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

• US 8,309,705 B2
• Filed: 03/29/2010
• Issued: 11/13/2012
• Est. Priority Date: 06/02/2003
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Zamore, Phillip D., Hutvagner, Gyorgy, Schwarz, Dianne, Simard, Martin
• Primary Examiner(s): MCDONALD, JENNIFER SUE PITRAK
• Application Number:
  US12/748,937
• Publication Number:
  US 20100184828A1
• Time in Patent Office: 960 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 small hairpin RNA (shRNA) comprising nucleotide sequence identical to the sense and antisense strand of 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, and 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 asymmetrical substituted siRNA duplex 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′

) and the sense strand 3′

end (S 3′

) relative to a base pairing strength within 5 base pairs from the antisense strand 3′

(AS 3′

) and the sense strand 5′

end (S 5′

), and

(b) synthesizing 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 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 of the substituted siRNA duplex 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 AS3′ and

the S 5′

end of the first siRNA duplex.

2. The shRNA of claim 1, wherein the nucleotide sequence identical to the sense strand is upstream of the nucleotide sequence identical to the antisense strand.

3. The shRNA of claim 1, wherein the nucleotide sequence identical to the antisense strand is upstream of the nucleotide sequence identical to the sense strand.

4. A vector encoding the shRNA of claim 1.

5. A cell comprising the vector of claim 4.

6. The cell of claim 5, which is a mammalian cell.

7. The cell of claim 5, which is a human cell.

8. A transgene encoding the shRNA of claim 1.

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

10. The shRNA of claim 9, wherein the mismatched base pair is selected from the group consisting of G:

A, C;

A, C;

U, G;

G, A;

A, C;

C and U;

U.

11. The shRNA of claim 9, wherein the mismatched base pair is selected from the group consisting of G:

A, C;

A, C;

T, G;

G, A;

A, C;

C and U;

T.

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

13. The shRNA of claim 12, wherein the wobble base pair is G:

U.

14. The shRNA of claim 12, wherein the wobble base pair is G:

T.

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

16. The shRNA of claim 15, wherein the rare nucleotide is inosine (I).

17. The shRNA of claim 16, wherein the base pair is selected from the group consisting of an I:

A, I;

U and I;

C.

18. The shRNA of claim 1, wherein the one or more substituted base pairs comprise at least one base pair comprising a modified amino-purine nucleotide.

19. The shRNA of claim 18, wherein the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

20. A pharmaceutical composition comprising the shRNA of claim 1, wherein the target mRNA is expressed in a human cell.

21. A pharmaceutical composition comprising the shRNA of claim 2, wherein the target mRNA is expressed in a human cell.

22. A pharmaceutical composition comprising the shRNA of claim 3, wherein the target mRNA is expressed in a human cell.

23. A pharmaceutical composition comprising the vector of claim 4, wherein the target mRNA is expressed in a human cell.

24. A pharmaceutical composition comprising the cell of claim 5, wherein the target mRNA is expressed in a human cell.

25. A pharmaceutical composition comprising the cell of claim 6, wherein the target mRNA is expressed in a human cell.

26. A pharmaceutical composition comprising the cell of claim 7, wherein the target mRNA is expressed in a human cell.

27. A pharmaceutical composition comprising the transgene of claim 8, wherein the target mRNA is expressed in a human cell.

28. A pharmaceutical composition comprising the shRNA of claim 9, wherein the target mRNA is expressed in a human cell.

29. A pharmaceutical composition comprising the shRNA of claim 10, wherein the target mRNA is expressed in a human cell.

30. A pharmaceutical composition comprising the shRNA of claim 11, wherein the target mRNA is expressed in a human cell.

31. A pharmaceutical composition comprising the shRNA of claim 12 wherein the target mRNA is expressed in a human cell.

32. A pharmaceutical composition comprising the shRNA of claim 13, wherein the target mRNA is expressed in a human cell.

33. A pharmaceutical composition comprising the shRNA of claim 14, wherein the target mRNA is expressed in a human cell.

34. A pharmaceutical composition comprising the shRNA of claim 15, wherein the target mRNA is expressed in a human cell.

35. A pharmaceutical composition comprising the shRNA of claim 16, wherein the target mRNA is expressed in a human cell.

36. A pharmaceutical composition comprising the shRNA of claim 17, wherein the target mRNA is expressed in a human cell.

37. A pharmaceutical composition comprising the shRNA of claim 18, wherein the target mRNA is expressed in a human cell.

38. A pharmaceutical composition comprising the shRNA of claim 19, wherein the target mRNA is expressed in a human cell.