
4 AD-14440 ugugcggcagugguugagc 1476-1494 ugugcggcagugguugagcTT 180
gcucaaccacugccgcacaTT 181 375 .+-. 34 AD-14441 caaccagcauaacccgggc
1896-1914 caaccagcauaacccgggcTT 182 gcccggguuaugcugguugTT 183 28
.+-. 4 AD-14442 uuuugagacuuuuccguug 281-299 uuuugagacuuuuccguugTT
184 caacggaaaagucucaaaaTT 185 75 .+-. 2 AD-14443
uuugagacuuuuccguugc 282-300 uuugagacuuuuccguugcTT 186
gcaacggaaaagucucaaaTT 187 98 .+-. 9 AD-14444 cucuuggcgcgacgcugcc
326-344 cucuuggcgcgacgcugccTT 188 ggcagcgucgcgccaagagTT 189 70 .+-.
4 AD-14445 ucugguaccagaucgcgcc 642-660 ucugguaccagaucgcgccTT 190
ggcgcgaucugguaccagaTT 191 50 .+-. 2 AD-14446 acaccagcccuguucgcgc
817-835 acaccagcccuguucgcgcTT 192 gcgcgaacagggcugguguTT 193 71 .+-.
4 AD-14447 cggccggccgcgggacuau 940-958 cggccggccgcgggacuauTT 194
auagucccgcggccggccgTT 195 38 .+-. 1 AD-14448 gguaccugaacccguguug
1296-1314 gguaccugaacccguguugTT 196 caacacggguucagguaccTT 197 21
.+-. 1 AD-14449 guaccugaacccguguugc 1297-1315 guaccugaacccguguugcTT
198 gcaacacggguucagguacTT 199 17 .+-. 1 AD-14450
auuccuggcgauaccucag 1397-1415 auuccuggcgauaccucagTT 200
cugagguaucgccaggaauTT 201 58 .+-. 6 AD-14451 auugagggcuuucgccuua
1507-1525 auugagggcuuucgccuuaTT 202 uaaggcgaaagcccucaauTT 203 31
.+-. 3 AD-14452 acaaccagcauaacccggg 1895-1913 acaaccagcauaacccgggTT
204 cccggguuaugcugguuguTT 205 87 .+-. 8 AD-14453
uguauuuaaggacacccgu 2118-2136 uguauuuaaggacacccguTT 206
acggguguccuuaaauacaTT 207 16 .+-. 2 AD-14454 gaggacugcggaucucugu
2174-2192 gaggacugcggaucucuguTT 208 acagagauccgcaguccucTT 209 77
.+-. 5 AD-14455 gggaacacuacuguaguua 2324-2342 gggaacacuacuguaguuaTT
210 uaacuacaguaguguucccTT 211 86 .+-. 4 AD-14456
accagcccuguucgcgcuc 819-837 accagcccuguucgcgcucTT 212
gagcgcgaacagggcugguTT 213 59 .+-. 0 AD-14457 agaggacugcggaucucug
2173-2191 agaggacugcggaucucugTT 214 cagagauccgcaguccucuTT 215 91
.+-. 3 AD-14458 guacuacgugggccgcaag 1968-1986 guacuacgugggccgcaagTT
216 cuugcggcccacguaguacTT 217 24 .+-. 1 AD-14459
gaucgcgcccaucuagguu 652-670 gaucgcgcccaucuagguuTT 218
aaccuagaugggcgcgaucTT 219 13 .+-. 1 AD-14460 aacgaaaucuaugacaagu
1219-1237 aacgaaaucuaugacaaguTT 220 acuugucauagauuucguuTT 221 16
.+-. 2 AD-14461 uucgccuuagcgcccacug 1517-1535 uucgccuuagcgcccacugTT
222 cagugggcgcuaaggcgaaTT 223 107 .+-. 6 AD-14462
aucaacggguucacuaccg 1573-1591 aucaacggguucacuaccgTT 224
cgguagugaacccguugauTT 225 107 .+-. 3 AD-14463 ccgcgcauccuagacccuu
559-577 ccgcgcauccuagacccuuTT 226 aagggucuaggaugcgcggTT 227 24 .+-.
3 AD-14464 gcgcauccuagacccuuuc 561-579 gcgcauccuagacccuuucTT 228
gaaagggucuaggaugcgcTT 229 15 .+-. 1 AD-14465 uuucgccuuagcgcccacu
1516-1534 uuucgccuuagcgcccacuTT 230 agugggcgcuaaggcgaaaTT 231 85
.+-. 6 AD-14466 cugguaccagaucgcgccc 641-661 cugguaccagaucgcgcccTT
232 gggcgcgaucugguaccagTT 233 54 .+-. 3 AD-14467
cagaucgcgcccaucuagg 650-668 cagaucgcgcccaucuaggTT 234
ccuagaugggcgcgaucugTT 235 25 .+-. 1 AD-14468 uccuaccuuuugccgggag
763-781 uccuaccuuuugccgggagTT 236 cucccggcaaaagguaggaTT 237 93 .+-.
5 AD-14469 cccuguucgcgcucucggc 824-842 cccuguucgcgcucucggcTT 238
gccgagagcgcgaacagggTT 239 16 .+-. 2 AD-14470 ccuguucgcgcucucggca
825-843 ccuguucgcgcucucggcaTT 240 ugccgagagcgcgaacaggTT 241 16 .+-.
3 AD-14471 cuguucgcgcucucggcag 826-844 cuguucgcgcucucggcagTT 242
cugccgagagcgcgaacagTT 243 32 .+-. 3 AD-14472 aagacuaucgacauggagc
967-985 aagacuaucgacauggagcTT 244 gcuccaugucgauagucuuTT 245 63 .+-.
9 AD-14473 cccgcgugcuaauggugga 1193-1211 cccgcgugcuaaugguggaTT 246
uccaccauuagcacgcgggTT 247 12 .+-. 2 AD-14474 cgugcuaaugguggaaacc
1197-1215 cgugcuaaugguggaaaccTT 248 gguuuccaccauuagcacgTT 249 13
.+-. 1 AD-14475 ccggaguugugcggcagug 1469-1487 ccggaguugugcggcagugTT
250 cacugccgcacaacuccggTT 251 52 .+-. 6 AD-14476
gcuuucgccuuagcgccca 1514-1532 gcuuucgccuuagcgcccaTT 252
ugggcgcuaaggcgaaagcTT 253 16 .+-. 0 AD-14477 agggauaacacacugcaag
1549-1567 agggauaacacacugcaagTT 254 cuugcaguguguuaucccuTT 255 22
.+-. 1 AD-14478 caucaacggguucacuacc 1572-1590 caucaacggguucacuaccTT
256 gguagugaacccguugaugTT 257 68 .+-. 1 AD-14479
uuuaaggacacccgugccc 2122-2140 uuuaaggacacccgugcccTT 258
gggcacggguguccuuaaaTT 259 92 .+-. 5 AD-14480 gacuuuuccguugccgcug
287-305 gacuuuuccguugccgcugTT 260 cagcggcaacggaaaagucTT 261 41 .+-.
5 AD-14481 uuuuccguugccgcuggga 290-308 uuuuccguugccgcugggaTT 262
ucccagcggcaacggaaaaTT 263 81 .+-. 3 AD-14482 gggaccucuuggcgcgacg
321-339 gggaccucuuggcgcgacgTT 264 cgucgcgccaagaggucccTT 265 26 .+-.
1 AD-14483 ggaccucuuggcgcgacgc 322-340 ggaccucuuggcgcgacgcTT 266
gcgucgcgccaagagguccTT 267 28 .+-. 3 AD-14484 gaccucuuggcgcgacgcu
323-341 gaccucuuggcgcgacgcuTT 268 agcgucgcgccaagaggucTT 269 33 .+-.
1 AD-14485 ccuacacggcgucccucag 419-437 ccuacacggcgucccucagTT 270
cugagggacgccguguaggTT 271 49 .+-. 1 AD-14486 cgcgcauccuagacccuuu
560-578 cgcgcauccuagacccuuuTT 272 aaagggucuaggaugcgcgTT 273 15 .+-.
2 AD-14487 cauccuagacccuuucucc 564-582 cauccuagacccuuucuccTT 274
ggagaaagggucuaggaugTT 275 33 .+-. 7 AD-14488 ugguaccagaucgcgccca
644-662 ugguaccagaucgcgcccaTT 276 ugggcgcgaucugguaccaTT 277 49 .+-.
5 AD-14489 uaccagaucgcgcccaucu 647-665 uaccagaucgcgcccaucuTT 278
agaugggcgcgaucugguaTT 279 59 .+-. 6 AD-14490 agaucgcgcccaucuaggu
651-669 agaucgcgcccaucuagguTT 280 accuagaugggcgcgaucuTT 281 19 .+-.
3 AD-14491 gcccaucuagguuauuucc 658-676 gcccaucuagguuauuuccTT 282
ggaaauaaccuagaugggcTT 283 25 .+-. 1 AD-14492 cuaccuuuugccgggagac
765-783 cuaccuuuugccgggagacTT 284 gucucccggcaaaagguagTT 285 55 .+-.
2 AD-14493 guucgcgcucucggcagug 828-846 guucgcgcucucggcagugTT 286
cacugccgagagcgcgaacTT 287 66 .+-. 9 AD-14494 uucgcgcucucggcagugc
829-847 uucgcgcacucggcagugcTT 288 gcacugccgagagcgcgaaTT 289 102
.+-. 12 AD-14495 accugcaagacuaucgaca 961-979 accugcaagacuaucgacaTT
290 ugucgauagucuugcagguTT 291 19 .+-. 1 AD-14496
cugcaagacuaucgacaug 963-981 cugcaagacuaucgacaugTT 292
caugucgauagucuugcagTT 293 14 .+-. 1 AD-14497 gcaagacuaucgacaugga
965-983 gcaagacuaucgacauggaTT 294 uccaugucgauagucuugcTT 295 15 .+-.
1 AD-14498 caagacuaucgacauggag 966-984 caagacuaucgacauggagTT 296
cuccaugucgauagucuugTT 297 37 .+-. 3
AD-14499 uguacaacagcacccgcga 1106-1124 uguacaacagcacccgcgaTT 298
ucgcgggugcuguuguacaTT 299 42 .+-. 2 AD-14500 ugaggccgacuacuacgcc
1164-1182 ugaggccgacuacuacgccTT 300 ggcguaguagucggccucaTT 301 122
.+-. 7 AD-14501 aaacccacaacgaaaucua 1211-1229 aaacccacaacgaaaucuaTT
302 uagauuucguuguggguuuTT 303 9 .+-. 1 AD-14502 ccacaacgaaaucuaugac
1215-1233 ccacaacgaaaucuaugacTT 304 gucauagauuucguuguggTT 305 24
.+-. 2 AD-14503 aguacacacagcauauaua 1246-1264 aguacacacagcauauauaTT
306 uauauaugcuguguguacuTT 307 8 .+-. 0 AD-14504 cgguaccugaacccguguu
1295-1313 cgguaccugaacccguguuTT 308 aacacggguucagguaccgTT 309 45
.+-. 4 AD-14505 uaccugaacccguguugcu 1298-1316 uaccugaacccguguugcuTT
310 agcaacacggguucagguaTT 311 29 .+-. 2 AD-14506
cccguguugcucucccggg 1336-1324 cccguguugcucucccgggTT 312
cccgggagagcaacacgggTT 313 107 .+-. 5 AD-14507 cugcgucugcugaggcuca
1330-1348 cugcgucugcugaggcucaTT 314 ugagccucagcagacgcagTT 315 9
.+-. 1 AD-14508 aggcucaaguuaaaagugg 1342-1360 aggcucaaguuaaaaguggTT
316 ccacuuuuaacuugagccuTT 317 20 .+-. 1 AD-14509
caacaauuccuggcgauac 1392-1410 caacaauuccuggcgauacTT 318
guaucgccaggaauuguugTT 319 14 .+-. 1 AD-14510 aacaauuccuggcgauacc
1393-1411 aacaauuccuggcgauaccTT 320 gguaucgccaggaauuguuTT 321 65
.+-. 3 AD-14511 uggcgauaccucagcaacc 1402-1420 uggcgauaccucagcaaccTT
322 gguugcugagguaucgccaTT 323 58 .+-. 5 AD-145l2
cgauaccucagcaaccggc 1405-1423 cgauaccucagcaaccggcTT 324
gccgguugcugagguaucgTT 325 20 .+-. 0 AD-14513 gauaccucagcaaccggcu
1406-1424 gauaccucagcaaccggcuTT 326 agccgguugcugagguaucTT 327 24
.+-. 1 AD-14514 cuggcacccagcgacucgc 1426-1444 cuggcacccagcgacucgcTT
328 gcgagucgcugggugccagTT 329 86 .+-. 13 AD-14515
gacucgccagagugguuau 1438-l456 gacucgccagagugguuauTT 330
auaaccacucuggcgagucTT 331 11 .+-. 1 AD-14516 acucgccagagugguuauc
1439-1457 acucgccagagugguuaucTT 332 gauaaccacucuggcgaguTT 333 47
.+-. 2 AD-14517 ugucaccggaguugugcgg 1464-1482 ugucaccggaguugugcggTT
334 ccgcacaacuccggugacaTT 335 115 .+-. 7 AD-14518
caccggaguugugcggcag 1467-1485 caccggaguugugcggcagTT 336
cugccgcacaacuccggugTT 337 44 .+-. 3 AD-14519 accggaguugugcggcagu
1468-1486 accggaguugugcggcaguTT 338 acugccgcacaacuccgguTT 339 28
.+-. 2 AD-14520 aaauugagggcuuucgccu 1505-1523 aaauugagggcuuucgccuTT
340 aggcgaaagcccucaauuuTT 341 46 .+-. 2 AD-14521
uugagggcuuucgccuuag 1508-1526 uugagggcuuucgccuuagTT 342
cuaaggcgaaagcccucaaTT 343 130 .+-. 15 AD-14522 gggcuuucgccuuagcgcc
1512-1530 gggcuuucgccuuagcgccTT 344 ggcgcuaaggcgaaagcccTT 345 14
.+-. 1 AD-14523 ccuuagcgcccacugcucc 1521-1539 ccuuagcgcccacugcuccTT
346 ggagcagugggcgcuaaggTT 347 30 .+-. 1 AD-14524
aaguggacaucaacggguu 1565-1583 aaguggacaucaacggguuTT 348
aacccguugauguccacuuTT 349 20 .+-. 1 AD-14525 caacggguucacuaccggc
1575-1593 caacggguucacuaccggcTT 350 gccgguagugaacccguugTT 351 73
.+-. 6 AD-14526 cuaccggccgccgagguga 1586-1604 cuaccggccgccgaggugaTT
352 ucaccucggcggccgguagTT 353 18 .+-. 1 AD-14527
caucugcaaagcucccggc 1675-1693 caucugcaaagcucccggcTT 354
gccgggagcuuugcagaugTT 355 37 .+-. 2 AD-14528 cguguacuacgugggccgc
1965-1983 cguguacuacgugggccgcTT 356 gcggcccacguaguacacgTT 357 80
.+-. 4 AD-14529 caacaugaucgugcgcucc 2007-2025 caacaugaucgugcgcuccTT
358 ggagcgcacgaucauguugTT 359 35 .+-. 2 AD-14530
cugugucauugggcgccug 2189-2207 cugugucauugggcgccugTT 360
caggcgcccaaugacacagTT 361 89 .+-. 6 AD-14531 ugccugucugcacuauucc
2273-2291 ugccugucugcacuauuccTT 362 ggaauagugcagacaggcaTT 363 97
.+-. 4 AD-14532 cacuauuccuuugcccggc 2283-2301 cacuauuccuuugcccggcTT
364 gccgggcaaaggaauagugTT 365 98 .+-. 8 AD-14533
aacacuacuguaguuagau 2327-2345 aacacuacuguaguuagauTT 366
aucuaacuacaguaguguuTT 367 89 .+-. 8 AD-14534 acacuacuguaguuagauc
2328-2346 acacuacuguaguuagaucTT 368 gaucuaacuacaguaguguTT 369 80
.+-. 2 AD-14535 gucugagacgagccgccgc 185-203 gucugagacgagccgccgcTT
370 gcggcggcucgucucagacTT 371 57 .+-. 4 AD-14536
cccuacacggcgucccuca 418-436 cccuacacggcgucccucaTT 372
ugagggacgccguguagggTT 373 50 .+-. 4 AC-14537 acggaucucucuccgaccu
591-609 acggaucucucuccgaccuTT 374 aggucggagagagauccguTT 375 23 .+-.
3 AD-14538 ggccggccgcgggacuauc 941-959 ggccggccgcgggacuaucTT 376
gauagucccgcggccggccTT 377 50 .+-. 2 aD-14539 cgcggccagauccugucca
1015-1033 cgcggccagauccuguccaTT 378 uggacaggaucuggccgcgTT 379 34
.+-. 3 AD-14540 ggaaacccacaacgaaauc 1209-1227 ggaaacccacaacgaaaucTT
380 gauuucguuguggguuuccTT 381 11 .+-. 0 AD-14541
acaauuccuggcgauaccu 1394-1412 acaauuccuggcgauaccuTT 382
agguaucgccaggaauuguTT 383 31 .+-. 2 AD-14542 agucugagacgagccgccg
184-202 agucugagacgagccgccgTT 384 cggcggcucgucucagacuTT 385 87 .+-.
1 AD-14543 cacggcgucccucaggcgc 423-441 cacggcgucccucaggcgcTT 386
gcgccugagggacgccgugTT 387 76 .+-. 3 AD-14544 acggcgucccucaggcgcc
424-442 acggcgucccucaggcgccTT 388 ggcgccugagggacgccguTT 389 68 .+-.
3 AD-14545 accagcccucgggagucgc 453-471 accagcccucgggagucgcTT 390
gcgacucccgagggcugguTT 391 96 .+-. 7 AD-14546 gcauccuagacccuuucuc
563-581 gcauccuagacccuuucucTT 392 gagaaagggucuaggaugcTT 393 22 .+-.
2 AD-14547 gacggaucucucuccgacc 590-608 gacggaucucucuccgaccTT 394
ggucggagagagauccgucTT 395 43 .+-. 2 AD-14548 gguaccagaucgcgcccau
645-663 gguaccagaucgcgcccauTT 396 augggcgcgaucugguaccTT 397 27 .+-.
3 AD-14549 uagguuauuuccgugggau 665-683 uagguuauuuccgugggauTT 398
aucccacggaaauaaccuaTT 399 22 .+-. 1 AD-14550 accuuuugccgggagaccc
767-785 accuuuugccgggagacccTT 400 gggucucccggcaaaagguTT 401 89 .+-.
2 AD-14551 ucgcgcucucggcagugcc 830-848 ucgcgcucucggcagugccTT 402
ggcacugccgagagcgcgaTT 403 95 .+-. 3 AD-14552 uauccaccugcaagacuau
956-974 uauccaccugcaagacuauTT 404 auagucuugcagguggauaTT 405 15 .+-.
1 AD-14553 ccgcggccagauccugucc 1014-1032 ccgcggccagauccuguccTT 406
ggacaggaucuggccgcggTT 407 53 .+-. 5 AD-14554 aacccacaacgaaaucuau
1212-1230 aacccacaacgaaaucuauTT 408 auagauuucguuguggguuTT 409 8
.+-. 1 AD-14555 agcgguaccugaacccgug 1293-1311 agcgguaccugaacccgugTT
410 cacggguucagguaccgcuTT 411 14 .+-. 2 AD-14556
uggcacccagcgacucgcc 1427-1445 uggcacccagcgacucgccTT 412
ggcgagucgcugggugccaTT 413 88 .+-. 9 AD-14557 cucgccagagugguuaucu
1440-1458 cucgccagagugguuaucuTT 414 agauaaccacucuggcgagTT 415 18
.+-. 2 AD-14558 cggcagugguugagccgug 1480-1498 cggcagugguugagccgugTT
416 cacggcucaaccacugccgTT 417 32 .+-. 2 AD-14559
gugguugagccguggaggg 1485-1503 gugguugagccguggagggTT 418
cccuccacggcucaaccacTT 419 24 .+-. 1 AD-14560 gagggcuuucgccuuagcg
1510-1528 gagggcuuucgccuuagcgTT 420 cgcuaaggcgaaagcccucTT 421 51
.+-. 2 AD-14561 agggcuuucgccuuagcgc 1511-1529 agggcuuucgccuuagcgcTT
422 gcgcuaaggcgaaagcccuTT 423 37 .+-. 1 AD-14562
ggugaccuggccaccauuc 1600-1618 ggugaccuggccaccauucTT 424
gaaugguggccaggucaccTT 425 51 .+-. 5 AD-14563 accauucauggcaugaacc
1612-1630 accauucauggcaugaaccTT 426 gguucaugccaugaaugguTT 427 29
.+-. 2 AD-14564 cauggcaugaaccggccuu 1618-1636 cauggcaugaaccggccuuTT
428 aaggccgguucaugccaugTT 429 25 .+-. 2 AD-14565
ccaacuauugcuucagcuc 1712-1730 ccaacuauugcuucagcucTT 430
gagcugaagcaauaguuggTT 431 11 .+-. 1 AD-14566 gaacugcugcgugcggcag
1740-1758 gaacugcugcgugcggcagTT 432 cugccgcacgcagcaguucTT 433 62
.+-. 3 AD-14567 gcccuguacaaccagcaua 1888-1906 gcccuguacaaccagcauaTT
434 uaugcugguuguacagggcTT 435 10 .+-. 0 AD-14568
cuguacaaccagcauaacc 1891-1909 cuguacaaccagcauaaccTT 436
gguuaugcugguuguacagTT 437 19 .+-. 0 AD-14569 guacaaccagcauaacccg
1893-1911 guacaaccagcauaacccgTT 438 cggguuaugcugguuguacTT 439 21
.+-. 1 AD-14570 cuauuccuuugcccggcau 2285-2303 cuauuccuuugcccggcauTT
440 augccgggcaaaggaauagTT 441 65 .+-. 2 AD-14571
agcggaggaaggagucgcc 143-161 agcggaggaaggagucgccTT 442
ggcgacuccuuccuccgcuTT 443 75 .+-. 2 AD-14572 ggaggaaggagucgccgag
146-164 ggaggaaggagucgccgagTT 444 cucggcgacuccuuccuccTT 445 43 .+-.
2 AD-14573 acuuuugagacuuuuccgu 279-297 acuuuugagacuuuuccguTT 446
acggaaaagucucaaaaguTT 447 32 .+-. 2 AD-14574 uugagacuuuuccguugcc
283-301 uugagacuuuuccguugccTT 448 ggcaacggaaaagucucaaTT 449 96 .+-.
4 AD-14575 accucuuggcgcgacgcug 324-342 accucuuggcgcgacgcugTT 450
cagcgucgcgccaagagguTT 451 67 .+-. 3 AD-14576 gacccggccucccgcaaag
473-491 gacccggccucccgcaaagTT 452 cuuugcgggaggccgggucTT 453 54 .+-.
3 AD-14577 cccggccucccgcaaagac 475-493 cccggccucccgcaaagacTT 454
gucuuugcgggaggccgggTT 455 48 .+-. 4 AD-14578 cuccuccaggagacggauc
579-597 cuccuccaggagacggaucTT 456 gauccgucuccuggaggagTT 457 25 .+-.
1 AD-14579 caccuucugguaccagauc 637-655 caccuucugguaccagaucTT 458
gaucugguaccagaaggugTT 459 25 .+-. 1 AD-14580 cuucugguaccagaucgcg
640-658 cuucugguaccagaucgcgTT 460 cgcgaucugguaccagaagTT 461 42 .+-.
2 AD-14581 uucugguaccagaucgcgc 641-659 uucugguaccagaucgcgcTT 462
gcgcgaucugguaccagaaTT 463 56 .+-. 2 AD-14582 gguuauuuccgugggauac
667-685 gguuauuuccgugggauacTT 464
guaucccacggaaauaaccTT 465 19 .+-. 2 AD-14583 accucagcuuucccucgag
740-758 accucagcuuucccucgagTT 466 cucgagggaaagcugagguTT 467 40 .+-.
4 AD-14584 ccucagcuuucccucgagg 741-759 ccucagcuuucccucgaggTT 468
ccucgagggaaagcugaggTT 469 47 .+-. 5 AD-14585 uaccuuuugccgggagacc
766-784 uaccuuuugccgggagaccTT 470 ggucucccggcaaaagguaTT 471 102
.+-. 7 AD-14586 ccaugccgcccuccgggcu 866-884 ccaugccgcccuccgggcuTT
472 agcccggagggcggcauggTT 473 54 .+-. 3 AD-14587
acuauccaccugcaagacu 954-972 acuauccaccugcaagacuTT 474
agucuugcagguggauaguTT 475 22 .+-. 2 AD-14588 cuauccaccugcaagacua
955-973 cuauccaccugcaagacuaTT 476 uagucuugcagguggauagTT 477 14 .+-.
1 AD-14589 guccaagcugcggcucgcc 1029-1047 guccaagcugcggcucgccTT 478
ggcgagccgcagcuuggacTT 479 31 .+-. 2 AD-14590 cgagccugaggccgacuac
1158-1176 cgagccugaggccgacuacTT 480 guagucggccucaggcucgTT 481 16
.+-. 1 AD-14591 ugguggaaacccacaacga 1205-1223 ugguggaaacccacaacgaTT
482 ucguuguggguuuccaccaTT 483 98 .+-. 4 AD-14592
acccacaacgaaaucuaug 1213-1231 acccacaacgaaaucuaugTT 484
cauagauuucguuguggguTT 485 14 .+-. 1 AD-14593 ucaagcagaguacacacag
1238-1256 ucaagcagaguacacacagTT 486 cuguguguacucugcuugaTT 487 67
.+-. 8 AD-14594 cagaguacacacagcauau 1243-1261 cagaguacacacagcauauTT
488 auaugcuguguguacucugTT 489 9 .+-. 1 AD-14595 ucuucaacacaucagagcu
1268-1286 ucuucaacacaucagagcuTT 490 agcucugauguguugaagaTT 491 23
.+-. 3 AD-14596 gcgguaccugaacccgugu 1294-1312 gcgguaccugaacccguguTT
492 acacggguucagguaccgcTT 493 33 .+-. 2 AD-14597
cugcugaggcucaaguuaa 1336-1354 cugcugaggcucaaguuaaTT 494
uuaacuugagccucagcagTT 495 8 .+-. 1 AD-14598 gcgauaccucagcaaccgg
1404-1422 gcgauaccucagcaaccggTT 496 ccgguugcugagguaucgcTT 497 15
.+-. 0 AD-14599 ggcacccagcgacucgcca 1428-1446 ggcacccagcgacucgccaTT
498 uggcgagucgcugggugccTT 499 20 .+-. 2 AD-14600
ccagcgacucgccagagug 1433-1451 ccagcgacucgccagagugTT 500
cacucuggcgagucgcuggTT 501 17 .+-. 2 AD-14601 gagugguuaucuuuugaug
1447-1465 gagugguuaucuuuugaugTT 502 caucaaaagauaaccacucTT 503 13
.+-. 1 AD-14602 uugugcggcagugguugag 1475-1493 uugugcggcagugguugagTT
504 cucaaccacugccgcacaaTT 505 132 .+-. 9 AD-14603
ugugacagcagggauaaca 1540-1558 ugugacagcagggauaacaTT 506
uguuaucccugcugucacaTT 507 16 .+-. 3 AD-14604 acagcagggauaacacacu
1544-1562 acagcagggauaacacacuTT 508 aguguguuaucccugcuguTT 509 19
.+-. 1 AD-14605 cagcagggauaacacacug 1545-1563 cagcagggauaacacacugTT
510 caguguguuaucccugcugTT 511 34 .+-. 2 AD-14606
uaacacacugcaaguggac 1554-1572 uaacacacugcaaguggacTT 512
guccacuugcaguguguuaTT 513 76 .+-. 2 AD-14607 guucacuaccggccgccga
1581-1599 guucacuaccggccgccgaTT 514 ucggcggccgguagugaacTT 515 23
.+-. 1 AD-14600 uggccaccauucauggcau 1607-1625 uggccaccauucauggcauTT
516 augccaugaaugguggccaTT 517 19 .+-. 1 AD-14609
cauucauggcaugaaccgg 1614-1632 cauucauggcaugaaccggTT 518
ccgguucaugccaugaaugTT 519 24 .+-. 2 AD-14610 cagcaucugcaaagcuccc
1672-1690 cagcaucugcaaagcucccTT 520 gggagcuuugcagaugcugTT 521 17
.+-. 2 AD-14611 gcaaagcucccggcaccgc 1680-1698 gcaaagcucccggcaccgcTT
522 gcggugccgggagcuuugcTT 523 73 .+-. 5 AD-14612
ggagaagaacugcugcgug 1734-1752 ggagaagaacugcugcgugTT 524
cacgcagcaguucuucuccTT 525 13 .+-. 0 AD-14613 gaagaacugcugcgugcgg
1737-1755 gaagaacugcugcgugcggTT 526 ccgcacgcagcaguucuucTT 527 62
.+-. 4 AD 14614 gcuggaaguggauccacga 1787-1805 gcuggaaguggauccacgaTT
528 ucguggauccacuuccagcTT 529 12 .+-. 1 AD-14615
uuuggagccuggacacgca 1853-1871 uuuggagccuggacacgcaTT 530
ugcguguccaggcuccaaaTT 531 35 .+-. 1 AD-14616 uuggagccuggacacgcag
1854-1872 uuggagccuggacacgcagTT 532 cugcguguccaggcuccaaTT 533 76
.+-. 7 AD-14617 gccgugcugcgugccgcag 1926-1944 gccgugcugcgugccgcagTT
534 cugcggcacgcagcacggcTT 535 71 .+-. 5 AD-14618
uguccaacaugaucgugcg 2003-2021 uguccaacaugaucgugcgTT 536
cgcacgaucauguuggacaTT 537 41 .+-. 2 AD-14619 cugcggaucucugugucau
2179-2197 cugcggaucucugugucauTT 538 augacacagagauccgcagTT 539 76
.+-. 7 AD-14620 gcacuauuccuuugcccgg 2282-2300 gcacuauuccuuugcccggTT
540 ccgggcaaaggaauagugcTT 541 89 .+-. 9 AD-14621
ccggccucccgcaaagacu 476-494 ccggccucccgcaaagacuTT 542
agucuuugcgggaggccggTT 543 48 .+-. 3 AD-14622 cuacuggugcugacgccug
919-937 cuacuggugcugacgccugTT 544 caggcgucagcaccaguagTT 545 14 .+-.
1 AD-14623 auucauggcaugaaccggc 1615-1633 auucauggcaugaaccggcTT 546
gccgguucaugccaugaauTT 547 103 .+-. 6 AD-14624 aacugcugcgugcggcagc
1741-1759 aacugcugcgugcggcagcTT 548 gcugccgcacgcagcaguuTT 549 82
.+-. 3 AD-14625 cggaggaaggagucgccga 145-163 cggaggaaggagucgccgaTT
550 ucggcgacuccuuccuccgTT 551 47 .+-. 3 AD-14626
ggagcgggaggagggacga 236-254 ggagcgggaggagggacgaTT 552
ucgucccuccucccgcuccTT 553 31 .+-. 1 AD-14627 gcccucgggagucgccgac
457-475 gcccucgggagucgccgacTT 554 gucggcgacucccgagggcTT 555 56 .+-.
6 AD-14628 uuucuccuccaggagacgg 576-594 uuucuccuccaggagacggTT 556
ccgucuccuggaggagaaaTT 557 90 .+-. 3 AD-14629 ucgaggcccuccuaccuuu
754-772 ucgaggcccuccuaccuuuTT 558 aaagguaggagggccucgaTT 559 25 .+-.
2 AD-14630 uacuggugcugacgccugg 920-938 uacuggugcugacgccuggTT 560
ccaggcgucagcaccaguaTT 561 34 .+-. 2 AD-14631 gacuauccaccugcaagac
953-971 gacuauccaccugcaagacTT 562 gucuugcagguggauagucTT 563 24 .+-.
3 AD-14632 uacaacagcacccgcgacc 1108-1126 uacaacagcacccgcgaccTT 564
ggucgcgggugcuguuguaTT 565 113 .+-. 4 AD-14633 ugcugaggcucaaguuaaa
1337-1355 ugcugaggcucaaguuaaaTT 566 uuuaacuugagccucagcaTT 567 8
.+-. 1 AD-14634 ccugugacagcagggauaa 1538-1556 ccugugacagcagggauaaTT
568 uuaucccugcugucacaggTT 569 14 .+-. 1 AD-14635
auaacacacugcaagugga 1553-1571 auaacacacugcaaguggaTT 570
uccacuugcaguguguuauTT 571 18 .+-. 1 AD-14636 uauugcuucagcuccacgg
1717-1735 uauugcuucagcuccacggTT 572 ccguggagcugaagcaauaTT 573 109
.+-. 5 AD-14637 gcuguccaacaugaucgug 2001-2019 gcuguccaacaugaucgugTT
574 cacgaucauguuggacagcTT 575 29 .+-. 1 AD-14638
gucuccaucccugacguuc 2214-2232 gucuccaucccugacguucTT 576
gaacgucagggauggagacTT 577 75 .+-. 11 .sup.1a, g, c, u:
ribonucleotide 5'-monophosphates (except where in 5'-most position,
see below); T: deoxythymidine 5'-monophosphate; all sequences given
5' .fwdarw. 3'; due to synthesis procedures, the 5'-most nucleic
acid is a nucleoside, i.e. does not bear a 5'-monophosphate group
Sequence CWU 1
1
577121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1ggucacccgc gugcuaaugt t
21221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2cauuagcacg cgggugacct t
21321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3ggucacccgc gugcuaaugt t
21421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 4cauuagcacg cgggugacct t
21521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 5gaggucaccc gcgugcuaat t
21621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6uuagcacgcg ggugaccuct t
21721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7gaggucaccc gcgugcuaat t
21821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 8uuagcacgcg ggugaccuct t
21921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 9agcacccgcg accggguggt t
211021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 10ccacccgguc gcgggugcut t
211121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 11agcacccgcg accggguggt t
211221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 12ccacccgguc gcgggugcut t
211321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 13uccacgagcc caagggcuat t
211421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 14uagcccuugg gcucguggat t
211521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 15uccacgagcc caagggcuat t
211621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 16uagcccuugg gcucguggat t
211721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 17auuccggacc agcccucggt t
211821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 18ccgagggcug guccggaaut t
211921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19auuccggacc agcccucggt t
212021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 20ccgagggcug guccggaaut t
212121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 21aggucacccg cgugcuaaut t
212221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 22auuagcacgc gggugaccut t
212321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 23aggucacccg cgugcuaaut t
212421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 24auuagcacgc gggugaccut t
212521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 25uuccggacca gcccucgggt t
212621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 26cccgagggcu gguccggaat t
212721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 27uuccggacca gcccucgggt t
212821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 28cccgagggcu gguccggaat t
212921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 29uguacaacag cacccgcgat t
213021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 30ucgcgggugc uguuguacat t
213121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 31uguacaacag cacccgcgat t
213221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 32ucgcgggugc uguuguacat t
213321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 33cacccgcgug cuaauggugt t
213421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 34caccauuagc acgcgggugt t
213521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 35cacccgcgug cuaauggugt t
213621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 36caccauuagc acgcgggugt t
213721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 37uccggaccag cccucgggat t
213821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 38ucccgagggc ugguccggat t
213921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 39uccggaccag cccucgggat t
214021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 40ucccgagggc ugguccggat t
214121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 41uggaagugga uccacgagct t
214221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 42gcucguggau ccacuuccat t
214321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 43uggaagugga uccacgagct t
214421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44gcucguggau ccacuuccat t
214521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 45ccaagggcua ccaugccaat t
214621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 46uuggcauggu agcccuuggt t
214721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 47ccaagggcua ccaugccaat t
214821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 48uuggcauggu agcccuuggt t
214921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49gucacccgcg ugcuaauggt t
215021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 50ccauuagcac gcgggugact t
215121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 51gucacccgcg ugcuaauggt t
215221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 52ccauuagcac gcgggugact t
215321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 53uacaacagca cccgcgacct t
215421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 54ggucgcgggu gcuguuguat t
215521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 55uacaacagca cccgcgacct t
215621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 56ggucgcgggu gcuguuguat t
215721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 57gcccaagggc uaccaugcct t
215821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 58ggcaugguag cccuugggct t
215921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 59gcccaagggc uaccaugcct t
216021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 60ggcaugguag cccuugggct t
216121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 61aaguggaucc acgagcccat t
216221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 62ugggcucgug gauccacuut t
216321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 63aaguggaucc acgagcccat t
216421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 64ugggcucgug gauccacuut t
216521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 65gggaggaggg acgagcuggt t
216621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 66ccagcucguc ccuccuccct t
216721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 67gggaggaggg acgagcuggt t
216821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 68ccagcucguc ccuccuccct t
216921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 69acaacagcac ccgcgaccgt t
217021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 70cggucgcggg ugcuguugut t
217121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 71acaacagcac ccgcgaccgt t
217221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 72cggucgcggg ugcuguugut t
217321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 73caagggcuac caugccaact t
217421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 74guuggcaugg uagcccuugt t
217521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 75caagggcuac caugccaact t
217621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 76guuggcaugg uagcccuugt t
217721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 77ccggaccagc ccucgggagt t
217821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 78cucccgaggg cugguccggt t
217921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 79ccggaccagc ccucgggagt t
218021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 80cucccgaggg cugguccggt t
218121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 81caacuauugc uucagcucct t
218221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 82ggagcugaag caauaguugt t
218321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 83caacuauugc uucagcucct t
218421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 84ggagcugaag caauaguugt t
218521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 85gugcggcagc uguacauugt t
218621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 86caauguacag cugccgcact t
218721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 87gugcggcagc uguacauugt t
218821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 88caauguacag cugccgcact t
218921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 89ggaaguggau ccacgagcct t
219021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 90ggcucgugga uccacuucct t
219121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 91ggaaguggau ccacgagcct t
219221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 92ggcucgugga uccacuucct t
219321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 93gaaguggauc cacgagccct t
219421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 94gggcucgugg auccacuuct t
219521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 95gaaguggauc cacgagccct t
219621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 96gggcucgugg auccacuuct t
219721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 97ccacgagccc aagggcuact t
219821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 98guagcccuug ggcucguggt t
219921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 99ccacgagccc aagggcuact t
2110021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 100guagcccuug ggcucguggt t
2110121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 101gagcccaagg gcuaccaugt t
2110221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 102caugguagcc cuugggcuct t
2110321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 103gagcccaagg gcuaccaugt t
2110421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 104caugguagcc cuugggcuct t
2110521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 105agcccaaggg cuaccaugct t
2110621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 106gcaugguagc ccuugggcut t
2110721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 107agcccaaggg cuaccaugct t
2110821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 108gcaugguagc ccuugggcut t
2110921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 109cgugcugcgu gccgcaggct t
2111021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 110gccugcggca cgcagcacgt t
2111121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 111cgugcugcgu gccgcaggct t
2111221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 112gccugcggca cgcagcacgt t
2111341DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 113gggctccggt tctgcactct tttttctctt
ggaaagaaag t 4111439DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 114cgcgggtgac ctccttggtt
tttctcttgg aaagaaagt 3911539DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 115gcagctctgc
ccgggagatt tttctcttgg aaagaaagt 3911644DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 116ttttaacttg agcctcagca gactttttct cttggaaaga aagt
4411741DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 117ggttgctgag gtatcgccag tttttctctt
ggaaagaaag t 4111838DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 118gctgggtgcc agcagccttt
ttctcttgga aagaaagt 3811946DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 119cgtagtagtc
ggcctcaggc tctttttagg cataggaccc gtgtct 4612045DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 120tgtgggtttc caccattagc atttttaggc ataggacccg
tgtct 4512146DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 121gcttctcgga gctctgatgt
gttttttagg cataggaccc gtgtct 4612244DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 122gcaacacggg ttcaggtacc tttttaggca taggacccgt gtct
4412349DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 123gaattgttgc tgtatttctg gtacattttt
aggcatagga cccgtgtct 4912425DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 124tgcttgaact
tgtcatagat ttcgt 2512529DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 125tgaagaacat
atatatgctg tgtgtactc 2912619DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 126gctccacgtg
ctgctccac 1912741DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 127gaatttgcca tgggtggaat
tttttctctt ggaaagaaag t 4112841DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 128ggagggatct
cgctcctgga tttttctctt ggaaagaaag t 4112940DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 129ccccagcctt ctccatggtt ttttctcttg gaaagaaagt
4013040DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 130gctcccccct gcaaatgagt ttttctcttg
gaaagaaagt 4013142DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 131agccttgacg gtgccatgtt
tttaggcata ggacccgtgt ct 4213245DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 132gatgacaagc
ttcccgttct ctttttaggc ataggacccg tgtct 4513346DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 133agatggtgat gggatttcca tttttttagg cataggaccc
gtgtct 4613444DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 134gcatcgcccc acttgatttt
tttttaggca taggacccgt gtct 4413543DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 135cacgacgtac
tcagcgccat ttttaggcat aggacccgtg tct 4313646DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 136ggcagagatg atgacccttt tgtttttagg cataggaccc
gtgtct 4613721DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 137ggtgaagacg ccagtggact c
2113821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 138aauuccuggc gauaccucat t
2113921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 139ugagguaucg ccaggaauut t
2114021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 140ucgcgcccau cuagguuaut t
2114121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 141auaaccuaga ugggcgcgat t
2114221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 142ggucacccgc gugcuaaugt t
2114321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 143cauuagcacg cgggugacct t
2114421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 144aggucacccg cgugcuaaut t
2114521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 145auuagcacgc gggugaccut t
2114621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 146cacccgcgug cuaauggugt t
2114721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 147caccauuagc acgcgggugt t
2114821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 148uacaaccagc auaacccggt t
2114921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 149ccggguuaug cugguuguat t
2115021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 150aucgcgccca ucuagguuat t
2115121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 151uaaccuagau gggcgcgaut t
2115221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 152guaccagauc gcgcccauct t
2115321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 153gaugggcgcg aucugguact t
2115421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 154agacggaucu cucuccgact t
2115521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 155gucggagaga gauccgucut t
2115621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 156cgcgcccauc uagguuauut t
2115721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 157aauaaccuag augggcgcgt t
2115821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 158cccgcgcauc cuagacccut t
2115921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 159agggucuagg augcgcgggt t
2116021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 160gcgcccaucu agguuauuut t
2116121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 161aaauaaccua gaugggcgct t
2116221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 162uccgugggau acugagacat t
2116321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 163ugucucagua ucccacggat t
2116421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 164cuuucgccuu agcgcccact t
2116521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 165gugggcgcua aggcgaaagt t
2116621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 166acccgcgugc uaaugguggt t
2116721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 167ccaccauuag cacgcgggut t
2116821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 168gcaacaauuc cuggcgauat t
2116921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 169uaucgccagg aauuguugct t
2117021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 170aacggguuca cuaccggcct t
2117121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 171ggccgguagu gaacccguut t
2117221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 172cggguucacu accggccgct t
2117321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 173gcggccggua gugaacccgt t
2117421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 174cuguauuuaa ggacacccgt t
2117521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 175cggguguccu uaaauacagt t
2117621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 176agguuauuuc cgugggauat t
2117721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 177uaucccacgg aaauaaccut t
2117821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 178cccucgggag ucgccgacct t
2117921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 179ggucggcgac ucccgagggt t
2118021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 180ugugcggcag ugguugagct t
2118121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 181gcucaaccac ugccgcacat t
2118221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 182caaccagcau aacccgggct t
2118321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 183gcccggguua ugcugguugt t
2118421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 184uuuugagacu uuuccguugt t
2118521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 185caacggaaaa gucucaaaat t
2118621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 186uuugagacuu uuccguugct t
2118721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 187gcaacggaaa agucucaaat t
2118821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 188cucuuggcgc gacgcugcct t
2118921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 189ggcagcgucg cgccaagagt t
2119021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 190ucugguacca gaucgcgcct t
2119121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 191ggcgcgaucu gguaccagat t
2119221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 192acaccagccc uguucgcgct t
2119321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 193gcgcgaacag ggcuggugut t
2119421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 194cggccggccg cgggacuaut t
2119521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 195auagucccgc ggccggccgt t
2119621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 196gguaccugaa cccguguugt t
2119721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 197caacacgggu ucagguacct t
2119821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 198guaccugaac ccguguugct t
2119921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 199gcaacacggg uucagguact t
2120021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 200auuccuggcg auaccucagt t
2120121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 201cugagguauc gccaggaaut t
2120221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 202auugagggcu uucgccuuat t
2120321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 203uaaggcgaaa gcccucaaut t
2120421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 204acaaccagca uaacccgggt t
2120521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 205cccggguuau gcugguugut t
2120621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 206uguauuuaag gacacccgut t
2120721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 207acgggugucc uuaaauacat t
2120821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic
oligonucleotide 208gaggacugcg gaucucugut t 2120921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 209acagagaucc gcaguccuct t 2121021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 210gggaacacua cuguaguuat t 2121121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 211uaacuacagu aguguuccct t 2121221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 212accagcccug uucgcgcuct t 2121321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 213gagcgcgaac agggcuggut t 2121421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 214agaggacugc ggaucucugt t 2121521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 215cagagauccg caguccucut t 2121621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 216guacuacgug ggccgcaagt t 2121721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 217cuugcggccc acguaguact t 2121821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 218gaucgcgccc aucuagguut t 2121921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 219aaccuagaug ggcgcgauct t 2122021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 220aacgaaaucu augacaagut t 2122121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 221acuugucaua gauuucguut t 2122221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 222uucgccuuag cgcccacugt t 2122321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 223cagugggcgc uaaggcgaat t 2122421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 224aucaacgggu ucacuaccgt t 2122521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 225cgguagugaa cccguugaut t 2122621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 226ccgcgcaucc uagacccuut t 2122721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 227aagggucuag gaugcgcggt t 2122821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 228gcgcauccua gacccuuuct t 2122921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 229gaaagggucu aggaugcgct t 2123021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 230uuucgccuua gcgcccacut t 2123121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 231agugggcgcu aaggcgaaat t 2123221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 232cugguaccag aucgcgccct t 2123321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 233gggcgcgauc ugguaccagt t 2123421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 234cagaucgcgc ccaucuaggt t 2123521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 235ccuagauggg cgcgaucugt t 2123621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 236uccuaccuuu ugccgggagt t 2123721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 237cucccggcaa aagguaggat t 2123821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 238cccuguucgc gcucucggct t 2123921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 239gccgagagcg cgaacagggt t 2124021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 240ccuguucgcg cucucggcat t 2124121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 241ugccgagagc gcgaacaggt t 2124221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 242cuguucgcgc ucucggcagt t 2124321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 243cugccgagag cgcgaacagt t 2124421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 244aagacuaucg acauggagct t 2124521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 245gcuccauguc gauagucuut t 2124621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 246cccgcgugcu aaugguggat t 2124721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 247uccaccauua gcacgcgggt t 2124821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 248cgugcuaaug guggaaacct t 2124921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 249gguuuccacc auuagcacgt t 2125021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 250ccggaguugu gcggcagugt t 2125121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 251cacugccgca caacuccggt t 2125221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 252gcuuucgccu uagcgcccat t 2125321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 253ugggcgcuaa ggcgaaagct t 2125421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 254agggauaaca cacugcaagt t 2125521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 255cuugcagugu guuaucccut t 2125621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 256caucaacggg uucacuacct t 2125721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 257gguagugaac ccguugaugt t 2125821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 258uuuaaggaca cccgugccct t 2125921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 259gggcacgggu guccuuaaat t 2126021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 260gacuuuuccg uugccgcugt t 2126121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 261cagcggcaac ggaaaaguct t 2126221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 262uuuuccguug ccgcugggat t 2126321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 263ucccagcggc aacggaaaat t 2126421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 264gggaccucuu ggcgcgacgt t 2126521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 265cgucgcgcca agagguccct t 2126621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 266ggaccucuug gcgcgacgct t 2126721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 267gcgucgcgcc aagaggucct t 2126821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 268gaccucuugg cgcgacgcut t 2126921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 269agcgucgcgc caagagguct t 2127021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 270ccuacacggc gucccucagt t 2127121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 271cugagggacg ccguguaggt t 2127221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 272cgcgcauccu agacccuuut t 2127321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 273aaagggucua ggaugcgcgt t 2127421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 274cauccuagac ccuuucucct t 2127521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 275ggagaaaggg ucuaggaugt t 2127621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 276ugguaccaga ucgcgcccat t 2127721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 277ugggcgcgau cugguaccat t 2127821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 278uaccagaucg cgcccaucut t 2127921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 279agaugggcgc gaucugguat t 2128021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 280agaucgcgcc caucuaggut t 2128121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 281accuagaugg gcgcgaucut t 2128221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 282gcccaucuag guuauuucct t 2128321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 283ggaaauaacc uagaugggct t 2128421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 284cuaccuuuug ccgggagact t 2128521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 285gucucccggc aaaagguagt t 2128621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 286guucgcgcuc ucggcagugt t 2128721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 287cacugccgag agcgcgaact t 2128821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 288uucgcgcucu cggcagugct t 2128921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 289gcacugccga gagcgcgaat t 2129021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 290accugcaaga cuaucgacat t 2129121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 291ugucgauagu cuugcaggut t 2129221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 292cugcaagacu aucgacaugt t 2129321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 293caugucgaua gucuugcagt t 2129421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 294gcaagacuau cgacauggat t 2129521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 295uccaugucga uagucuugct t 2129621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 296caagacuauc gacauggagt t 2129721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 297cuccaugucg auagucuugt t 2129821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 298uguacaacag cacccgcgat t 2129921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 299ucgcgggugc uguuguacat t 2130021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 300ugaggccgac uacuacgcct t 2130121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 301ggcguaguag ucggccucat t 2130221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 302aaacccacaa cgaaaucuat t 2130321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 303uagauuucgu uguggguuut t 2130421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 304ccacaacgaa aucuaugact t 2130521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 305gucauagauu ucguuguggt t 2130621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 306aguacacaca gcauauauat t 2130721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 307uauauaugcu guguguacut t 2130821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 308cgguaccuga acccguguut t
2130921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 309aacacggguu cagguaccgt t
2131021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 310uaccugaacc cguguugcut t
2131121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 311agcaacacgg guucagguat t
2131221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 312cccguguugc ucucccgggt t
2131321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 313cccgggagag caacacgggt t
2131421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 314cugcgucugc ugaggcucat t
2131521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 315ugagccucag cagacgcagt t
2131621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 316aggcucaagu uaaaaguggt t
2131721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 317ccacuuuuaa cuugagccut t
2131821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 318caacaauucc uggcgauact t
2131921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 319guaucgccag gaauuguugt t
2132021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 320aacaauuccu ggcgauacct t
2132121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 321gguaucgcca ggaauuguut t
2132221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 322uggcgauacc ucagcaacct t
2132321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 323gguugcugag guaucgccat t
2132421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 324cgauaccuca gcaaccggct t
2132521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 325gccgguugcu gagguaucgt t
2132621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 326gauaccucag caaccggcut t
2132721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 327agccgguugc ugagguauct t
2132821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 328cuggcaccca gcgacucgct t
2132921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 329gcgagucgcu gggugccagt t
2133021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 330gacucgccag agugguuaut t
2133121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 331auaaccacuc uggcgaguct t
2133221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 332acucgccaga gugguuauct t
2133321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 333gauaaccacu cuggcgagut t
2133421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 334ugucaccgga guugugcggt t
2133521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 335ccgcacaacu ccggugacat t
2133621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 336caccggaguu gugcggcagt t
2133721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 337cugccgcaca acuccggugt t
2133821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 338accggaguug ugcggcagut t
2133921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 339acugccgcac aacuccggut t
2134021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 340aaauugaggg cuuucgccut t
2134121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 341aggcgaaagc ccucaauuut t
2134221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 342uugagggcuu ucgccuuagt t
2134321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 343cuaaggcgaa agcccucaat t
2134421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 344gggcuuucgc cuuagcgcct t
2134521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 345ggcgcuaagg cgaaagccct t
2134621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 346ccuuagcgcc cacugcucct t
2134721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 347ggagcagugg gcgcuaaggt t
2134821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 348aaguggacau caacggguut t
2134921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 349aacccguuga uguccacuut t
2135021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 350caacggguuc acuaccggct t
2135121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 351gccgguagug aacccguugt t
2135221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 352cuaccggccg ccgaggugat t
2135321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 353ucaccucggc ggccgguagt t
2135421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 354caucugcaaa gcucccggct t
2135521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 355gccgggagcu uugcagaugt t
2135621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 356cguguacuac gugggccgct t
2135721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 357gcggcccacg uaguacacgt t
2135821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 358caacaugauc gugcgcucct t
2135921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 359ggagcgcacg aucauguugt t
2136021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 360cugugucauu gggcgccugt t
2136121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 361caggcgccca augacacagt t
2136221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 362ugccugucug cacuauucct t
2136321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 363ggaauagugc agacaggcat t
2136421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 364cacuauuccu uugcccggct t
2136521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 365gccgggcaaa ggaauagugt t
2136621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 366aacacuacug uaguuagaut t
2136721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 367aucuaacuac aguaguguut t
2136821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 368acacuacugu aguuagauct t
2136921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 369gaucuaacua caguagugut t
2137021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 370gucugagacg agccgccgct t
2137121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 371gcggcggcuc gucucagact t
2137221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 372cccuacacgg cgucccucat t
2137321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 373ugagggacgc cguguagggt t
2137421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 374acggaucucu cuccgaccut t
2137521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 375aggucggaga gagauccgut t
2137621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 376ggccggccgc gggacuauct t
2137721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 377gauagucccg cggccggcct t
2137821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 378cgcggccaga uccuguccat t
2137921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 379uggacaggau cuggccgcgt t
2138021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 380ggaaacccac aacgaaauct t
2138121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 381gauuucguug uggguuucct t
2138221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 382acaauuccug gcgauaccut t
2138321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 383agguaucgcc aggaauugut t
2138421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 384agucugagac gagccgccgt t
2138521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 385cggcggcucg ucucagacut t
2138621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 386cacggcgucc cucaggcgct t
2138721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 387gcgccugagg gacgccgugt t
2138821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 388acggcguccc ucaggcgcct t
2138921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 389ggcgccugag ggacgccgut t
2139021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 390accagcccuc gggagucgct t
2139121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 391gcgacucccg agggcuggut t
2139221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 392gcauccuaga cccuuucuct t
2139321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 393gagaaagggu cuaggaugct t
2139421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 394gacggaucuc ucuccgacct t
2139521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 395ggucggagag agauccguct t
2139621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 396gguaccagau cgcgcccaut t
2139721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 397augggcgcga ucugguacct t
2139821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 398uagguuauuu ccgugggaut t
2139921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 399aucccacgga aauaaccuat t
2140021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 400accuuuugcc gggagaccct t
2140121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 401gggucucccg gcaaaaggut t
2140221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 402ucgcgcucuc ggcagugcct t
2140321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 403ggcacugccg agagcgcgat t
2140421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 404uauccaccug caagacuaut t
2140521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 405auagucuugc agguggauat t
2140621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 406ccgcggccag auccugucct t
2140721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 407ggacaggauc uggccgcggt t
2140821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 408aacccacaac gaaaucuaut t
2140921DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 409auagauuucg
uuguggguut t 2141021DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 410agcgguaccu gaacccgugt t
2141121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 411cacggguuca gguaccgcut t
2141221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 412uggcacccag cgacucgcct t
2141321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 413ggcgagucgc ugggugccat t
2141421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 414cucgccagag ugguuaucut t
2141521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 415agauaaccac ucuggcgagt t
2141621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 416cggcaguggu ugagccgugt t
2141721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 417cacggcucaa ccacugccgt t
2141821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 418gugguugagc cguggagggt t
2141921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 419cccuccacgg cucaaccact t
2142021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 420gagggcuuuc gccuuagcgt t
2142121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 421cgcuaaggcg aaagcccuct t
2142221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 422agggcuuucg ccuuagcgct t
2142321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 423gcgcuaaggc gaaagcccut t
2142421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 424ggugaccugg ccaccauuct t
2142521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 425gaaugguggc caggucacct t
2142621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 426accauucaug gcaugaacct t
2142721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 427gguucaugcc augaauggut t
2142821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 428cauggcauga accggccuut t
2142921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 429aaggccgguu caugccaugt t
2143021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 430ccaacuauug cuucagcuct t
2143121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 431gagcugaagc aauaguuggt t
2143221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 432gaacugcugc gugcggcagt t
2143321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 433cugccgcacg cagcaguuct t
2143421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 434gcccuguaca accagcauat t
2143521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 435uaugcugguu guacagggct t
2143621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 436cuguacaacc agcauaacct t
2143721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 437gguuaugcug guuguacagt t
2143821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 438guacaaccag cauaacccgt t
2143921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 439cggguuaugc ugguuguact t
2144021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 440cuauuccuuu gcccggcaut t
2144121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 441augccgggca aaggaauagt t
2144221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 442agcggaggaa ggagucgcct t
2144321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 443ggcgacuccu uccuccgcut t
2144421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 444ggaggaagga gucgccgagt t
2144521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 445cucggcgacu ccuuccucct t
2144621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 446acuuuugaga cuuuuccgut t
2144721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 447acggaaaagu cucaaaagut t
2144821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 448uugagacuuu uccguugcct t
2144921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 449ggcaacggaa aagucucaat t
2145021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 450accucuuggc gcgacgcugt t
2145121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 451cagcgucgcg ccaagaggut t
2145221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 452gacccggccu cccgcaaagt t
2145321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 453cuuugcggga ggccggguct t
2145421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 454cccggccucc cgcaaagact t
2145521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 455gucuuugcgg gaggccgggt t
2145621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 456cuccuccagg agacggauct t
2145721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 457gauccgucuc cuggaggagt t
2145821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 458caccuucugg uaccagauct t
2145921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 459gaucugguac cagaaggugt t
2146021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 460cuucugguac cagaucgcgt t
2146121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 461cgcgaucugg uaccagaagt t
2146221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 462uucugguacc agaucgcgct t
2146321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 463gcgcgaucug guaccagaat t
2146421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 464gguuauuucc gugggauact t
2146521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 465guaucccacg gaaauaacct t
2146621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 466accucagcuu ucccucgagt t
2146721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 467cucgagggaa agcugaggut t
2146821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 468ccucagcuuu cccucgaggt t
2146921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 469ccucgaggga aagcugaggt t
2147021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 470uaccuuuugc cgggagacct t
2147121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 471ggucucccgg caaaagguat t
2147221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 472ccaugccgcc cuccgggcut t
2147321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 473agcccggagg gcggcauggt t
2147421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 474acuauccacc ugcaagacut t
2147521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 475agucuugcag guggauagut t
2147621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 476cuauccaccu gcaagacuat t
2147721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 477uagucuugca gguggauagt t
2147821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 478guccaagcug cggcucgcct t
2147921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 479ggcgagccgc agcuuggact t
2148021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 480cgagccugag gccgacuact t
2148121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 481guagucggcc ucaggcucgt t
2148221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 482ugguggaaac ccacaacgat t
2148321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 483ucguuguggg uuuccaccat t
2148421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 484acccacaacg aaaucuaugt t
2148521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 485cauagauuuc guugugggut t
2148621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 486ucaagcagag uacacacagt t
2148721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 487cuguguguac ucugcuugat t
2148821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 488cagaguacac acagcauaut t
2148921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 489auaugcugug uguacucugt t
2149021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 490ucuucaacac aucagagcut t
2149121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 491agcucugaug uguugaagat t
2149221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 492gcgguaccug aacccgugut t
2149321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 493acacggguuc agguaccgct t
2149421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 494cugcugaggc ucaaguuaat t
2149521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 495uuaacuugag ccucagcagt t
2149621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 496gcgauaccuc agcaaccggt t
2149721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 497ccgguugcug agguaucgct t
2149821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 498ggcacccagc gacucgccat t
2149921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 499uggcgagucg cugggugcct t
2150021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 500ccagcgacuc gccagagugt t
2150121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 501cacucuggcg agucgcuggt t
2150221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 502gagugguuau cuuuugaugt t
2150321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 503caucaaaaga uaaccacuct t
2150421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 504uugugcggca gugguugagt t
2150521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 505cucaaccacu gccgcacaat t
2150621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 506ugugacagca gggauaacat t
2150721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 507uguuaucccu gcugucacat t
2150821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 508acagcaggga uaacacacut t
2150921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 509aguguguuau
cccugcugut t 2151021DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 510cagcagggau aacacacugt t
2151121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 511caguguguua ucccugcugt t
2151221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 512uaacacacug caaguggact t
2151321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 513guccacuugc aguguguuat t
2151421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 514guucacuacc ggccgccgat t
2151521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 515ucggcggccg guagugaact t
2151621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 516uggccaccau ucauggcaut t
2151721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 517augccaugaa ugguggccat t
2151821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 518cauucauggc augaaccggt t
2151921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 519ccgguucaug ccaugaaugt t
2152021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 520cagcaucugc aaagcuccct t
2152121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 521gggagcuuug cagaugcugt t
2152221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 522gcaaagcucc cggcaccgct t
2152321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 523gcggugccgg gagcuuugct t
2152421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 524ggagaagaac ugcugcgugt t
2152521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 525cacgcagcag uucuucucct t
2152621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 526gaagaacugc ugcgugcggt t
2152721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 527ccgcacgcag caguucuuct t
2152821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 528gcuggaagug gauccacgat t
2152921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 529ucguggaucc acuuccagct t
2153021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 530uuuggagccu ggacacgcat t
2153121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 531ugcgugucca ggcuccaaat t
2153221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 532uuggagccug gacacgcagt t
2153321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 533cugcgugucc aggcuccaat t
2153421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 534gccgugcugc gugccgcagt t
2153521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 535cugcggcacg cagcacggct t
2153621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 536uguccaacau gaucgugcgt t
2153721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 537cgcacgauca uguuggacat t
2153821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 538cugcggaucu cugugucaut t
2153921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 539augacacaga gauccgcagt t
2154021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 540gcacuauucc uuugcccggt t
2154121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 541ccgggcaaag gaauagugct t
2154221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 542ccggccuccc gcaaagacut t
2154321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 543agucuuugcg ggaggccggt t
2154421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 544cuacuggugc ugacgccugt t
2154521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 545caggcgucag caccaguagt t
2154621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 546auucauggca ugaaccggct t
2154721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 547gccgguucau gccaugaaut t
2154821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 548aacugcugcg ugcggcagct t
2154921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 549gcugccgcac gcagcaguut t
2155021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 550cggaggaagg agucgccgat t
2155121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 551ucggcgacuc cuuccuccgt t
2155221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 552ggagcgggag gagggacgat t
2155321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 553ucgucccucc ucccgcucct t
2155421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 554gcccucggga gucgccgact t
2155521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 555gucggcgacu cccgagggct t
2155621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 556uuucuccucc aggagacggt t
2155721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 557ccgucuccug gaggagaaat t
2155821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 558ucgaggcccu ccuaccuuut t
2155921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 559aaagguagga gggccucgat t
2156021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 560uacuggugcu gacgccuggt t
2156121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 561ccaggcguca gcaccaguat t
2156221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 562gacuauccac cugcaagact t
2156321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 563gucuugcagg uggauaguct t
2156421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 564uacaacagca cccgcgacct t
2156521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 565ggucgcgggu gcuguuguat t
2156621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 566ugcugaggcu caaguuaaat t
2156721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 567uuuaacuuga gccucagcat t
2156821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 568ccugugacag cagggauaat t
2156921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 569uuaucccugc ugucacaggt t
2157021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 570auaacacacu gcaaguggat t
2157121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 571uccacuugca guguguuaut t
2157221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 572uauugcuuca gcuccacggt t
2157321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 573ccguggagcu gaagcaauat t
2157421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 574gcuguccaac augaucgugt t
2157521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 575cacgaucaug uuggacagct t
2157621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 576gucuccaucc cugacguuct t
2157721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 577gaacgucagg gauggagact t 21
* * * * *
References