Novel Structurally Designed shRNAs

Abstract

Provided is an improved design of shRNA based on structural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogenesis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Ago2 because of its unique catalytic activity. These data demonstrate that this newly identified small RNA biogenesis pathway can be exploited in vivo to produce active molecules.

1 - 27 . (canceled) 
     
     
         28 . A short hairpin RNA (shRNA) having the structure 
       
         
           
           
               
               
           
         
       
       wherein X 2  to X 22  are nucleotides complementary to a sequence in a target gene, and are in a sequence other than the mature sequence of miR-451; 
       Y 4  to Y 20  are nucleotides complementary to X 2  to X 18 ; and 
       X 1 , Y 1 , Y 2 , and Y 3  are nucleotides that are present, 
       wherein, X 1  and Y 3  are not complementary. 
     
     
         29 . An expression vector comprising a sequence encoding the shRNA according to  claim 28  operably linked to an RNA polymerase promoter. 
     
     
         30 . A library of expression vectors, each expression vector encoding the shRNA according to  claim 28  operably linked to an RNA polymerase promoter. 
     
     
         31 . An isolated mammalian cell comprising the shRNA according to  claim 28 . 
     
     
         32 . A method of attenuating expression of a target gene in a mammalian cell, the method comprising introducing into the mammalian cell an expression vector comprising a sequence encoding the short hairpin RNA molecule (shRNA) of  claim 28 , 
       wherein the shRNA molecule is expressed in the mammalian cell in an amount sufficient to attenuate expression of the target gene in a sequence specific manner, whereby expression of the target gene is inhibited. 
     
     
         33 . A short hairpin RNA (shRNA) comprising
 (i) a first sequence of 22 nucleotides complementary to a sequence in a target gene, in a sequence other than the mature sequence of miR-451,   (ii) a second sequence directly following the first sequence, wherein the entire second sequence is fully complementary to the sequence of the first 18 nucleotides counted from the 5′ end of the first sequence,   
       wherein the first sequence and the second sequence form the shRNA, and wherein the last 4 nucleotides of the first sequence form a loop region in the shRNA. 
     
     
         34 . A short hairpin RNA (shRNA) comprising
 (i) a first sequence of 21 nucleotides complementary to a sequence in a target gene, in a sequence other than the mature sequence of miR-451   (ii) a second sequence directly following the first sequence, wherein the entire second sequence is fully complementary to the sequence of the first 17 nucleotides counted from the 5′ end of the first sequence,   
       wherein the first sequence and the second sequence form the shRNA, and wherein the last 4 nucleotides of the first sequence form a loop region in the shRNA. 
     
     
         35 . The short hairpin RNA (shRNA) of  claim 28 , wherein X 2  to X 22  comprise a sequence complimentary to a sequence with known involvement in ataxia. 
     
     
         36 . The short hairpin RNA (shRNA) of  claim 33 , wherein X 2  to X 22  comprise a sequence complimentary to a sequence with known involvement in ataxia. 
     
     
         37 . The short hairpin RNA (shRNA) of  claim 34 , wherein X 2  to X 22  comprise a sequence complimentary to a sequence with known involvement in ataxia. 
     
     
         38 . The method of  claim 32 , wherein the expression vector encoding the shRNA is operably linked to an RNA polymerase promoter. 
     
     
         39 . The method of  claim 38 , wherein the RNA polymerase promoter is a pol II promoter. 
     
     
         40 . The method of  claim 32 , wherein the expression vector encoding the shRNA is based on a CMV-based backbone. 
     
     
         41 . The method of  claim 32 , wherein the expression vector is an adeno-associated virus (AAV) vector. 
     
     
         42 . The method of  claim 32 , wherein X 2  to X 22  are complementary to a sequence in an mRNA molecule encoded by the gene, wherein the sequence in the mRNA molecule is present in the sequence of the target gene. 
     
     
         43 . The method of  claim 32 , wherein X 2  to X 22  are complementary to a sequence that includes both a translated region and a portion of the 3′ untranslated region (UTR) sequence in an mRNA molecule encoded by the gene, wherein the 3′ UTR sequence in the mRNA molecule is present in the sequence of the target gene. 
     
     
         44 . The method of  claim 32 , wherein the shRNA further comprises 5′ and 3′ flanking sequences comprising restriction sites. 
     
     
         45 . The method of  claim 32 , wherein X 2  to X 22  comprise a sequence complimentary to a sequence with known involvement in ataxia. 
     
     
         46 . The method of  claim 43 , wherein X 2  to X 22  comprise a sequence complimentary to a sequence with known involvement in ataxia.