Compositions for RNA interference and methods of use thereof

• US 20040203145A1
• Filed: 08/07/2003
• Published: 10/14/2004
• Est. Priority Date: 08/07/2002
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Haley, Benjamin, Zamore, Phillip D., Tang, Guiliang, Hutvagner, Gyorgy, Schwarz, Dianne
• Granted Patent: US 8,729,036 B2
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2330/50 Biochemical production, i.e. in a transformed host cell

Abstract

The present invention provides compositions for RNA interference and methods of use thereof. In particular, the invention provides single-stranded small interfering RNAs. Functional and genomic and proteomic methods are featured. Therapeutic methods are also featured.

Claims

1. An isolated, single-stranded small interfering molecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the 5′

nucleotide is 5′

phosphorylated or is capable of being 5′

phosphorylated in situ or in vivo.

2. The ss-siRNA of claim 1 which is sufficiently complementary to a target mRNA, said target mRNA specifying the amino acid sequence of a cellular protein.

3. The ss-siRNA of claim 1 which is sufficiently complementary to a target mRNA, said target mRNA specifying the amino acid sequence of a viral protein.

4. The ss-siRNA of any one of claims 1-3, which is modified such that the ss-siRNA has increased in situ or in vivo stability as compared to a corresponding unmodified ss-siRNA.

5. The modified ss-siRNA of claim 4, which is modified by the substitution of at least one nucleotide with a modified nucleotide.

6. The modified ss-siRNA of claim 5, wherein the modified nucleotide is a sugar-modified nucleotide.

7. The modified ss-siRNA of claim 6, wherein the modified nucleotide has a 2′

—

OH replaced by a moiety selected from the group consisting of H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ and CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

8. The modified ss-siRNA of claim 6, wherein the modified nucleotide is a 3′

most nucleotide.

9. The modified ss-siRNA of claim 8, wherein the 3′

most nucleotide has a 2′

—

OH replaced by a moiety selected from the group consisting of H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ and CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

10. The modified ss-siRNA of claim 8, wherein the 3′

most nucleotide has a 3′

—

OH replaced by a moiety selected from the group consisting of H, OR, R, halo, SH, SR¹, NH₂, NHR, NR₂ and CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

11. The modified ss-siRNA of claim 8, wherein the modified nucleotide is a backbone-modified nucleotide.

12. The modified ss-siRNA of claim 11, wherein the backbone-modified nucleotide contains a phosphorothioate group.

13. The ss-siRNA of any one of claims 1-3, comprising between about 10 and 50 nucleotides.

14. The ss-siRNA of any one of claims 1-3, comprising between about 15 and 45 nucleotides.

15. The ss-siRNA of any one of claims 1-3, comprising between about 19 and 40 nucleotides.

16. The ss-siRNA of any one of claims 1-3 which is chemically synthesized.

17. A transgene that encodes the ss-siRNA of any one of claims 1-3.

18. A composition comprising the ss-siRNA molecule of any one of claims 1-3 and a pharmaceutically acceptable carrier.

19. A method of activating target-specific RNA interference (RNAi) in a cell comprising introducing into said cell a single-stranded small interfering RNA molecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the 5′

nucleotide is 5′

phosphorylated or is capable of being 5′

phosphorylated in situ or in vivo, said ss-siRNA being introduced in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the cell.

20. The method of claim 19, wherein the ss-siRNA is introduced into the cell by contacting the cell with the ss-siRNA.

21. The method of claim 20, wherein the ss-siRNA is introduced into the cell by contacting the cell with a composition comprising the ss-siRNA and a lipophillic carrier.

22. The method of claim 19, wherein the ss-siRNA is introduced into the cell by transfecting or infecting the cell with a vector comprising nucleic acid sequences capable of producing the ss-siRNA when transcribed in situ.

23. The method of claim 19, wherein the ss-siRNA is introduced into the cell by injecting into the cell a vector comprising nucleic acid sequences capable of producing the ss-siRNA when transcribed in situ.

24. The method of claim 23, wherein the vector comprises transgene nucleic acid sequences.

25. The method of claim 19, wherein the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder.

26. A cell obtained by the method of claim 19.

27. The cell of claim 26 which is of mammalian origin.

28. The cell of claim 26 which is of murine origin.

29. The cell of claim 26 which is of human origin.

30. The cell of claim 26, which is an embryonic stem cell.

31. An organism derived from the cell of claim 30.

41. The method of claim 19-24 and 32-34, wherein degradation of the target mRNA is such that the protein specified by said target mRNA is decreased by at least 10%.

32. A method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism a single-stranded small interfering RNA molecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the 5′

nucleotide is 5′

phosphorylated or is capable of being 5′

phosphorylated in situ or in vivo, said ss-siRNA being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism.

33. The method of claim 32, wherein the ss-siRNA is administered by an intravenous or intraperitoneal route.

34. The method of claim 32, wherein the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder.

35. An organism obtained by the method of claim 32.

36. The organism of claim 35 which is of mammalian origin.

37. The organism of claim 35 which is of murine origin.

38. The organism of claim 35 which is of human origin.

39. The organism of any one of claims 35-38, wherein the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder.

40. The organism of any one of claims 35-38, wherein degradation of the target mRNA produces a loss-of-function phenotype.

42. A method of treating a disease or disorder associated with the activity of a protein specified by a target mRNA in a subject, comprising administering to said subject a single-stranded small interfering RNA molecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule is sufficiently complementary to the target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the 5′

nucleotide is 5′

phosphorylated or is capable of being 5′

phosphorylated in situ or in vivo, said ss-siRNA being administered in an amount sufficient for degradation of the target mRNA to occur, thereby treating the disease or disorder associated with the protein.

43. A method for deriving information about the function of a gene in a cell or organism comprising:

(a) introducing into said cell or organism a single-stranded small interfering RNA molecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the 5′

nucleotide is 5′

phosphorylated or is capable of being 5′

phosphorylated in situ or in vivo;

(b) maintaining the cell or organism under conditions such that target-specific RNAi can occur;

(c) determining a characteristic or property of said cell or organism; and

(d) comparing said characteristic or property to a suitable control, the comparison yielding information about the function of the gene.

44. A method of validating a candidate protein as a suitable target for drug discovery comprising:

(a) introducing into a cell or organism a single-stranded small interfering RNA molecule (ss-siRNA), wherein the sequence of said ss-siRNA molecule is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the 5′

nucleotide is 5′

phosphorylated or is capable of being 5′

phosphorylated in situ or in vivo, said target mRNA specifying the amino acid sequence of the candidate protein;

(b) maintaining the cell or organism under conditions such that target-specific RNAi can occur;

(c) determining a characteristic or property of said cell or organism; and

(d) comparing said characteristic or property to a suitable control, the comparison yielding information about whether the candidate protein is a suitable target for drug discovery.

45. A kit comprising reagents for activating target-specific RNA interference (RNAi) in a cell or organism, said kit comprising:

(a) an isolated, single-stranded small interfering (ss-siRNA) molecule, wherein the sequence of said ss-siRNA molecule is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the 5′

nucleotide is 5′

phosphorylated or is capable of being 5′

phosphorylated in situ or in vivo; and

(b) instructions for use.