Methods and compositions for enhancing the efficacy and specificity of RNAi
First Claim
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1. A method of producing a siRNA duplex wherein entry of an antisense strand of the siRNA duplex into a RISC complex is promoted, the method comprising:
- (a) selecting a first siRNA duplex comprising a sense strand and an antisense strand, each strand having a 5′
end 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 between 5 base pairs from the antisense strand 5′
end (AS 5′
) and the sense strand 3′
end (S 3′
) relative to a base pairing strength between 5 base pairs from the antisense strand 3′
end (AS 3′
) and the sense strand 5′
end (S 5′
), and(b) synthesizing a 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′
end 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, the substituted siRNA duplex having a base pairing strength between 5 base pairs from the AS 5′ and
the S 3′
relative to a base pairing strength between 5 base pairs from the AS 3′ and
the S 5′
, wherein the one or more substituted base pairs comprise at least one mismatched base pair and wherein the one or more substituted base pairs are within the 5 base pairs from the AS 5′ and
the S 3′
,such that the base pairing strength between the 5 base pairs from the AS 5′ and
the S 3′
of the substituted siRNA duplex relative to the base pairing strength between the 5 base pairs from the AS 3′ and
the S ′
5 of the substituted siRNA duplex is lessened as compared to the base pairing strength between the AS 5′ and
the S 3′
of the first siRNA duplex relative to the base pairing strength between the AS3′ and
the S 5′
of the first siRNA duplex,such that entry of the antisense strand of the substituted siRNA duplex into the RISC complex is promoted relative to the antisense strand of the first siRNA duplex.
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Abstract
The present invention provides methods of enhancing the efficacy and specificity of RNAi. The invention also provides compositions for mediating RNAi. In particular, the invention provides siRNAs, shRNAs, vectors and transgenes having improved specificity and efficacy in mediating silencing of a target gene. Therapeutic methods are also featured.
98 Citations
31 Claims
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1. A method of producing a siRNA duplex wherein entry of an antisense strand of the siRNA duplex into a RISC complex is promoted, the method comprising:
-
(a) selecting a first siRNA duplex comprising a sense strand and an antisense strand, each strand having a 5′
end 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 between 5 base pairs from the antisense strand 5′
end (AS 5′
) and the sense strand 3′
end (S 3′
) relative to a base pairing strength between 5 base pairs from the antisense strand 3′
end (AS 3′
) and the sense strand 5′
end (S 5′
), and(b) synthesizing a 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′
end 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, the substituted siRNA duplex having a base pairing strength between 5 base pairs from the AS 5′ and
the S 3′
relative to a base pairing strength between 5 base pairs from the AS 3′ and
the S 5′
, wherein the one or more substituted base pairs comprise at least one mismatched base pair and wherein the one or more substituted base pairs are within the 5 base pairs from the AS 5′ and
the S 3′
,such that the base pairing strength between the 5 base pairs from the AS 5′ and
the S 3′
of the substituted siRNA duplex relative to the base pairing strength between the 5 base pairs from the AS 3′ and
the S ′
5 of the substituted siRNA duplex is lessened as compared to the base pairing strength between the AS 5′ and
the S 3′
of the first siRNA duplex relative to the base pairing strength between the AS3′ and
the S 5′
of the first siRNA duplex,such that entry of the antisense strand of the substituted siRNA duplex into the RISC complex is promoted relative to the antisense strand 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, 30, 31)
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20. A method of enhancing silencing of a desired target mRNA by a siRNA duplex in a cell in vitro, the method comprising:
-
(a) selecting a first siRNA duplex comprising a sense strand and an antisense strand, each strand having a 5′
end and a 3′
end, wherein the first siRNA duplex directs cleavage by a RISC complex at a phosphodiester bond within the target mRNA, the first siRNA duplex having a base pairing strength between 5 base pairs from the antisense strand 5′
end (AS 5′
) and the sense strand 3′
end (S 3′
) relative to a base pairing strength between 5 base pairs from the antisense strand 3′
end (AS 3′
) and the sense strand 5′
end (S 5′
); and(b) contacting the cell with a 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′
end 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, the substituted siRNA duplex having a base pairing strength between 5 base pairs from the AS 5′ and
S 3′
relative to a base pairing strength between 5 base pairs from the AS 3′ and
the S 5′
, wherein the one or more substituted base pairs comprise at least one mismatched base pair and wherein the one or more substituted base pairs are within the 5 base pairs from the AS 5′ and
the S 3′
,such that the base pairing strength between the 5 base pairs from the AS 5′ and
the S 3′
of the substituted siRNA duplex relative to the base pairing strength between the 5 base pairs from the AS 3′ and
the S ′
5 of the substituted siRNA duplex is lessened as compared to the base pairing strength between the AS 5′ and
the S 3′
of the first siRNA duplex relative to the base pairing strength between the AS3′ and
the S 5′
of the first siRNA duplex,such that entry of the antisense strand of the substituted siRNA duplex into the RISC complex is promoted relative to the antisense strand of the first siRNA duplex; such that silencing of the target mRNA in the cell by the substituted siRNA duplex is enhanced relative to silencing of the target mRNA by the first siRNA duplex. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29)
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21. A method of enhancing silencing of a desired target mRNA by a siRNA duplex in a subject, the method comprising:
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(a) selecting a first siRNA duplex comprising a sense strand and an antisense strand, each strand having a 5′
end and a 3′
end, wherein the first siRNA duplex directs cleavage by a RISC complex at a phosphodiester bond within the desired target mRNA, the first siRNA duplex having a base pairing strength between 5 base pairs from the antisense strand 5′
end (AS 5′
) and the sense strand 3′
end (S 3′
) relative to a base pairing strength between 5 base pairs from the antisense strand 3′
end (AS 3′
) and the sense strand 5′
end (S 5′
); and(b) administering to the subject a pharmaceutical composition comprising a 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′
end 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, the substituted siRNA duplex having a base pairing strength between 5 base pairs from the AS 5′ and
the S 3′
relative to a base pairing strength between 5 base pairs from the AS 3′ and
the S′
, wherein the one or more substituted base pairs comprise at least one mismatched base pair and wherein the one or more substituted base pairs are within 5 base pairs from the AS 5′ and
the S 3′
, such that the base pairing strength between the 5 base pairs from the AS 5′ and
the S 3′
of the substituted siRNA duplex relative to the base pairing strength between the 5 base pairs from the AS 3′ and
the S ′
5 of the substituted siRNA duplex is lessened as compared to the base pairing strength between the AS 5′ and
the S 3′
of the first siRNA duplex relative to the base pairing strength between the AS3′ and
the S 5′
of the first siRNA duplex,such that entry of the antisense strand of the substituted siRNA duplex into the RISC complex is promoted relative to the antisense strand of the first siRNA duplex; such that silencing of the target mRNA in the subject by the substituted siRNA duplex is enhanced relative to silencing of the desired target mRNA by the first siRNA duplex.
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Current AssigneeUniversity Of Massachusetts
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Original AssigneeUniversity Of Massachusetts
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InventorsZamore, Phillip D., Hutvagner, Gyorgy, Schwarz, Dianne, Simard, Martin
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Primary Examiner(s)MCDONALD, JENNIFER SUE PITRAK
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Application NumberUS10/859,321Publication NumberTime in Patent Office3,086 DaysField of SearchNoneUS Class Current536/25.3CPC Class CodesC12N 15/111 General methods applicable ...C12N 2310/111 spanning the whole gene, or...C12N 2310/14 interfering N.A.C12N 2310/331 Universal or degenerate baseC12N 2310/53 partially self-complementar...C12N 2320/11 for the determination of ta...C12N 2320/50 Methods for regulating/modu...
