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.

This application claims the benefit of U.S. Provisional Application No. 60/782,829, filed Mar. 16, 2006, and U.S. Ser. No. 11/724,790, filed Mar. 15, 2007, which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

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.

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 hypoxemic, 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 SEQ SEQ sequence in Duplex ID antisense ID NM_000660.3 identifier sense strand sequence1 NO: 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

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