RNA interference suppresion 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:13) or shSCA1.F11 (SEQ ID NO:14), 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 first strand of RNA is encoded by shSCA1.F10.  
     
     
         3 . The RNA duplex of  claim 1 , wherein the second strand of RNA is encoded by shSCA1.F11.  
     
     
         4 . An 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 
 (a) shHDEx2.1 (SEQ ID NO:7),    (b) shHDEx2.2 19 nt (SEQ ID NO:8),    (c) shHDEx2.2 21 nt (SEQ ID NO:9),    (d) shHDEx3.1 19 nt (SEQ ID NO:10),    (e) shHDEx3.1 21 nt (SEQ ID NO:11),    (f) siEX58#1 (SEQ ID NO:12), or    (g) siEX58#2 (SEQ ID NO:17),    and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand.    
     
     
         5 . The RNA duplex of  claim 4 , wherein the first strand of RNA is encoded by shHDEx2.1.  
     
     
         6 . The RNA duplex of  claim 4 , wherein the first strand of RNA is encoded by shHDEx2.2 19 nt.  
     
     
         7 . The RNA duplex of  claim 4 , wherein the first strand of RNA is encoded by shHDEx2.2 21 nt.  
     
     
         8 . The RNA duplex of  claim 4 , wherein the first strand of RNA is encoded by shHDEx3.1 19 nt.  
     
     
         9 . The RNA duplex of  claim 4 , wherein the first strand of RNA is encoded by shHDEx3.1 21 nt.  
     
     
         10 . The RNA duplex of  claim 4 , wherein the first strand of RNA is encoded by siEX58#2.  
     
     
         11 . The RNA duplex of  claim 4 , wherein the first strand of RNA is encoded by siEX58#1.  
     
     
         12 . The RNA duplex of  claim 1 , wherein the duplex is between 15 and 30 base pairs in length.  
     
     
         13 . The RNA duplex of  claim 1 , wherein the duplex is between 19 and 25 base pairs in length.  
     
     
         14 . The RNA duplex of  claim 1 , wherein the first and/or second strand further comprises an overhang region.  
     
     
         15 . 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.  
     
     
         16 . The RNA duplex of  claim 14 , wherein the overhang region is from 1 to 10 nucleotides in length.  
     
     
         17 . 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.  
     
     
         18 . The RNA duplex of  claim 17 , wherein the loop structure contains from 4 to 10 nucleotides.  
     
     
         19 . The RNA duplex of  claim 17 , wherein the loop structure contains 4, 5 or 6 nucleotides.  
     
     
         20 . An expression cassette comprising a nucleic acid encoding at least one strand of the RNA duplex of  claim 1 .  
     
     
         21 . The expression cassette of  claim 20 , further comprising a promoter.  
     
     
         22 . The expression cassette of  claim 21 , wherein the promoter is a regulatable promoter.  
     
     
         23 . The expression cassette of  claim 21 , wherein the promoter is a constitutive promoter.  
     
     
         24 . The expression cassette of  claim 21 , wherein the promoter is a CMV, RSV, pol II or pol III promoter.  
     
     
         25 . The expression cassette of  claim 20 , wherein the expression cassette further comprises a polyadenylation signal.  
     
     
         26 . The expression cassette of  claim 25 , wherein the polyadenylation signal is a synthetic minimal polyadenylation signal.  
     
     
         27 . The expression cassette of  claim 20 , further comprising a marker gene.  
     
     
         28 . A vector comprising the expression cassette of  claim 20 .  
     
     
         29 . 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 .  
     
     
         30 . A cell comprising the expression cassette of  claim 20 .  
     
     
         31 . The cell of  claim 30 , wherein the cell is a mammalian cell.  
     
     
         32 . A non-human mammal comprising the expression cassette of  claim 20 .  
     
     
         33 . 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.  
     
     
         34 . The method of  claim 1  in which accumulation of huntingtin or ataxin-1 is suppressed by at least 10%.  
     
     
         35 . 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.  
     
     
         36 . 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.  
     
     
         37 . 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.    
     
     
         38 . The method of  claim 36  or  37 , in which expression of huntingtin or ataxin-1 is inhibited by at least 10%.  
     
     
         39 . The method of  claim 37 , wherein the mammal is a human.  
     
     
         40 . The method of any of claims  35  or  36 , wherein the cell is located in vivo in a mammal.  
     
     
         41 . A viral vector comprising a promoter and an miRNA shuttle containing an embedded siRNA that specificly targets a sequence associated with a condition amenable to siRNA therapy.  
     
     
         42 . The vector of  claim 41 , wherein the promoter is an inducible promoter.  
     
     
         43 . The vector of  claim 41 , wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector.  
     
     
         44 . The vector of  claim 41 , wherein the vector is an adenoviral viral vector.  
     
     
         45 . The vector of  claim 44 , wherein the condition amenable to siRNA therapy is a neurodegenerative disease.  
     
     
         46 . The vector of  claim 45 , wherein the neurodegenerative disease is a trinucleotide-repeat disease.  
     
     
         47 . The vector of  claim 46 , wherein the trinucleotide-repeat disease is a disease associated with polyglutamine repeats.  
     
     
         48 . The vector of  claim 47 , wherein the trinucleotide-repeat disease is Huntington's disease or a spinocerebellar ataxia (SCA).  
     
     
         49 . The vector of  claim 48 , wherein the SCA is SCA1, SCA2, SCA3, SCA6, SCA7,or SCA17.  
     
     
         50 . The vector of  claim 40 , wherein the target sequence is a sequence encoding ataxin-1 or huntingtin.  
     
     
         51 . A method of preventing cytotoxic effects of neurodegenerative disease in a mammal in need thereof, comprising introducing the vector of  claim 40  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.  
     
     
         52 . 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 40  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.  
     
     
         53 . 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 41 , thereby inhibiting expression of the huntingtin or ataxin-1 gene.