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

• US 8,304,530 B2
• Filed: 03/23/2010
• Issued: 11/06/2012
• 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 Pitrak
• Application Number:
  US12/729,892
• Publication Number:
  US 20100184826A1
• Time in Patent Office: 959 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. An asymmetrical substituted dsRNA silencing agent comprising one or more mismatched base pairs, the dsRNA silencing agent comprising a sense strand and an antisense strand, each strand having a 5′

end and a 3′

end, wherein the one or more mismatched base pairs are within 5 base pairs from the antisense strand 5′

(AS 5′

) and the sense strand 3′

(S 3′

) end, 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 dsRNA silencing agent is made by;

(a) selecting a first dsRNA silencing agent comprising a sense strand and an antisense strand, each strand having a 5′ and

a 3′

end, wherein the first dsRNA silencing agent directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA, the first dsRNA silencing agent 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 dsRNA silencing agent comprising one or more substituted base pairs with respect to the first dsRNA silencing agent, wherein the substituted dsRNA silencing agent comprises a sense strand and an antisense strand, each strand having a 5′ and

a 3′

end, and wherein the substituted dsRNA silencing agent directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA as the first dsRNA silencing agent, the substituted dsRNA silencing agent 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 comprise at least one mismatched base pair and are within 5 base pairs from the AS 5′ and

the S 3′

end of the substituted dsRNA silencing agent,

such that the base pairing strength within 5 base pairs from the AS 5′ and

the S 3′

end of the substituted dsRNA silencing agent relative to the base pairing strength within 5 base pairs from the AS 3′ and

the S ′

5 end of the substituted dsRNA silencing agent is lessened as compared to the base pairing strength within AS 5′ and

the S 3′

end of the first dsRNA silencing agent relative to the base pairing strength between the AS3′ and

the S 5′

end of the first dsRNA silencing agent.

2. The substituted dsRNA silencing agent of claim 1, 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.

3. The substituted dsRNA silencing agent of claim 1, 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.

4. A composition comprising the substituted dsRNA silencing agent of claim 1, formulated to facilitate entry of the substituted dsRNA silencing agent into a cell.

5. A pharmaceutical composition comprising the substituted dsRNA silencing agent of claim 1.

6. The substituted dsRNA silencing agent of claim 1, wherein the substituted dsRNA silencing agent is derived from an engineered precursor.

7. The substituted dsRNA silencing agent of claim 1, wherein the substituted dsRNA silencing agent is chemically synthesized.

8. The substituted dsRNA silencing agent of claim 1, wherein the substituted dsRNA silencing agent is enzymatically synthesized.

9. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA specifies the amino acid sequence of a cellular protein.

10. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA specifies the amino acid sequence of an extracellular protein.

11. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA specifies the amino acid sequence of a protein associated with a pathological condition.

12. The substituted dsRNA silencing agent of claim 11, wherein the protein is a pathogen-associated protein.

13. The substituted dsRNA silencing agent of claim 11, wherein the protein is a host protein.

14. The substituted dsRNA silencing agent of claim 11, wherein the protein is a tumor-associated protein.

15. The substituted dsRNA silencing agent of claim 11, wherein the protein is an autoimmune disease-associated protein.

16. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA specifies the amino acid sequence of an endogenous protein.

17. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA specifies the amino acid sequence of a heterologous protein expressed in a recombinant cell or a genetically altered organism.

18. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA specifies the amino acid sequence of a protein encoded by a transgene.

19. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA is expressed in a plant cell.

20. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA is expressed in a mammalian cell.

21. The substituted dsRNA silencing agent of claim 1, wherein the desired target mRNA is expressed in a human cell.

22. The substituted dsRNA silencing agent of claim 1, wherein the substituted dsRNA silencing agent is a siRNA duplex.

23. A composition comprising the siRNA duplex of claim 22, formulated to facilitate entry of the siRNA duplex into a cell.

24. A pharmaceutical composition comprising the siRNA duplex of claim 22.

25. An engineered pre-miRNA comprising the siRNA duplex of claim 22.

26. A vector encoding the pre-miRNA of claim 25.

27. A cell comprising the vector of claim 26.

28. A pri-miRNA comprising the pre-miRNA of claim 25.

29. A vector encoding the pri-miRNA of claim 28.

30. A cell comprising the vector of claim 29.

31. A small hairpin RNA (shRNA) comprising nucleotide sequence identical to the sense and antisense strand of the siRNA duplex of claim 22.

32. The shRNA of claim 31, wherein the nucleotide sequence identical to the sense strand is upstream of the nucleotide sequence identical to the antisense strand.

33. The shRNA of claim 31, wherein the nucleotide sequence identical to the antisense strand is upstream of the nucleotide sequence identical to the sense strand.

34. A vector encoding the shRNA of claim 31.

35. A cell comprising the vector of claim 34.

36. A transgene encoding the shRNA of claim 31.

37. The substituted dsRNA silencing agent of claim 1, wherein the substituted dsRNA silencing agent is a siRNA-like duplex, wherein the antisense strand is a miRNA strand and the sense strand is a miRNA* strand.

38. A composition comprising the siRNA-like duplex of claim 37, formulated to facilitate entry of the siRNA duplex into a cell.

39. A pharmaceutical composition comprising the siRNA-like duplex of claim 37.

40. A small hairpin RNA (shRNA) comprising nucleotide sequence identical to the miRNA and miRNA* strand of the siRNA-like duplex of claim 37.