METHODS AND COMPOSITIONS FOR ENHANCING THE EFFICACY AND SPECIFICITY OF SINGLE AND DOUBLE BLUNT-ENDED siRNA

Abstract

The present invention provides methods of enhancing the efficacy and specificity of RNAi using single or double blunt-ended siRNA. The invention also provides single and double-blunt ended siRNA compositions, vectors, and transgenes containing the same for mediating silencing of a target gene. Therapeutic methods are also featured.

1 . A method of enhancing the ability of a first strand of a RNAi agent to act as a guide strand in mediating RNAi, the RNAi agent derived from an RNA duplex having at least one blunt end, comprising lessening the base pair strength between the 5′ end of the first strand and the 3′ end of a second strand of the duplex as compared to the base pair strength between the 3′ end of the first strand and the 5′ end of the second strand. 
     
     
         2 . A method of enhancing the efficacy of a siRNA duplex having at least one blunt end, the siRNA duplex comprising a sense and an antisense strand, comprising lessening the base pair strength between the antisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) as compared to the base pair strength between the antisense strand 3′ end (AS 3′) and the sense strand 5′ end (S ′5), such that efficacy is enhanced. 
     
     
         3 .- 4 . (canceled) 
     
     
         5 . A method of promoting entry of a desired strand of an siRNA duplex having at least one blunt end into a RISC complex, comprising enhancing the asymmetry of the siRNA duplex, such that entry of the desired strand is promoted. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 5 , wherein asymmetry is enhanced by lessening the base pair strength between the 5′ end of the desired strand and the 3′ end of a complementary strand of the duplex as compared to the base pair strength between the 3′ end of the desired strand and the 5′ end of the complementary strand. 
     
     
         8 . The method of any one of  claims 1  or  2 , wherein the base-pair strength is less due to fewer G:C base pairs between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand than between the 3′ end of the first or antisense strand and the 5′ end of the second or sense strand. 
     
     
         9 . The method of any one of  claims 1  or  2 , wherein the base pair strength is less due to at least one mismatched base pair between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand. 
     
     
         10 . The method of  claim 9 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U, and I:C. 
     
     
         11 . The method of any one of  claims 1  or  2 , wherein the base pair strength is less due to at least one wobble base pair between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand. 
     
     
         12 . The method of  claim 11 , wherein the wobble base pair is G:U. 
     
     
         13 . The method of  claim 11 , wherein the wobble base pair is G:T. 
     
     
         14 . The method of any one of  claims 1  or  2 , wherein the base pair strength is less due to:
 (a) at least one mismatched base pair between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand; and 
 (b) at least one wobble base pair between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand. 
 
     
     
         15 . The method of  claim 14 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. 
     
     
         16 . The method of  claim 14 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:T, G:G, A:A, C:C and U:T. 
     
     
         17 . The method of  claim 14 , wherein the wobble base pair is G:U. 
     
     
         18 . The method of  claim 14 , wherein the wobble base pair is G:T. 
     
     
         19 . The method of any one of  claims 1  or  2 , wherein the base pair strength is less due to at least one base pair comprising a rare nucleotide. 
     
     
         20 . The method of  claim 19 , wherein the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A. 
     
     
         21 . The method of  claim 1 , wherein the RNAi agent is a siRNA duplex. 
     
     
         22 . The method of any one of  claims 1  or  2 , wherein the RNAi agent or siRNA duplex is chemically synthesized. 
     
     
         23 . The method of any one of  claims 1  or  2 , wherein the RNAi agent or siRNA duplex is enzymatically synthesized. 
     
     
         24 . The method of any one of  claims 1  or  2 , wherein the RNAi agent or siRNA duplex is derived from an engineered precursor. 
     
     
         25 . A method of enhancing silencing of a target mRNA, comprising contacting a cell having an RNAi pathway with the RNAi agent or siRNA duplex of any one of the preceding claims under conditions such that silencing is enhanced. 
     
     
         26 . A method of enhancing silencing of a target mRNA in a subject, comprising administering to the subject a pharmaceutical composition comprising the RNAi agent or siRNA duplex of any one of the preceding claims such that silencing is enhanced. 
     
     
         27 . A method of decreasing silencing of an inadvertent target mRNA by a dsRNAi agent, the dsRNAi agent comprising a sense strand, an antisense strand, and having at least one blunt end comprising:
 (a) detecting a significant degree of complementarity between the sense strand and the inadvertent target; and   (b) enhancing the base pair strength between the 5′ end of the sense strand and the 3′ end of the antisense strand relative to the base pair strength between the 3′ end of the sense strand and the 5′ end of the antisense strand;   such that silencing of the inadvertent target mRNA is decreased.   
     
     
         28 . The method of  claim 27 , wherein silencing of the inadvertent target mRNA is decreased relative to silencing of a desired target mRNA. 
     
     
         29 . An siRNA duplex having 5′ and 3′ blunt ends comprising a sense strand and an antisense strand, wherein the base pair strength between the antisense strand 3′ end (AS 3′) and the sense strand 5′ end (S 5′) is less than the base pair strength between the antisense strand 5′ end (AS 5′) and the sense strand 3′ end (S ′3), such that the antisense strand preferentially guides cleavage of a target mRNA. 
     
     
         30 . The siRNA duplex of  claim 29 , wherein the base-pair strength is less due to fewer G:C base pairs between the AS 5′ and the S 3′ than between the AS 3′ and the S 5′. 
     
     
         31 . The siRNA duplex of  claim 29 , wherein the base pair strength is less due to at least one mismatched base pair between the AS 5′ and the S 3′. 
     
     
         32 . The siRNA duplex of  claim 31 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:T, U:T, C:U, G:G, A:A, C:C, U:U, I:A, I:U, and I:C. 
     
     
         33 . The siRNA duplex of  claim 29 , wherein the base pair strength is less due to at least one wobble base pair between the AS 5′ and the S 3′. 
     
     
         34 . The siRNA duplex of  claim 29 , wherein the wobble base pair is G:U. 
     
     
         35 . The siRNA duplex of  claim 29 , wherein the wobble base pair is G:T. 
     
     
         36 . The siRNA duplex of  claim 29 , wherein the base pair strength is less due to at least one base pair comprising a modified nucleotide. 
     
     
         37 . The siRNA duplex of  claim 36 , wherein the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A. 
     
     
         38 . A composition comprising the RNAi agent or siRNA duplex of  claim 29 , formulated to facilitate entry of the RNAi agent or siRNA duplex into a cell. 
     
     
         39 . A pharmaceutical composition comprising the RNAi agent or siRNA duplex of  claim 29 . 
     
     
         40 . An engineered pre-miRNA comprising the RNAi agent or siRNA duplex of  claim 29 . 
     
     
         41 . A vector encoding the pre-miRNA of  claim 40 . 
     
     
         42 . A pre-miRNA comprising the pre-miRNA of  claim 41 . 
     
     
         43 . A vector encoding the pre-miRNA of  claim 42 . 
     
     
         44 . A small hairpin RNA (shRNA) comprising nucleotide sequence identical to the sense and antisense strand of the siRNA duplex of  claim 29 . 
     
     
         45 . The shRNA of  claim 44 , wherein the nucleotide sequence identical to the sense strand is upstream of the nucleotide sequence identical to the antisense strand. 
     
     
         46 . The shRNA of  claim 44 , wherein the nucleotide sequence identical to the antisense strand is upstream of the nucleotide sequence identical to the sense strand. 
     
     
         47 . A vector encoding the shRNA of  claim 44 . 
     
     
         48 . A cell comprising the vector of  claim 47 . 
     
     
         49 . The cell of  claim 48 , which is a mammalian cell. 
     
     
         50 . The cell of  claim 48 , which is a human cell. 
     
     
         51 . A transgene encoding the shRNA of  claim 44 .