Rna Interference Suppression of Neurodegenerative Diseases and Methods of Use Thereof

Abstract

The present invention is directed to small interfering RNA molecules (siRNA) targeted against nucleic acid sequence that encodes huntingtin or ataxin-1, and methods of using these siRNA 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 shSCA1.F10 (SEQ ID NO:102), shSCA1.F11 (SEQ ID NO:103), SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, or SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:96 through SEQ ID NO:101 or SEQ ID NO:106 through SEQ ID NO:142, and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand. 
     
     
         2 . The RNA duplex of  claim 1  or, wherein the duplex is between 15 and 30 base pairs in length. 
     
     
         3 . The RNA duplex of  claim 1 , 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 an overhang region. 
     
     
         5 . 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. 
     
     
         6 . The RNA duplex of  claim 4 , wherein the overhang region is from 1 to 10 nucleotides in length. 
     
     
         7 . 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. 
     
     
         8 . The RNA duplex of  claim 7 , wherein the loop structure contains from 4 to 10 nucleotides. 
     
     
         9 . The RNA duplex of  claim 7 , wherein the loop structure contains 4, 5 or 6 nucleotides. 
     
     
         10 . The RNA duplex of  claim 7 , wherein the loop structure corresponds to SEQ ID NO:61 or SEQ ID NO:64. 
     
     
         11 . An expression cassette comprising a nucleic acid encoding at least one strand of the RNA duplex of  claim 1 . 
     
     
         12 . The expression cassette of  claim 11 , further comprising a promoter. 
     
     
         13 . The expression cassette of  claim 12 , wherein the promoter is a regulatable promoter. 
     
     
         14 . The expression cassette of  claim 12 , wherein the promoter is a constitutive promoter. 
     
     
         15 . The expression cassette of  claim 12 , wherein the promoter is a CMV, RSV, pol II or pol III promoter. 
     
     
         16 . The expression cassette of  claim 11 , wherein the expression cassette further comprises a polyadenylation signal. 
     
     
         17 . The expression cassette of  claim 16 , wherein the polyadenylation signal is a synthetic minimal polyadenylation signal. 
     
     
         18 . The expression cassette of  claim 11 , further comprising a marker gene. 
     
     
         19 . A vector comprising the expression cassette of  claim 11 . 
     
     
         20 . A vector comprising two expression cassettes, a first expression cassette comprising a nucleic acid encoding the first strand of the RNA duplex of  claim 1  and a second expression cassette comprising a nucleic acid encoding the second strand of the RNA duplex of  claim 1 . 
     
     
         21 . A cell comprising the expression cassette of  claim 11 . 
     
     
         22 . The cell of  claim 21 , wherein the cell is a mammalian cell. 
     
     
         23 . The mammalian cell of  claim 22 , wherein the expression cassette is contained in a vector. 
     
     
         24 . The mammalian cell of  claim 23 , wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector. 
     
     
         25 . A non-human mammal comprising the expression cassette of  claim 11 . 
     
     
         26 . A method of suppressing the accumulation of huntingtin or ataxin-1 in a cell comprising introducing a ribonucleic acid (RNA) of  claim 1  into the cell in an amount sufficient to suppress accumulation of huntingtin or ataxin-1 in the cell. 
     
     
         27 . The method of  claim 26  in which accumulation of huntingtin or ataxin-1 is suppressed by at least 10%. 
     
     
         28 . A method of preventing cytotoxic effects of mutant huntingtin or ataxin-1 in a cell comprising introducing a ribonucleic acid (RNA) of  claim 1  into the cell in an amount sufficient to suppress accumulation of huntingtin or ataxin-1, and wherein the RNA prevents cytotoxic effects of huntingtin or ataxin-1 in the cell. 
     
     
         29 . A method to inhibit expression of a huntingtin or ataxin-1 gene in a cell comprising introducing a ribonucleic acid (RNA) of  claim 1  into the cell in an amount sufficient to inhibit expression of the huntingtin or ataxin-1, and wherein the RNA inhibits expression of the huntingtin or ataxin-1 gene. 
     
     
         30 . A method to inhibit expression of a huntingtin or ataxin-1 gene in a mammal comprising:
 (a) providing a mammal containing a neuronal cell, wherein the neuronal cell contains the huntingtin or ataxin-1 gene and the neuronal cell is susceptible to RNA interference, and the huntingtin or ataxin-1 gene is expressed in the neuronal cell; and   (b) contacting the mammal with a ribonucleic acid (RNA) of  claim 1  or a vector of  claim 27 , thereby inhibiting expression of the huntingtin or ataxin-1 gene.   
     
     
         31 . The method of  claim 29  or  30 , in which expression of huntingtin or ataxin-1 is inhibited by at least 10%. 
     
     
         32 . The method of  claim 30 , wherein the mammal is a human. 
     
     
         33 . The method of any of  claims 28 ,  29  or  30 , wherein the cell is located in vivo in a mammal. 
     
     
         34 . A viral vector comprising a promoter and an miRNA shuttle containing an embedded siRNA that specifically targets a target sequence associated with a condition amenable to siRNA therapy. 
     
     
         35 . The vector of  claim 34 , wherein the promoter is an inducible promoter. 
     
     
         36 . The vector of  claim 34 , wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector. 
     
     
         37 . The vector of  claim 34 , wherein the vector is an adenoviral viral vector. 
     
     
         38 . The vector of  claim 37 , wherein the condition amenable to siRNA therapy is a neurodegenerative disease. 
     
     
         39 . The vector of  claim 38 , wherein the neurodegenerative disease is a trinucleotide-repeat disease. 
     
     
         40 . The vector of  claim 39 , wherein the trinucleotide-repeat disease is a disease associated with polyglutamine repeats. 
     
     
         41 . The vector of  claim 40 , wherein the trinucleotide-repeat disease is Huntington's disease or a spinocerebellar ataxia (SCA). 
     
     
         42 . The vector of  claim 41 , wherein the SCA is SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17. 
     
     
         43 . The vector of  claim 34 , wherein the target sequence is a sequence encoding ataxin-1 or huntingtin. 
     
     
         44 . A method of preventing cytotoxic effects of neurodegenerative disease in a mammal in need thereof, comprising introducing the vector of  claim 34  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. 
     
     
         45 . 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 34  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. 
     
     
         46 . A method to inhibit expression of huntingtin or ataxin-1 in a mammal in need thereof comprising:
 (a) providing a mammal containing a neuronal cell, wherein the neuronal cell contains the huntingtin or ataxin-1 gene and the neuronal cell is susceptible to RNA interference, and the huntingtin or ataxin-1 gene is expressed in the neuronal cell; and   (b) contacting the mammal the vector of  claim 34 , thereby inhibiting expression of the huntingtin or ataxin-1 gene.   
     
     
         47 . 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. 
     
     
         48 . The siRNA of  claim 47 , wherein the first and/or second strand further comprises a 3′ overhang region, a 5′ overhang region, or both 3′ and 5′ overhang regions. 
     
     
         49 . The siRNA of  claim 48 , wherein the overhang region or regions is from 1 to 10 nucleotides in length. 
     
     
         50 . The siRNA of  claim 47 , 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. 
     
     
         51 . The siRNA of  claim 50 , wherein the loop structure contains from 4 to 10 nucleotides. 
     
     
         52 . The siRNA of  claim 50 , wherein the loop structure corresponds to SEQ ID NO:61 or SEQ ID NO:64. 
     
     
         53 . The siRNA of  claim 47 , wherein the first strand corresponds to SEQ ID NO:60 and the second strand corresponds to SEQ ID NO:62. 
     
     
         54 . A vector comprising two expression cassettes, a first expression cassette comprising a nucleic acid encoding a first strand of an RNA duplex corresponding to SEQ ID NO:60 and a second expression cassette comprising a nucleic acid encoding a second strand of the RNA duplex corresponding to SEQ ID NO:62. 
     
     
         55 . The vector of  claim 54 , wherein the vector is a viral vector. 
     
     
         56 . The vector of  claim 55 , wherein the viral vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector. 
     
     
         57 . The vector of  claim 55 , wherein the vector is an AAV vector. 
     
     
         58 . A vector comprising an expression cassette, wherein the expression cassette encodes a nucleic acid SEQ ID NO:59. 
     
     
         59 . The vector of  claim 58 , wherein the vector is a viral vector. 
     
     
         60 . The vector of  claim 59 , wherein the viral vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector. 
     
     
         61 . The vector of  claim 60 , wherein the vector is an AAV vector.