Methods and compositions for enhancing the efficacy and specificity of rna silencing

Abstract

The present invention provides methods of enhancing the efficacy and specificity of RNA silencing. The invention also provides compositions for mediating RNA silencing. In particular, the invention provides siRNAs, siRNA-like molecules, shRNAs, vectors and transgenes having improved specificity and efficacy in mediating silencing of a target gene. Therapeutic methods are also featured.

1 . A method of enhancing silencing of a target mRNA in a cell, comprising contacting said cell with an engineered RNA precursor, wherein said engineered RNA precursor is selected from the group consisting of an shRNA and a pre-miR and wherein upon contacting the engineered RNA precursor is processed to a double stranded RNA (dsRNA), said dsRNA comprising a sense and an antisense strand, wherein the base pair strength between the antisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) is less than the base pair strength between the antisense strand 3′ end (AS 3′) and the sense strand 5′ end (S ′5). 
     
     
         2 . The method of  claim 1 , wherein the first four base pairs between the AS 5′ and the S 3′ comprise a ΔG that is at least 0.3 kilocalories per mol (kcal/mol) more than the ΔG of the first four base pairs between the AS 3′ and the S ′5. 
     
     
         3 . The method of either of  claim 1 , wherein said dsRNA comprises at least one mismatched tail. 
     
     
         4 . The method of  claim 3 , wherein said at least one mismatched tail comprises two mismatched tails comprising two unpaired deoxythymidine (dT) nucleotides on the AS 3′ and the S 3′. 
     
     
         5 . The method of  claim 1 , wherein said target mRNA comprises superoxide dismutase 1 (SOD1) mRNA. 
     
     
         6 . The method of  claim 5 , wherein said sense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 15, 75, 84, 85 and 86 and wherein said antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 20, 76, 77 and 78. 
     
     
         7 . The method of  claim 5 , wherein said SOD1 mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 11 and 73. 
     
     
         8 . The method of  claim 7 , wherein said sense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 15, 75, 84, 85 and 86 and wherein said antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 20, 76, 77 and 78. 
     
     
         9 . The method of  claim 8 , wherein said engineered RNA precursor is encoded by a transgene. 
     
     
         10 . The method of  claim 9 , wherein said transgene is carried by a viral vector. 
     
     
         11 . The method of  claim 10 , wherein said viral vector comprises an adeno-associated virus. 
     
     
         12 . The method of  claim 1 , wherein said target mRNA comprises Huntingtin (htt) mRNA. 
     
     
         13 . The method of  claim 12 , wherein said sense strand comprises the sequence of SEQ ID NO: 82 and wherein said antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 81 and 83. 
     
     
         14 . The method of  claim 12 , wherein said htt mRNA comprises the sequence of SEQ ID NO: 78. 
     
     
         15 . The method of  claim 14 , wherein said sense strand comprises the sequence of SEQ ID NO: 82 and wherein said antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 81 and 83. 
     
     
         16 . The method of  claim 15 , wherein said engineered RNA precursor is encoded by a transgene. 
     
     
         17 . The method of  claim 16 , wherein said transgene is carried by a viral vector. 
     
     
         18 . The method of  claim 17 , wherein said viral vector comprises an adeno-associated virus.