Methods and compositions for controlling efficacy of RNA silencing
First Claim
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1. A method of enhancing RNA silencing of a target mRNA, the method comprising:
- (a) selecting a target sequence in an mRNA expressed in a mammalian cell or plant cell; and
(b) contacting the mammalian cell or the plant cell with an RNAi agent, the RNAi agent comprising an antisense strand that is complementary to the target sequence, wherein at least one nucleotide within 5 or fewer nucleotides from the 3′
end of the antisense strand is substituted with a nucleotide that forms a G;
U wobble base pair when the antisense strand is base paired with the target sequence, wherein the mammalian cell or plant cell is contacted under conditions such that RNA silencing of the target mRNA is enhanced, and wherein the RNAi agent is chemically or enzymatically synthesized.
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Reexamination
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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.
109 Citations
64 Claims
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1. A method of enhancing RNA silencing of a target mRNA, the method comprising:
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(a) selecting a target sequence in an mRNA expressed in a mammalian cell or plant cell; and (b) contacting the mammalian cell or the plant cell with an RNAi agent, the RNAi agent comprising an antisense strand that is complementary to the target sequence, wherein at least one nucleotide within 5 or fewer nucleotides from the 3′
end of the antisense strand is substituted with a nucleotide that forms a G;
U wobble base pair when the antisense strand is base paired with the target sequence, wherein the mammalian cell or plant cell is contacted under conditions such that RNA silencing of the target mRNA is enhanced, and wherein the RNAi agent is chemically or enzymatically synthesized. - View Dependent Claims (2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33)
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5. A method of enhancing RNA silencing of a target mRNA, the method comprising:
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(a) selecting a target sequence in an mRNA expressed in a mammalian cell or plant cell; and (b) contacting the mammalian cell or the plant cell with an RNAi agent, the RNAi agent comprising an antisense strand that is complementary to the target sequence, wherein three, four or five nucleotides within 5 or fewer nucleotides from the 3′
end of the antisense strand are substituted with a nucleotide which does not form a Watson-Crick base pair when the antisense strand is base paired with the target sequence, wherein the mammalian cell or plant cell is contacted under conditions such that silencing of the target mRNA is enhanced. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63)
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32. A method of enhancing RNA silencing of a target mRNA in a mammalian subject, comprising administering to the subject a pharmaceutical composition comprising an RNAi agent, the RNAi agent comprising an antisense strand that is complementary to a target sequence selected from an mRNA expressed in the subject, 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, wherein the pharmaceutical composition is administered such that silencing of the target mRNA is enhanced.
- end of the antisense strand is substituted with a nucleotide which forms a G;
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64. A method of enhancing RNA silencing of a target mRNA in a mammalian subject, comprising administering to the subject a pharmaceutical composition comprising an RNAi agent, the RNAi agent comprising an antisense strand that is complementary to a target sequence selected from an mRNA expressed in the subject, wherein three, four or five nucleotides within 5 or fewer nucleotides from the 3′
- end of the antisense strand is substituted with a nucleotide that forms a G;
U wobble base pair when the antisense strand is base paired with the target sequence, wherein the pharmaceutical composition is administered such that silencing of the target mRNA is enhanced.
- end of the antisense strand is substituted with a nucleotide that forms a G;
Specification