# Patent

## Title
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.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 60/782,829, filed Mar. 16, 2006, which is hereby incorporated by reference in its entirety.


TECHNICAL FIELD
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
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.
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.
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.
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.
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 hypoxemia, right-heart failure, heart attack, stroke, blood clot (embolism) in the lungs, or lung infection brought on by the disease.
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
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.
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%.
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).
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.
In yet another aspect, the instant invention provides a vector encoding an iRNA agent of the invention.
In yet another aspect, the instant invention provides a cell comprising an iRNA agent or a vector of the invention.
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.
In yet another aspect, the instant invention provides a pharmaceutical composition comprising an iRNA agent of the invention and a pharmaceutically acceptable carrier.
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.
Table 1 provides exemplary iRNA agents of the invention.







TABLE 1







Oligonucleotide sequences of siRNAs specific for TGF-beta













Position of








target


sequence in
Duplex

SEQ ID

SEQ ID


NM_000660.3
identifier
sense strand sequence1
NO:
antisense strand sequence1
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
cmacmcmcmgcmgumgcmumaaumggumgTT
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-6856
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
cmaacmumaumumgcmumummagcmumcmcmTT
83
ggagcugaagcmaaumaguugTT
84


 


1748-1770
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





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′ → 3′; due to synthesis procedures, the 5′-most nucleic acid is anucleoside, i.e. does not bear a 5′-monophosate group






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.
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”).
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”).
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”).
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”).
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-1.4527, 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”).
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”).
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”).
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”).
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).
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.
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.
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.
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.
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.
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.
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.
In one aspect, methods of treating any disease or disorder associated with undesired TGF-beta signaling are provided.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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




  
    
      
        
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(“mRNA” may be replaced by another parameter used to assess TGF-beta gene expression, where applicable)
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.
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.
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° C. or 70° 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.
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.
“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.
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.
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).
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.
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.
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.
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.
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.
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.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
Design and Selection of iRNA Agents
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.
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.
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”).
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′-O-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 hybrized 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Evaluation of Candidate iRNA Agents
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.
Stability Testing, Modification, and Retesting of iRNA Agents
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.
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.
In Vivo Testing
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.
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.
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 35S, 32P, 33P, or 3H; gold particles; or antigen particles for immunohistochemistry).
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.
iRNA Chemistry
Described herein are isolated iRNA agents, e.g., ds RNA agents, that mediate RNAi to inhibit expression of a TGF-beta gene.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Enhanced Nuclease Resistance
An iRNA agent, e.g., an iRNA agent that targets TGF-beta, can have enhanced resistance to nucleases.
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.
Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “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(CH2)nAMINE, (e.g., AMINE=NH2; 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), (OCH2CH2OCH3, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.
“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. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; 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.
Preferred substituents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.
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′-ca-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′-ca-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′-ca-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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 β-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.
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-hydrosuccinimide 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.
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.
Representative hydroxyl protecting groups, for example, are disclosed by Beaucage et al. (Tetrahedron, 1992, 48:2223-23.11). 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.
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.
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. Trav. Chim. Pays-Bas, 1987, 107:621).
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.
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.
Ligand Conjugates
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.
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.
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—(CH2)nNH2 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.
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.
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.
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 moleculeses, 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.
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.
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.
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-κB.
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, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
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, neproxin 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.
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.
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.
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.
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 that or antennapedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, 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.
Backbone Modifications
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.
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.
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, salts and free-acid forms are also included.
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.
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, 0, S and CH2 component parts.
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.
5′-Terminal Modifications
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 ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)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.
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 Nykänen 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.
Disorders and Diseases Associated with Undesired TGF-Beta Signaling
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.
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.
Delivery of iRNA Agents to Tissues and Cells
Formulation
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.
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.
The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.
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.
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.
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.
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.
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.
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.
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.
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 Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
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.
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.
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.
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.
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.
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.
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.
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.
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 (WPS 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).
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.
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; Ojwang et 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).
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 intramuscular 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).
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.
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.
Preservatives include, for example, without limitation, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
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, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.
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.
Treatment Methods and Delivery
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.
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.
Suitable enteral administration routes include oral delivery.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Dosage. The dosage can be an amount effective to treat or prevent a disorder or disease associated with undesired TGF-beta signaling.
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.
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.
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, intracistemal or intracapsular), or reservoir may be advisable.
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.
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.
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.
Dosage levels on the order of about 1 μg/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.
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 4.4×1016 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.
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.
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 μg 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.
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
siRNA design was carried out to identify siRNAs targeting human and mouse TGF-beta 1 genes. The siRNA in silico selection resulted in 28 siRNAs satisfying our selection criteria.
Human (NM—000660.3; May 4, 2005) and mouse (NM—011577.1; Dec. 17, 2003) mRNA sequences to TGF-beta were downloaded from NCBI resource on the worldwide web at ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide.
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 the worldwide web at mbio.ncsu.edu/BioEdit/bioedit.html and installed on a windows 2000 computer.
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.
The siRNA design web interface available at the Whitehead Institute for Biomedical Research (on the worldwide web at 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.
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:
1) high specificity can be predicted by weak identity to off-target hits
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)
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.
The off-target score was calculated as follows: identity score−0.2*mismatches in seed region.
siRNAs were sorted according to off-target score (ascending). The top 28 siRNAs according to the above criteria were selected and synthesized.
Designing siRNAs Against TGF-Beta mRNA
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—000660.3.
Example 2
TGF-Beta siRNA In Vitro Screening Protocol
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.
For transfections, A549 cells (ATCC, Manassas, USA, ATCC #: CCL-185) were seeded at 1.5×104 cells/well on 96-well cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 100 μl growth medium (RPMI 1640, 10% fetal calf serum, 100 u/ml penicillin 100 μg/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 μl Lipofectamine 2000 were mixed with 12.5 μl 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 μl of a 5 μM solution of siRNA in annealing buffer per well were mixed with 10 μl Opti-MEM, per well. To achieve a final siRNA concentration of 10 nM, 2.5 μl of a 0.5 μM solution of siRNA in annealing buffer were mixed with 10 μl 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 μl/well of fresh medium. 25 μl of the siRNA-Lipofectamine2000-complex solution were applied to the cells to a final volume of 125 μl per well and cells were incubated for 24 h at 37° C. and 5% CO2 in a humidified incubator (Heraeus GmbH, Hanau).
Following incubation, TGF-beta mRNA levels were determined by QuantiGene bDNA-kit (Genospectra, Fremont, USA). The incubation mixture was replaced by 100 μl fresh growth medium and cells were lysed by applying 50 μl of lysis mixture from the QuantiGene bDNA-kit and incubation for 30 min at 53° C. Cell lysates were incubated in parallel with bDNA probes specific to hGAPDH (10 μl lysate) and hTGF-beta (40 μl 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%.
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).
bDNA Probes Used in bDNA Assay for TGF-β (Generated with Bayer QuantiGene Probeset Software):








TABLE 2







TGF-β-specific probes detecting hsTGF-β1



(Genbank accession no. NM_000660.3)











FPL
Func-

SEQ



Name
tion
Sequence
ID NO





hsTGFb 001
CE
GGGCTCCGGTTCTGCACTCTTTTT
113





TCTCTTGGAAAGAAAGT


 


hsTGFb 002
CE
CGCGGGTGACCTCCTTGGTTTTTC
114




TCTTGGAAAGAAAGT


 


hsTGFb 003
CE
GCAGCTCTGCCCGGGAGATTTTTC
115




TCTTGGAAAGAAAGT


 


hsTGFb 004
CE
TTTTAACTTGAGCCTCAGCAGACT
116




TTTTCTCTTGGAAAGAAAGT


 


hsTGFb 005
CE
GGTTGCTGAGGTATCGCCAGTTTT
117




TCTCTTGGAAAGAAAGT


 


hsTGFb 006
CE
GCTGGGTGCCAGCAGCCTTTTTCT
118




CTTGGAAAGAAAGT


 


hsTGFb 007
LE
CGTAGTAGTCGGCCTCAGGCTCTT
119




TTTAGGCATAGGACCCGTGTCT


 


hsTGFb 008
LE
TGTGGGTTTCCACCATTAGCATTT
120




TTAGGCATAGGACCCGTGTCT


 


hsTGFb 009
LE
GCTTCTCGGAGCTCTGATGTGTTT
121




TTTAGGCATAGGACCCGTGTCT


 


hsTGFb 010
LE
GCAACACGGGTTCAGGTACCTTTT
122




TAGGCATAGGACCCGTGTCT


 


hsTGFb 011
LE
GAATTGTTGCTGTATTTCTGGTAC
123




ATTTTTAGGCATAGGACCCGTGTC




T


 


hsTGFb 012
BL
TGCTTGAACTTGTCATAGATTTCG
124




T


 


hsTGFb 013
BL
TGAAGAACATATATATGCTGTGTG
125




TACTC


 


hsTGFb 014
BL
GCTCCACGTGCTGCTCCAC
126

















TABLE 3







GAPDH-specific probes detecting hsGAPDH



(Genbank accession no. NM_002046.2):











FPL
Func-

SEQ



Name
tion
Sequence
ID NO





hGAP001
CE
GAATTTGCCATGGGTGGAATTTTTTCT
127





CTTGGAAAGAAAGT


 


hGAP002
CE
GGAGGGATCTCGCTCCTGGATTTTTCT
128




CTTGGAAAGAAAGT


 


hGAP003
CE
CCCCAGCCTTCTCCATGGTTTTTTCTC
129




TTGGAAAGAAAGT


 


hGAP004
CE
GCTCCCCCCTGCAAATGAGTTTTTCTC
130




TTGGAAAGAAAGT


 


hGAP005
LE
AGCCTTGACGGTGCCATGTTTTTAGGC
131




ATAGGACCCGTGTCT


 


hGAP006
LE
GATGACAAGCTTCCCGTTCTCTTTTTA
132




GGCATAGGACCCGTGTCT


 


hGAP007
LE
AGATGGTGATGGGATTTCCATTTTTTT
133




AGGCATAGGACCCGTGTCT


 


hGAP008
LE
GCATCGCCCCACTTGATTTTTTTTTAG
134




GCATAGGACCCGTGTCT


 


hGAP009
LE
CACGACGTACTCAGCGCCATTTTTAGG
135




CATAGGACCCGTGTCT


 


hGAP010
LE
GGCAGAGATGATGACCCTTTTGTTTTT
136




AGGCATAGGACCCGTGTCT


 


hGAP011
BL
GGTGAAGACGCCAGTGGACTC
137









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 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











at 100 nM
at 10 nM



Duplex identifier
duplex conc.
duplex conc.
IC(50) [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%









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
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 guanosines) targeting 19 mer target sequences in NM—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.
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 5







Additional RNAi agents targeting hTGF-beta






















Remaining










TGF-beta









mRNA




target

SEQ

SEQ
in % of


Duplex
19 mer target
sequence in

ID
Antisense strand
ID
controls ±


identifier
sequence
NM_000660.3
Sense strand sequence
NO:
sequence
NO:
stand. 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
aggucacccgcgugcuaauTT
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
172
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-14499
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
643-661
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
235
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
1157-1215
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
uucgcgcucucggcagugcTT
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
1306-1324
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-14512
cgauaccucagcaaccggc
1405-1423
cgauaccucagcaaccggcTT
324
gccgguugcugagguaucgTT
32S
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
1438-1456
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
cugugucauugggcgccugTl
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


 


AD-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-S81
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
485
 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-14608
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





1a, g, c, u: ribonucleotide 5′-monophosphates (except where in 5′-most position, see below); T: deoxythymidine 5′-monophosphate; all sequences given 5′ → 3′; due to synthesis procedures, the 5′-most nucleic acid is a nucleoside, i.e. does not bear a 5′-monophosphate group

## Claims

1. An isolated iRNA agent, comprising an antisense strand and a sense strand complementary to said antisense strand, wherein the antisense strand comprises SEQ ID NO:88 (cmaaugumacmagcugccgcmacTT*), wherein cm is 2′-O-methylcytidine-5′-monophosphate, wherein um is 2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine 5′-monothiophosphate.

2. The iRNA agent of claim 1, wherein said iRNA agent mediates the cleavage of a TGF-beta mRNA within the target sequence iRNA agent AL-DP-6858.

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 sense and/or antisense strand is further stabilized against nucleolytic degradation.

5. The iRNA agent of claim 1, wherein the sense strand further comprises at least one 3′-overhang wherein said 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.

7. The iRNA agent of claim 6, further comprising a phosphorothioate at the first internucleotide linkage at the 3′ end of the sense strand.

8. The iRNA agent of claim 1, further comprising a 2′-modified nucleotide in the sense 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 said sense strand is 21 contiguous nucleotides of the sense sequence of iRNA agent AL-DP-6858 (SEQ ID NO:85).

11. The iRNA agent of claim 1, wherein said sense strand comprises SEQ ID NO:85.

12. The iRNA agent of claim 1, wherein said sense strand consists of SEQ ID NO:85.

13. The iRNA agent of claim 1, wherein the iRNA agent is AL-DP-6271.

14. The iRNA agent of claim 1, wherein the antisense consists of SEQ ID NO:88 (cmaaugumacmagcugccgcmacTT*), wherein cm is 2′-O-methylcytidine-5′-monophosphate, wherein um is 2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine 5′-monothiophosphate.

15. The iRNA agent of claim 1, wherein the iRNA agent comprises AL-DP-6271.

16. An isolated iRNA agent, comprising an antisense strand and a sense strand complementary to said antisense strand, wherein the sense strand comprises SEQ ID NO:87 (gumgcmggcmagcmumgumacmaumumgTT*), wherein cm is 2′-O-methylcytidine-5′-monophosphate, wherein um is 2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine 5′-monothiophosphate.

17. The iRNA agent of claim 16, wherein the sense strand consists of SEQ ID NO:87 (gumgcmggcmagcmumgumacmaumumgTT*), wherein cm is 2′-O-methylcytidine-5′-monophosphate, wherein um is 2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine 5′-monothiophosphate.

18. An isolated iRNA agent, comprising an antisense strand and a sense strand complementary to said antisense strand, wherein the sense strand comprises SEQ ID NO:87 (gumgcmggcmagcmumgumacmaumumgTT*) and the antisense strand comprises SEQ ID NO:88 (cmaaugumacmagcugccgcmacTT*), wherein cm is 2′-O-methylcytidine-5′-monophosphate, wherein um is 2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine 5′-monothiophosphate.

19. The iRNA agent of claim 18, wherein the sense strand consists of SEQ ID NO:87 (gumgcmggcmagcmumgumacmaumumgTT*) and the antisense strand consists of SEQ ID NO:88 (cmaaugumacmagcugccgcmacTT*), wherein cm is 2′-O-methylcytidine-5′-monophosphate, wherein um is 2′-O-methyl-uridine-5′-monophosphate, and wherein T* is deoxythymidine 5′-monothiophosphate.

20. A vector encoding the iRNA agent of claim 1.

21. A cell comprising the iRNA agent of claim 1 or the vector of claim 20.

22. 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.

23. A pharmaceutical composition comprising the iRNA agent of claim 1 and a pharmaceutically acceptable carrier.

24. 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.

25. The method of claim 24, wherein the human is diagnosed as having idiopathic pulmonary fibrosis, diabetic nephropathy or chronic liver disease.

26. 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.

27. The method of claim 26 wherein said iRNA agent mediates the cleavage of a TGF-beta mRNA within the target sequence of iRNA agent AL-DP-6858.

28. The method of claim 26, wherein said iRNA agent is administered to the subject.

29. The method of claim 28, wherein said administration comprises pulmonary administration.

30. The method of claim 26, wherein the level of TGF-beta protein and/or TGF-beta mRNA is reduced by at least 20%.

31. The method of claim 26, wherein said sense strand is 21 contiguous nucleotides of the sense sequence of iRNA agent AL-DP-6858 (SEQ ID NO:85).

32. 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 the iRNA agent of claim 1.