Methods and compositions for enhancing the efficacy and specificity of RNA silencing
First Claim
Patent Images
1. An engineered pre-miRNA comprising an asymmetrical substituted siRNA duplex comprising one or more substituted base pairs, the duplex comprising a sense strand and an antisense strand, each strand having a 5′
- end and a 3′
end, and wherein the one or more substituted base pairs are within 5 base pairs from the antisense strand 5′
(AS 5′
) end and the sense strand 3′
(S 3′
) end and are selected from the group consisting of a mismatched base pair, a wobble base pair, a base pair comprising a rare nucleotide and a base pair comprising a modified amino-purine nucleotide, such that entry of the antisense strand into a RISC complex is promoted relative to the sense strand;
such that the antisense strand preferentially guides cleavage of a desired target mRNA by the RISC complex, wherein the engineered pre-miRNA is made by;
(a) selecting a first siRNA duplex comprising a sense strand and an antisense strand, each strand having a 5′ and
a 3′
end, wherein the first siRNA duplex directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA, the first siRNA duplex having a base pairing strength within 5 base pairs from the antisense strand 5′
(AS 5′
) end and the sense strand 3′
(S 3′
) end relative to a base pairing strength within 5 base pairs from the antisense strand 3′
(AS 3′
) and the sense strand 5′
(S 5′
) end, and(b) synthesizing the engineered pre-miRNA comprising the substituted siRNA duplex comprising one or more substituted base pairs with respect to the first siRNA duplex, wherein the substituted siRNA duplex comprises a sense strand and an antisense strand, each strand having a 5′ and
a 3′
end, and wherein the substituted siRNA duplex directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA as the first siRNA duplex, the substituted siRNA duplex having a base pairing strength within 5 base pairs from the AS 5′ and
the S 3′
end and a base pairing strength within 5 base pairs from the AS 3′ and
the S 5′
end, wherein the one or more substituted base pairs are within 5 base pairs from the AS 5′ and
the S 3′
end and are selected from the group consisting of a mismatched base pair, a wobble base pair, and a base pair comprising a rare nucleotide and a base pair comprising a modified amino-purine nucleotide, such that the base pairing strength within 5 base pairs from the AS 5′ and
the S 3′
end of the substituted siRNA duplex relative to the base pairing strength within 5 base pairs from the AS 3′ and
the S '"'"'5 end of the substituted siRNA duplex is lessened as compared to the base pairing strength within AS 5′ and
the S 3′
end of the first siRNA duplex relative to the base pairing strength between the AS 3′ and
the S 5′
end of the first siRNA duplex.
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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.
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Citations
36 Claims
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1. An engineered pre-miRNA comprising an asymmetrical substituted siRNA duplex comprising one or more substituted base pairs, the duplex comprising a sense strand and an antisense strand, each strand having a 5′
- end and a 3′
end, and wherein the one or more substituted base pairs are within 5 base pairs from the antisense strand 5′
(AS 5′
) end and the sense strand 3′
(S 3′
) end and are selected from the group consisting of a mismatched base pair, a wobble base pair, a base pair comprising a rare nucleotide and a base pair comprising a modified amino-purine nucleotide, such that entry of the antisense strand into a RISC complex is promoted relative to the sense strand;
such that the antisense strand preferentially guides cleavage of a desired target mRNA by the RISC complex, wherein the engineered pre-miRNA is made by;(a) selecting a first siRNA duplex comprising a sense strand and an antisense strand, each strand having a 5′ and
a 3′
end, wherein the first siRNA duplex directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA, the first siRNA duplex having a base pairing strength within 5 base pairs from the antisense strand 5′
(AS 5′
) end and the sense strand 3′
(S 3′
) end relative to a base pairing strength within 5 base pairs from the antisense strand 3′
(AS 3′
) and the sense strand 5′
(S 5′
) end, and(b) synthesizing the engineered pre-miRNA comprising the substituted siRNA duplex comprising one or more substituted base pairs with respect to the first siRNA duplex, wherein the substituted siRNA duplex comprises a sense strand and an antisense strand, each strand having a 5′ and
a 3′
end, and wherein the substituted siRNA duplex directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA as the first siRNA duplex, the substituted siRNA duplex having a base pairing strength within 5 base pairs from the AS 5′ and
the S 3′
end and a base pairing strength within 5 base pairs from the AS 3′ and
the S 5′
end, wherein the one or more substituted base pairs are within 5 base pairs from the AS 5′ and
the S 3′
end and are selected from the group consisting of a mismatched base pair, a wobble base pair, and a base pair comprising a rare nucleotide and a base pair comprising a modified amino-purine nucleotide, such that the base pairing strength within 5 base pairs from the AS 5′ and
the S 3′
end of the substituted siRNA duplex relative to the base pairing strength within 5 base pairs from the AS 3′ and
the S '"'"'5 end of the substituted siRNA duplex is lessened as compared to the base pairing strength within AS 5′ and
the S 3′
end of the first siRNA duplex relative to the base pairing strength between the AS 3′ and
the S 5′
end of the first siRNA duplex. - View Dependent Claims (2, 3, 4, 5, 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, 32, 33, 34, 35, 36)
- end and a 3′
Specification