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 .- 11 . (canceled) 
     
     
         12 . An RNAi agent comprising an antisense strand that is complementary to a target sequence selected from an mRNA expressed in a mammalian cell or plant cell, wherein at least one nucleotide within 5 or fewer nucleotides from the 3′ end of the antisense strand is substituted with a nucleotide which forms a G:U wobble base pair when the antisense strand is base paired with the target sequence. 
     
     
         13 . The RNAi agent of  claim 12 , wherein the at least one substitution is an A→G substitution, the G forming a G:U wobble base pair with a U in the target mRNA sequence. 
     
     
         14 . The RNAi agent of  claim 13 , wherein the at least one substitution is a C→U substitution, the U forming a G:U wobble base pair with a G in the target mRNA sequence. 
     
     
         15 .- 16 . (canceled) 
     
     
         17 . The RNAi agent of  claim 12 , wherein at least two nucleotides within 5 or fewer nucleotides from the 3′ end of the antisense strand are substituted. 
     
     
         18 .- 20 . (canceled) 
     
     
         21 . The RNAi agent of  claim 12 , wherein at least three, four or five nucleotides are substituted within 5 or fewer nucleotides from the 3′ end of the antisense strand. 
     
     
         22 . The RNAi agent of  claim 12 , wherein the agent is chemically synthesized. 
     
     
         23 . The RNAi agent of  claim 12 , wherein the agent is chemically synthesized. 
     
     
         24 . The RNAi agent of  claim 12 , wherein the agent is derived from an engineered precursor. 
     
     
         25 .- 26 . (canceled) 
     
     
         27 . A composition comprising the RNAi agent of  claim 12 , formulated to facilitate entry of the agent into a cell. 
     
     
         28 . A pharmaceutical composition comprising the RNAi agent of  claim 12 . 
     
     
         29 . An engineered pre-miRNA comprising the RNAi agent of  claim 12 . 
     
     
         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  claim 12 . 
     
     
         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 .- 44 . (canceled) 
     
     
         45 . The RNAi agent of  claim 12 , wherein the RNAi agent is a small interfering RNA (siRNA). 
     
     
         46 . The RNAi agent of  claim 45 , wherein the at least one substitution is an A→G substitution, the G forming a G:U wobble base pair with a U in the target mRNA sequence. 
     
     
         47 . The RNAi agent of  claim 45 , wherein the at least one substitution is a C→U substitution, the U forming a G:U wobble base pair with a G in the target mRNA sequence. 
     
     
         48 . The RNAi agent of  claim 12 , wherein the RNAi agent is a microRNA (miRNA). 
     
     
         49 . The RNAi agent of  claim 48 , wherein the RNAi agent is a plant miRNA. 
     
     
         50 . The RNAi agent of  claim 48 , wherein the RNAi agent is an animal miRNA. 
     
     
         51 . The RNAi agent of  claim 24 , wherein the engineered precursor is a miRNA precursor (pre-miRNA). 
     
     
         52 . The RNAi agent of  claim 51 , wherein the pre miRNA is encoded by a vector. 
     
     
         53 . The RNAi agent of  claim 24 , wherein the engineered precursor is a small hairpin RNA (shRNA) comprising a stem portion comprising the antisense strand. 
     
     
         54 . The RNAi agent of  claim 53 , wherein the at least one substitution is an A→G substitution, the G forming the G:U wobble base pair with a U in the target mRNA sequence. 
     
     
         55 . The RNAi agent of  claim 53 , wherein the at least one substitution is a C→U substitution, the U forming the G:U wobble base pair with a G in the target mRNA sequence. 
     
     
         56 . The RNAi agent of  claim 53 , wherein the shRNA is encoded by a vector. 
     
     
         57 . The RNAi agent of  claim 12 , wherein the cell is a mammalian cell. 
     
     
         58 . The RNAi agent of  claim 12 , wherein the cell is a human cell. 
     
     
         59 . The RNAi agent of  claim 12 , wherein the cell is a plant cell. 
     
     
         60 . The RNAi agent of  claim 12 , wherein the mRNA specifies the amino acid sequence of a cellular protein. 
     
     
         61 . The RNAi agent of  claim 12 , wherein the mRNA specifies the amino acid sequence of an extracellular protein. 
     
     
         62 . The RNAi agent of  claim 12 , wherein the mRNA specifies the amino acid sequence of a pathogen-associated protein. 
     
     
         63 . The RNAi agent of  claim 62 , wherein the pathogen-associated protein is a viral protein. 
     
     
         64 . The RNAi agent of  claim 62 , wherein the pathogen-associated protein is expressed by a host of a pathogen. 
     
     
         65 . The RNAi agent of  claim 62 , wherein the mRNA specifies the amino acid sequence of an endogenous protein. 
     
     
         66 . The RNAi agent of  claim 12 , wherein the mRNA specifies the amino acid sequence of a heterologous protein expressed in a recombinant cell or a genetically altered organism. 
     
     
         67 . The RNAi agent of  claim 12 , wherein the mRNA specifies the amino acid sequence of a protein encoded by a transgene. 
     
     
         68 . The RNAi agent of  claim 12 , wherein the mRNA specifies the amino acid sequence of a protein encoded by a pathogen genome which is capable of infecting a cell or an organism from which the cell is derived.