RNAi Modulation of the BCR-ABL Fusion Gene and Uses Thereof

Abstract

The invention relates to compositions and methods for modulating the expression of Bcr-Abl, and more particularly to the down-regulation of Bcr-Abl mRNA and Bcr-Abl protein levels by oligonucleotides via RNA interference, e.g., chemically modified oligonucleotides.

1 . A dsRNA comprising an antisense strand and a sense strand each 15-30 nucleotides in length, wherein the antisense strand and the sense strand form a duplex region, and wherein the antisense strand comprises a sequence that is complementary to at least 15 contiguous nucleotides of SEQ ID NOs: 5, 13, 15, or 19. 
     
     
         2 . The dsRNA of  claim 1 , wherein the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NOs: 24, 26, 27, or 28. 
     
     
         3 . The dsRNA of  claim 1 , wherein the antisense strand comprises SEQ ID NOs: 24, 26, 27, or 28. 
     
     
         4 . The dsRNA of  claim 1 , wherein the antisense strand consists of SEQ ID NOs: 24, 26, 27, or 28. 
     
     
         5 . The dsRNA of  claim 1 , wherein the sense strand comprises SEQ ID NOs: 5, 13, 15, or 19. 
     
     
         6 . The dsRNA of  claim 1 , wherein the sense strand consists of SEQ ID NOs: 5, 13, 15, or 19. 
     
     
         7 . The dsRNA of  claim 1 , wherein the sense strand comprises SEQ ID NOs: 5, 13, 15, or 19 and the antisense strand comprises SEQ ID NOs: 24, 26, 27, or 28. 
     
     
         8 . The dsRNA of  claim 1 , wherein the sense strand consists of SEQ ID NOs: 5, 13, 15, or 19 and the antisense strand consists of SEQ ID NOs: 24, 26, 27, or 28. 
     
     
         9 . The dsRNA of  claim 1 , wherein the sense strand consists of SEQ ID NO:19 and the antisense strand consists of SEQ ID NO: 28. 
     
     
         10 . The dsRNA of  claim 1 , wherein the dsRNA significantly reduces the amount of BCR-ABL fusion protein levels present in cultured human cells after incubation with the agent compared to cells which have not been incubated with the agent, wherein the cells are 32Dp210/e14a2, 32Dp210-T315I, 32Dp210-H396P, 32Dp210/e13a2, 32Dp190/e1a2, M07p210/e14a2, K562, MEG-01, or SUP-B15, or wherein the cells have been isolated from a leukemic patient. 
     
     
         11 . The dsRNA of  claim 1 , wherein at least one nucleotide of the dsRNA comprises a modified nucleotide that causes the dsRNA to have increased stability in a biological sample. 
     
     
         12 . The dsRNA of  claim 11 , wherein the modified nucleotide is a phosphorothioate or a 2′-modified nucleotide. 
     
     
         13 . The dsRNA of  claim 12 , comprising at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide wherein the uridine 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′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide; or at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide. 
     
     
         14 . The dsRNA of  claim 12 , 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). 
     
     
         15 . The dsRNA of  claim 1 , wherein the dsRNA comprises a nucleotide overhang having 1 to 4 unpaired nucleotides. 
     
     
         16 . The dsRNA of  claim 15 , wherein the nucleotide overhang comprises 2 unpaired nucleotides. 
     
     
         17 . The dsRNA of  claim 16 , wherein the nucleotide overhang is at the 3′-end of the antisense strand of the dsRNA. 
     
     
         18 . The dsRNA of  claim 1 , wherein the duplex region is 15 to 30 base pairs in length. 
     
     
         19 . The dsRNA of  claim 1 , wherein the duplex region is 19 to 21 base pairs in length. 
     
     
         20 . The dsRNA of  claim 1 , wherein the sense strand is 21 nucleotides in length. 
     
     
         21 . The dsRNA of  claim 1 , wherein the antisense strand is 23 nucleotides in length. 
     
     
         22 . The dsRNA of  claim 1 , further comprising a ligand. 
     
     
         23 . The dsRNA of  claim 22 , wherein the ligand is conjugated to the 3′-end of the sense strand of the dsRNA. 
     
     
         24 . A pharmaceutical composition, comprising:
 the dsRNA of  claims 1 , and   a pharmaceutically acceptable carrier.   
     
     
         25 . A method of reducing the amount of BCR-ABL RNA in a cell of a subject, comprising contacting the cell with the dsRNA of  claim 1 . 
     
     
         26 . The method of  claim 25 , wherein the dsRNA, upon contact with the cell expressing the BCR-ABL RNA, inhibits expression of a BCR-ABL fusion gene by at least 20%. 
     
     
         27 . A method of treating a human having or at risk for developing a proliferative disorder comprising administering the dsRNA of  claim 1 . 
     
     
         28 . The method of  claim 27 , wherein the proliferative disorder is leukemia. 
     
     
         29 . A cell comprising the dsRNA of  claim 1 . 
     
     
         30 . A vector for inhibiting the expression of BCR-ABL RNA, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of  claim 1 . 
     
     
         31 . A cell comprising the vector of  claim 30 .