Compositions for RNA interference and methods of use thereof

• US 9,611,472 B2
• Filed: 03/20/2014
• Issued: 04/04/2017
• Est. Priority Date: 08/07/2002
• Status: Expired due to Term

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Zamore, Phillip D., Hutvagner, Gyorgy, Schwarz, Dianne, Tang, Guiliang, Haley, Benjamin
• Primary Examiner(s): SHIN, DANA H
• Application Number:
  US14/220,388
• Publication Number:
  US 20140373188A1
• Time in Patent Office: 1,111 Days
• 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) of 19-21 nucleotides in length, wherein the sequence of said ss-siRNA molecule is sufficiently complementary to a target mRNA sequence in a mammalian cell 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 with a phosphate group or phosphate group analog, and further wherein the ss-siRNA comprises at least one modified nucleotide.

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.

3. 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.

4. The modified ss-siRNA of claim 1, wherein the modified nucleotide is a sugar-modified nucleotide.

5. The modified ss-siRNA of claim 4, wherein the modified nucleotide has a 2′

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

6. The modified ss-siRNA of claim 4, wherein the modified nucleotide is a 3′

most nucleotide.

7. The modified ss-siRNA of claim 6, wherein the 3′

most nucleotide has a 2′

-OH replaced by a moiety selected from the group consisting of H, OR, R, halo, SH, SR, 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 3′

most nucleotide has a 3′

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

9. The modified ss-siRNA of claim 6, wherein the modified nucleotide is a backbone-modified nucleotide.

10. The modified ss-siRNA of claim 9, wherein the backbone-modified nucleotide contains a phosphorothioate group.

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

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

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

14. 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.

15. The isolated ss-siRNA of claim 1, wherein the target mRNA sequence is mammalian.

16. An isolated, single-stranded small interfering molecule (ss-siRNA) of 19-21 nucleotides in length, wherein the sequence of said ss-siRNA molecule is complementary to a target mRNA sequence in a mammalian cell 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 with a phosphate group or phosphate group analog, and further wherein the ss-siRNA comprises at least one modified nucleotide.