Rna interference suppression of neurodegenerative diseases and methods of use thereof

Abstract

The present invention is directed to RNA interference (RNAi) molecules targeted against a nucleic acid sequence that encodes poly-glutamine repeat diseases, and methods of using these RNAi molecules.

1 . An isolated RNA duplex comprising a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand. 
     
     
         2 . The RNA duplex of  claim 1 , wherein the duplex is between 15 and 30 base pairs in length. 
     
     
         3 . The RNA duplex of  claim 2 , wherein the duplex is between 19 and 25 base pairs in length. 
     
     
         4 . The RNA duplex of  claim 1 , wherein the first and/or second strand further comprises a 3′ overhang region, a 5′ overhang region, or both 3′ and 5′ overhang regions. 
     
     
         5 . The RNA duplex of  claim 4 , wherein the 3′ overhang region, a 5′ overhang region, or both 3′ and 5′ overhang regions is from 1 to 10 nucleotides in length. 
     
     
         6 . The RNA duplex of  claim 1 , wherein the first strand and the second strand are operably linked by means of an RNA loop strand to form a hairpin structure comprising a duplex structure and a loop structure. 
     
     
         7 . The RNA duplex of  claim 6 , wherein the loop structure contains from 4 to 50 nucleotides. 
     
     
         8 . An expression cassette comprising a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8). 
     
     
         9 . The expression cassette of  claim 8 , further comprising a promoter. 
     
     
         10 . The expression cassette of  claim 9 , wherein the promoter is a CMV, RSV, pol II or pol III promoter. 
     
     
         11 . The expression cassette of  claim 8 , wherein the expression cassette further comprises a polyadenylation signal. 
     
     
         12 . The expression cassette of  claim 8 , further comprising a marker gene. 
     
     
         13 . A vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8). 
     
     
         14 . The vector of  claim 13 , wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector. 
     
     
         15 . A vector comprising a promoter and an miRNA shuttle comprising an embedded siRNA, wherein the miRNA shuttle encodes
 (a) an isolated first strand of RNA of 15 to 30 nucleotides in length and an isolated second strand of RNA of 15 to 30 nucleotides in length, wherein the first strand comprises at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand; or   (b) miRNA comprising miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8).   
     
     
         16 . A vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first nucleic acid encoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the second expression cassette comprises a second nucleic acid encoding at least 12 contiguous nucleotides complementary to the first strand. 
     
     
         17 . A cell comprising:
 (a) an expression cassette comprising a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8;   (b) a vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); or   (c) a vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first nucleic acid encoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the second expression cassette comprises a second nucleic acid encoding at least 12 contiguous nucleotides complementary to the first strand.   
     
     
         18 . A method of suppressing the accumulation of huntingtin in a cell comprising introducing an RNA duplex or vector into the cell in an amount sufficient to suppress accumulation of huntingtin in the cell, wherein the RNA duplex or vector comprises:
 (a) an isolated or purified miRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8);   (b) a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand;   (c) a vector comprising an expression cassettes comprising a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); or   (d) a vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first nucleic acid encoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the second expression cassette comprises a second nucleic acid encoding at least 12 contiguous nucleotides complementary to the first strand.   
     
     
         19 . A method of preventing cytotoxic effects of mutant huntingtin in a cell comprising introducing an RNA duplex or vector into the cell in an amount sufficient to suppress accumulation of huntingtin, wherein the RNA prevents cytotoxic effects of huntingtin in the cell, and wherein the RNA duplex or vector comprises:
 (a) an isolated or purified miRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8);   (b) a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand;   (c) a vector comprising an expression cassettes comprising a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); or   (d) a vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first nucleic acid encoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the second expression cassette comprises a second nucleic acid encoding at least 12 contiguous nucleotides complementary to the first strand.   
     
     
         20 . A method to inhibit expression of a huntingtin gene in a cell comprising introducing an RNA duplex or vector into the cell in an amount sufficient to inhibit expression of the huntingtin, and wherein the RNA inhibits expression of the huntingtin gene, and wherein the RNA duplex or vector comprises:
 (a) an isolated or purified miRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8);   (b) a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand;   (c) a vector comprising an expression cassettes comprising a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); or   (d) a vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first nucleic acid encoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the second expression cassette comprises a second nucleic acid encoding at least 12 contiguous nucleotides complementary to the first strand.   
     
     
         21 . A method to inhibit expression of a huntingtin gene in a mammal comprising:
 (a) providing a mammal comprising a neuronal cell, wherein the neuronal cell comprises the huntingtin gene and the neuronal cell is susceptible to RNA interference, and the huntingtin gene is expressed in the neuronal cell; and   (b) contacting the mammal with
 (i) an isolated or purified miRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); 
 (ii) a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand; 
 (iii) a vector comprising an expression cassettes comprising a nucleic acid encoding HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3), HDAS 20 (SEQ ID NO:4), miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8); or 
 (iv) a vector comprising a first expression cassette and a second expression cassette, wherein the first expression cassette comprises a first nucleic acid encoding at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4), and the second expression cassette comprises a second nucleic acid encoding at least 12 contiguous nucleotides complementary to the first strand, 
   
       thereby inhibiting expression of the huntingtin gene. 
     
     
         22 . A method of preventing cytotoxic effects of neurodegenerative disease in a mammal in need thereof, comprising introducing the vector of  claim 15  into a cell in an amount sufficient to suppress accumulation of a protein associated with the neurodegenerative disease, and wherein the RNA prevents cytotoxic effects of neurodegenerative disease. 
     
     
         23 . A method to inhibit expression of a protein associated with the neurodegenerative disease in a mammal in need thereof, comprising introducing the vector of  claim 15  into a cell in an amount sufficient to inhibit expression of the protein associated with the neurodegenerative disease, wherein the RNA inhibits expression of the protein associated with the neurodegenerative disease. 
     
     
         24 . A method to inhibit expression of huntingtin in a mammal in need thereof comprising:
 (a) providing a mammal comprising a neuronal cell, wherein the neuronal cell comprises the huntingtin gene and the neuronal cell is susceptible to RNA interference, and the huntingtin gene is expressed in the neuronal cell; and   (b) contacting the mammal the vector of  claim 15 , thereby inhibiting expression of the huntingtin gene.   
     
     
         25 . An AAV-1 expressed siRNA comprising an isolated first strand of RNA of 15 to 30 nucleotides in length and an isolated second strand of RNA of 15 to 30 nucleotides in length, wherein the first or second strand comprises a sequence that is complementary to a nucleotide sequence encoding a mutant Huntington's Disease protein, wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a small interfering RNA (siRNA) duplex under physiological conditions, and wherein the siRNA silences the expression of the nucleotide sequence encoding the mutant Huntington's Disease protein in the cell, wherein the first strand of RNA comprises at least 15 contiguous nucleotides encoded by HDAS 07 (SEQ ID NO:1), HDAS18 (SEQ ID NO:2), HDAS19 (SEQ ID NO:3) or HDAS 20 (SEQ ID NO:4). 
     
     
         26 . The siRNA of  claim 25 , wherein the first and/or second strand further comprises a 3′ overhang region, a 5′ overhang region, or both 3′ and 5′ overhang regions. 
     
     
         27 . The siRNA of  claim 26 , wherein the overhang region or regions is from 1 to 10 nucleotides in length. 
     
     
         28 . The siRNA of  claim 25 , wherein the first strand and the second strand are operably linked by means of an RNA loop strand to form a hairpin structure comprising a duplex structure and a loop structure. 
     
     
         29 . An isolated or purified miRNA consisting of miHD7A-1 (SEQ ID NO:5), miHD7A-2 (SEQ ID NO:6), miHD7B-1 (SEQ ID NO:7), or miHD7B-2 (SEQ ID NO:8).