Methods and compositions for enhancing the efficacy and specificity of RNA silencing

• US 7,750,144 B2
• Filed: 08/04/2004
• Issued: 07/06/2010
• Est. Priority Date: 06/02/2003
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Simard, Martin, Hutvagner, Gyorgy, Schwarz, Dianne, Zamore, Phillip D.
• Primary Examiner(s): Vivlemore; Tracy
• Assistant Examiner(s): MCDONALD, JENNIFER SUE PITRAK
• Application Number:
  US10/912,440
• Publication Number:
  US 20050186586A1
• Time in Patent Office: 2,162 Days
• Field of Search: None
• US Class Current: 536/25.3
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/336 Modified G
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

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.

Claims

1. A method of producing a dsRNAi agent having decreased off-target silencing activity, the method comprising:

(a) identifying an off-target silencing activity mediated by a sense strand of a first dsRNAi agent, wherein the first dsRNAi agent directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA; and

(b) synthesizing a substituted dsRNAi agent comprising one or more substituted base pairs with respect to the first dsRNAi agent, wherein the substituted dsRNAi agent comprises a sense and an antisense strand, each strand having a 5′

end and a 3′

end, wherein the substituted dsRNAi agent directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′

end of the antisense strand (AS 5′

) and the 3′

end of the sense strand (S 3′

) 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 base-modified nucleotide, such that the sense strand of the substituted dsRNAi agent is less effective than the sense strand of the first dsRNAi agent at entering the RISC complex;

such that the substituted dsRNAi agent has decreased off-target silencing activity relative to the off-target silencing activity of the first dsRNAi agent.

2. The method of claim 1, wherein the one or more substituted base pairs comprise at least one mismatched base pair.

3. The method of claim 2, wherein the mismatched base pair is selected from the group consisting of G:

A, C;

A, C;

T, C;

U, G;

G, A;

A, C;

C, U;

T, and U;

U.

4. The method of claim 1, wherein the one or more substituted base pairs comprise at least one wobble base pair.

5. The method of claim 4, wherein the wobble base pair is selected from the group consisting of a G:

U or G;

T.

6. The method of claim 1, wherein the one or more substituted base pairs comprise at least one base pair comprising a rare nucleotide.

7. The method of claim 6, wherein the rare nucleotide is inosine (I).

8. The method of claim 6, wherein the base pair is selected from the group consisting of an I:

A, I;

U and I;

C.

9. The method of claim 1, wherein the one or more substituted base pairs comprise at least one base pair comprising a base-modified nucleotide.

10. The method of claim 9, wherein the base-modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

12. The method of claim 1 or 11, wherein the dsRNAi agent is a siRNA duplex.

13. The method of claim 1 or 11, wherein the dsRNAi agent is derived from an engineered precursor.

26. The method of claim 1, further comprising contacting a cell or subject having an RNAi pathway and an inadvertent target mRNA with the substituted dsRNAi agent produced by step (b) such that silencing of the inadvertent target mRNA in the cell or subject by the substituted dsRNAi agent is decreased relative to silencing of the inadvertent target mRNA by the first dsRNAi agent.

11. A method of decreasing off-target silencing of an inadvertent target mRNA by a dsRNAi agent, the method comprising:

(a) identifying off-target silencing of an inadvertent target mRNA by a sense strand of a first dsRNAi agent, wherein the first dsRNAi agent directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA; and

(b) contacting a cell or subject having an RNAi pathway and the inadvertent target mRNA with a substituted dsRNAi agent comprising one or more substituted base pairs with respect to the first dsRNAi agent, wherein the substituted dsRNAi agent comprises a sense strand and an antisense strand, each strand having a 5′

end and a 3′

end, and wherein the substituted dsRNAi agent directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′

end of the antisense strand (AS 5′

) and the 3′

end of the sense strand (S 3′

) 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 base-modified nucleotide,

such that the sense strand of the substituted dsRNAi agent is less effective than the sense strand of the first dsRNAi agent at entering the RISC complex;

such that off-target silencing of the inadvertent target mRNA by the substituted dsRNAi agent is decreased relative to the off-target silencing of the inadvertent target mRNA by the first dsRNAi agent.

28. The method of claim 11 or 14, wherein the one or more substituted base pairs comprise at least one mismatched base pair.

29. The method of claim 28, wherein the mismatched base pair is selected from the group consisting of G:

A, C;

A, C;

T, C;

U, G;

G, A;

A, C;

C, U;

T, and U;

U.

30. The method of claim 11 or 14, wherein the one or more substituted base pairs comprise at least one wobble base pair.

31. The method of claim 30, wherein the wobble base pair is selected from the group consisting of a G:

U or G;

T.

32. The method of claim 11 or 14, wherein the one or more substituted base pairs comprise at least one base pair comprising a rare nucleotide.

33. The method of claim 32, wherein the rare nucleotide is inosine (I).

34. The method of claim 32, wherein the base pair is selected from the group consisting of an I:

A, I;

U and I;

C.

35. The method of claim 11 or 14, wherein the one or more substituted base pairs comprise at least one base pair comprising a base-modified nucleotide.

36. The method of claim 35, wherein the base-modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

14. A method of producing an siRNA-like duplex having decreased off-target silencing activity, the method comprising:

(a) identifying an off-target silencing activity mediated by a miRNA* strand of a first siRNA-like duplex, wherein the first siRNA-like duplex directs silencing by a RISC complex of a desired target mRNA; and

(b) synthesizing a substituted siRNA-like duplex comprising one or more substituted base pairs with respect to the first siRNA-like duplex, wherein the substituted siRNA-like duplex comprises a miRNA* strand and a miRNA strand, each strand having a 5′

end and a 3′

end, wherein the substituted siRNA-like duplex directs silencing by the RISC complex of the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′

end of the miRNA strand (miRNA 5′

) and the 3′

end of the miRNA* strand (miRNA* 3′

) 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 base-modified nucleotide,

such that the miRNA* strand of the substituted siRNA-like duplex is less effective than the miRNA* strand of the first siRNA-duplex at entering the RISC complex;

such that the substituted siRNA-like duplex has decreased off-target silencing activity relative to the off-targeting silencing activity of the first siRNA-like duplex.

15. The method of claim 14, wherein the one or more substituted base pairs comprise at least one mismatched base pair.

16. The method of claim 14, wherein the mismatched base pair is selected from the group consisting of G:

A, C;

A, C;

T, C;

U, G;

G, A;

A, C;

C, U;

T, and U;

U.

17. The method of claim 14, wherein the one or more substituted base pairs comprise at least one wobble base pair.

18. The method of claim 17, wherein the wobble base pair is selected from the group consisting of a G:

U or G;

T.

19. The method of claim 14, wherein the one or more substituted base pairs comprise at least one base pair comprising a rare nucleotide.

20. The method of claim 19, wherein the rare nucleotide is inosine (I).

21. The method of claim 19, wherein the base pair is selected from the group consisting of an I:

A, I;

U and I;

C.

22. The method of claim 14, wherein the one or more substituted base pairs comprise at least one base pair comprising a base-modified nucleotide.

23. The method of claim 22, wherein the base-modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

25. The method of claim 14 or 24, wherein the siRNA like duplex silencing agent is derived from an engineered precursor.

27. The method of claim 14, further comprising

contacting a cell or subject having an RNAi pathway and an inadvertent target mRNA with the substituted siRNA-like duplex of step (b) such that silencing of the inadvertent target mRNA in the cell or subject by the substituted siRNA-like duplex is decreased relative to silencing of the inadvertent target mRNA by the first dsRNAi agent.

24. A method of decreasing off-target silencing of inadvertent target mRNA by a siRNA-like duplex, the method comprising:

(a) identifying an off-target silencing activity mediated by a miRNA* strand of a first siRNA-like duplex, wherein the first siRNA-like duplex directs silencing by a RISC complex of a desired target mRNA; and

(b) contacting a cell or subject having an RNAi pathway, with a substituted siRNA-like duplex comprising one or more substituted base pairs with respect to the first siRNA-like duplex, wherein the substituted siRNA-like duplex comprises a miRNA strand and a miRNA* strand, each strand having a 5′

end and a 3′

end, and wherein the substituted siRNA-like duplex directs silencing by the RISC complex of the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′

end of the miRNA strand (miRNA 5′

) and the 3′

end of the miRNA* strand (miRNA* 3′

) and are selected from the group consisting of a wobble base pair, a base pair comprising a rare nucleotide, and a base pair comprising a base-modified nucleotide,

such that the miRNA* strand of the substituted siRNA-like duplex is less effective than the miRNA* strand of the first siRNA-like duplex at entering the RISC complex;

such that off-target silencing of the inadvertent target mRNA by the substituted siRNA-like duplex is decreased relative to the off-target silencing of the inadvertent target mRNA by the first siRNA-like duplex.