Methods and sequences to suppress primate huntington gene expression

Abstract

Disclosed herein are sequences, molecules and methods used to suppress the expression of HD genes encoding for huntingtin protein in primates including Macaca mulatta and Homo sapiens. These sequences, molecules and methods aid in the study of the pathogenesis of HD and can also provide a treatment for this disease.

1 . A nucleic acid molecule comprising a first strand and a second strand wherein said first strand comprises a nucleotide sequence and wherein said second strand comprises the reverse complement of said first strand and wherein said nucleic acid molecule suppresses the expression of both  Macaca mulatta  and  Homo sapiens  mRNA sequences that encode for huntingtin.  
     
     
         2 . An isolated nucleic acid duplex comprising a first strand of nucleic acid and a second strand of nucleic acid, wherein the first strand comprises at least 19 contiguous nucleotides encoded by the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 and wherein the second strand is complementary to at least 15 contiguous nucleotides of the first strand.  
     
     
         3 . An isolated nucleic acid duplex comprising a first strand of nucleic acid and a second strand of nucleic acid, wherein the first strand comprises at least 27 contiguous nucleotides encoded by the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 and wherein the second strand is complementary to at least 23 contiguous nucleotides of the first strand.  
     
     
         4 . A nucleic acid duplex according to  claim 2 , wherein said duplex is between 19 and 30 base pairs in length.  
     
     
         5 . A nucleic acid duplex according to  claim 2  or  3 , wherein said first and/or said second strand further comprises an overhang region.  
     
     
         6 . A nucleic acid duplex according to  claim 2  or  3 , wherein said first and/or second strand further comprises a 3′ overhang region, a 5′ overhang region, or both 3′ and 5′ overhang regions.  
     
     
         7 . A nucleic acid duplex according to  claim 5 , wherein said overhang region is from 1 to 10 nucleotides in length.  
     
     
         8 . A nucleic acid duplex according to  claim 2  or  3 , wherein said first strand and said second strand are operably linked by means of a nucleic acid loop strand to form a hairpin structure comprising a duplex structure and a loop structure.  
     
     
         9 . A nucleic acid duplex according to  claim 8 , wherein said loop structure contains from 4 to 13 nucleotides.  
     
     
         10 . An expression cassette comprising a nucleic acid encoding at least one strand of the nucleic acid duplex of  claim 2  or  3 .  
     
     
         11 . An expression cassette according to  claim 10 , further comprising a promoter.  
     
     
         12 . An expression cassette according to  claim 11 , wherein said promoter is a regulatable promoter.  
     
     
         13 . An expression cassette according to  claim 11 , wherein said promoter is a constitutive promoter.  
     
     
         14 . An expression cassette according to  claim 11 , wherein said promoter is a CMV, RSV, pol II or pot III promoter.  
     
     
         15 . An expression cassette according to  claim 10 , wherein said expression cassette further comprises a polyadenylation signal.  
     
     
         16 . An expression cassette according to  claim 15 , wherein said polyadenylation signal is a synthetic minimal polyadenylation signal.  
     
     
         17 . An expression cassette according to  claim 10 , further comprising a marker gene.  
     
     
         18 . A vector comprising the expression cassette of  claim 10 .  
     
     
         19 . A vector comprising two expression cassettes, a first expression cassette comprising a nucleotide sequence encoding said first strand of said nucleic acid duplex of  claim 2  and a second expression cassette comprising a nucleotide sequence encoding the second strand of the nucleic acid duplex of  claim 2 .  
     
     
         20 . A vector comprising two expression cassettes, a first expression cassette comprising a nucleotide sequence encoding said first strand of said nucleic acid duplex of  claim 3  and a second expression cassette comprising a nucleotide sequence encoding the second strand of the nucleic acid duplex of  claim 3 .  
     
     
         21 . A vector according to  claim 19  or  20 , wherein said vector is a viral vector.  
     
     
         22 . A vector according to  claim 21 , wherein said vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, herpes simplex virus, feline immunodeficiency virus or murine Maloney-based viral vector.  
     
     
         23 . A vector according to  claim 19  or  20 , wherein said vector further comprises a promoter.  
     
     
         24 . A vector according to  claim 23 , wherein said promoter is an inducible promoter.  
     
     
         25 . A cell comprising the expression cassette of  claim 10 .  
     
     
         26 . A cell of  claim 25 , wherein said cell is a mammalian cell.  
     
     
         27 . A non-human mammal comprising the expression cassette of  claim 10 .  
     
     
         28 . A method of suppressing the accumulation of huntingtin in a cell comprising: 
 introducing a nucleic acid duplex of  claim 2  or  3  into said cell in an amount sufficient to suppress accumulation of huntingtin in said cell.    
     
     
         29 . A method according to  claim 28 , wherein said accumulation of said huntingtin is suppressed by at least 10%.  
     
     
         30 . A method of preventing cytotoxic effects of mutant huntingtin in a cell comprising: 
 introducing a nucleic acid duplex of  claim 2  or  3  into said cell in an amount sufficient to suppress accumulation of said mutant huntingtin and wherein said nucleic acid duplex prevents cytotoxic effects of said mutant huntingtin in said cell.    
     
     
         31 . A method to inhibit expression of a huntingtin gene in a cell comprising: 
 introducing a nucleic acid duplex of  claim 2  or  3  into said cell in an amount sufficient to inhibit expression of said huntingtin protein, and wherein said nucleic acid duplex inhibits expression of said huntingtin gene.    
     
     
         32 . A method to inhibit expression of huntingtin in  Macaca mulatta  and  Homo sapiens  comprising: 
 providing a  Macaca mulatta  or  Homo sapiens  containing a neuronal cell, wherein said neuronal cell contains the huntingtin gene and said neuronal cell is susceptible to nucleic acid interference and said huntingtin gene is expressed in said neuronal cell; and    contacting said  Macaca mulatta  or  Homo sapiens  with a nucleic acid duplex of  claim 2  or  3 , thereby inhibiting expression of said huntingtin gene.    
     
     
         33 . A method according to  claim 32  wherein expression of said huntingtin is inhibited by at least 10%.  
     
     
         34 . A method of preventing cytotoxic effects of Huntington's disease (“HD”) in a  Macaca mulatta  or  Homo sapiens  comprising: 
 introducing the vector of  claim 18  into a cell in an amount sufficient to suppress accumulation of a protein associated with HD, and wherein the nucleic acid duplex prevents cytotoxic effects of HD.    
     
     
         35 . A method to inhibit expression of huntingtin in a  Macaca mulatta  or  Homo sapiens  comprising: 
 introducing the vector of  claim 18  info a cell in an amount sufficient to inhibit expression of huntingtin wherein said nucleic acid duplex inhibits expression of the protein associated with HD.    
     
     
         36 . A method to inhibit expression of huntingtin in a  Macaca mulatta  or  Homo sapiens  comprising: 
 providing a  Macaca mulatta  or  Homo sapiens  containing a neuronal cell, wherein said neuronal cell contains the huntingtin gene and said neuronal cell is susceptible to nucleic acid interference, and the huntingtin gene is expressed in said neuronal cell; and    contacting said  Macaca mulatta  or  Homo sapiens  with the vector of  claim 19  or  20 , thereby inhibiting expression of the huntingtin gene.