Methods and compositions for controlling efficacy of RNA silencing

Abstract

Based at least in part on an understanding of the mechanisms by which small RNAs (e.g., naturally-occurring miRNAs) mediate RNA silencing in plants, rules have been established for determining, for example, the degree of complementarity required between an RNAi-mediating agent and its target, i.e., whether mismatches are tolerated, the number of mismatches tolerated, the effect of the position of the mismatches, etc. Such rules are useful, in particular, in the design of improved RNAi-mediating agents which allow for more exact control of the efficacy of RNA silencing.

1 . A method of enhancing the efficacy of an RNAi agent, comprising substituting at least one terminal nucleotide of the RNAi agent with a nucleotide which does not form a Watson-Crick base pair with the corresponding nucleotide in a target mRNA, such that efficacy is enhanced. 
     
     
         2 . The method of  claim 1 , wherein the substituted nucleotide forms a G:U wobble base pair with the target mRNA. 
     
     
         3 . The method of  claim 2 , wherein the substitution is an A→G substitution, the G forming a G:U wobble base pair with a U in the corresponding target mRNA. 
     
     
         4 . The method of  claim 2 , wherein the substitution is a C→U substitution, the U forming a G:U wobble base pair with a G in the corresponding target mRNA. 
     
     
         5 . The method of  claim 1 , wherein the terminal nucleotide is within 5 or fewer nucleotides from the 5′ end of the RNAi agent. 
     
     
         6 . The method of  claim 1 , wherein the terminal nucleotide is within 5 or fewer nucleotides from the 3′ end of the RNAi agent. 
     
     
         7 . The method of  claim 1 , wherein at least two terminal nucleotides are substituted. 
     
     
         8 . The method of  claim 7 , wherein the two terminal nucleotides substituted are at the 5′ end of the RNAi agent. 
     
     
         9 . The method of  claim 7 , wherein the two terminal nucleotides substituted are at the 3′ end of the RNAi agent. 
     
     
         10 . The method of  claim 7 , wherein a first terminal nucleotide substituted is at the 5′ end of the RNAi agent and a second terminal nucleotide substituted is at the 3′ end of the RNAi agent. 
     
     
         11 . The method of  claim 1 , wherein at least three, four or five terminal nucleotides are substituted. 
     
     
         12 . An RNAi agent having at least one terminal nucleotide of the RNAi agent substituted with a nucleotide which forms a G:U wobble base pair with the corresponding nucleotide in a target mRNA. 
     
     
         13 . The RNAi agent of  claim 12 , wherein the substitution is an A→G substitution, the G forming a G:U wobble base pair with a U in the corresponding target mRNA. 
     
     
         14 . The RNAi agent of  claim 13 , wherein the substitution is a C→U substitution, the U forming a G:U wobble base pair with a G in the corresponding target mRNA. 
     
     
         15 . The RNAi agent of  claim 14 , wherein the terminal nucleotide is within 5 or fewer nucleotides from the 5′ end of the RNAi agent. 
     
     
         16 . The RNAi agent of  claim 15 , wherein the terminal nucleotide is within 5 or fewer nucleotides from the 3′ end of the RNAi agent. 
     
     
         17 . The RNAi agent of  claim 12 , wherein at least two terminal nucleotides are substituted. 
     
     
         18 . The RNAi agent of  claim 17 , wherein the two terminal nucleotides substituted are at the 5′ end of the RNAi agent. 
     
     
         19 . The RNAi agent of  claim 17 , wherein the two terminal nucleotides substituted are at the 3′ end of the RNAi agent. 
     
     
         20 . The RNAi agent of  claim 17 , wherein a first terminal nucleotide substituted is at the 5′ end of the RNAi agent and a second terminal nucleotide substituted is at the 3′ end of the RNAi agent. 
     
     
         21 . The RNAi agent of  claim 12 , wherein at least three, four or five terminal nucleotides are substituted. 
     
     
         22 . The RNAi agent of any one of the preceding claims, wherein the agent is chemically synthesized. 
     
     
         23 . The RNAi agent of any one of the preceding claims, wherein the agent is enzymatically synthesized. 
     
     
         24 . The RNAi agent of any one of the preceding claims, wherein the agent 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 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 of any one of the preceding claims such that silencing is enhanced. 
     
     
         27 . A composition comprising the RNAi agent of any one of the preceding claims, formulated to facilitate entry of the agent into a cell. 
     
     
         28 . A pharmaceutical composition comprising the RNAi agent of any one of the preceding claims. 
     
     
         29 . An engineered pre-miRNA comprising the RNAi agent of any one of the preceding claims. 
     
     
         30 . A vector encoding the pre-miRNA of  claim 29 . 
     
     
         31 . A pri-miRNA comprising the pre-miRNA of  claim 30 . 
     
     
         32 . A vector encoding the pre-miRNA of  claim 31 . 
     
     
         33 . A small hairpin RNA (shRNA) comprising nucleotide sequence identical to the RNAi agent of any one of the preceding claims. 
     
     
         34 . A vector encoding the shRNA of  claim 33 . 
     
     
         35 . A cell comprising the vector of  claim 32  or  34 . 
     
     
         36 . The cell of  claim 35 , which is a mammalian cell. 
     
     
         37 . The cell of  claim 36 , which is a human cell. 
     
     
         38 . A transgene encoding the shRNA of  claim 33 . 
     
     
         39 . An isolated  Arabidopsis thaliana  Dicer-like enzyme capable of cleaving a long dsRNA substrate into short, 23-24 nucleotide dsRNA products, the activity of said enzyme being inhibited in the presence of said dsRNA products. 
     
     
         40 . A method of generating a RNAi agent 23-24 nucleotides in length, comprising incubating a dsRNA substrate with the enzyme of  claim 38 , such that the agent is generated. 
     
     
         41 . A wheat germ cell-free extract comprising the enzyme of  claim 38 . 
     
     
         42 . A cauliflower cell-free extract comprising the enzyme of  claim 38 . 
     
     
         43 . A method of generating a RNAi agent 23-24 nucleotides in length, comprising incubating a dsRNA substrate with the extract of  claim 39 , such that the agent is generated. 
     
     
         44 . A kit for use in mediating RNAi, comprising the enzyme of  claim 38  or the extract of  claim 40 , and instructions for use.