COMPOSITIONS FOR RNA INTERFERENCE AND METHODS OF USE THEREOF

• US 20140373188A1
• Filed: 03/20/2014
• Published: 12/18/2014
• Est. Priority Date: 08/07/2002
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: ZAMORE, Phillip D., HUTVAGNER, Gyorgy, SCHWARZ, Dianne, TANG, Guiliang, HALEY, Benjamin
• Granted Patent: US 9,611,472 B2
• Time in Patent Office: Days
• US Class Current: 800/13
• 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 or a viral protein.

4. The ss-siRNA of claim 1, 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 claim 1, comprising between about 10 and 50 nucleotides.

14. The ss-siRNA of claim 1, comprising between about 15 and 45 nucleotides.

15. The ss-siRNA of claim 1, comprising between about 19 and 40 nucleotides.

16. The ss-siRNA of claim 1 which is chemically synthesized.

17. A transgene that encodes the ss-siRNA of claim 1.

18. A composition comprising the ss-siRNA molecule of claim 1 and a pharmaceutically acceptable carrier.

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 the ss-siRNA of claim 1, 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 the ss-siRNA of claim 1;

(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 the ss-siRNA of claim 1;

(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) the ss-siRNA of claim 1; and

(b) instructions for use.

3. (canceled)

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.

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

31. An organism derived from the cell of claim 26.

20-25. -25. (canceled)

27-30. -30. (canceled)

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.

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

33-34. -34. (canceled)

36-41. -41. (canceled)