RNA interference mediating small RNA molecules

Patent metadata

Field	Value
Publication number	US-10633656-B2
Application number	US-201615388681-A
Country	US
Kind code	B2
Filing date	Dec 22, 2016
Priority date	Dec 1, 2000
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

Abstract

Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.

First claim

The invention claimed is:

1. A method of mediating target-specific RNA interference in a cell or an organism comprising contacting said cell or organism with an isolated double-stranded RNA molecule, said contacting step comprising administration of the RNA molecule under conditions wherein target-specific RNA interference can occur, wherein the RNA molecule comprises:
(i) a sense strand and an antisense strand;
(ii) each strand consists of 19-52 nucleotides;
(iii) the RNA molecule comprises at least one 3′-overhang;
(iv) the RNA molecule is synthetic;
(v) the RNA molecule comprises at least one nucleotide analogue; and
(vi) a strand of said RNA molecule is at least 85% identical in the double-stranded portion of the RNA molecule to a target mRNA.

2. The method of claim 1 , wherein the contacting step occurs in a cell.

3. The method of claim 1 , wherein the contacting step occurs in an organism.

4. The method of claim 1 , wherein the RNA molecule is administered using a carrier-mediated delivery.

5. The method of claim 4 , wherein said carrier-mediated delivery comprises a liposomal carrier or a lipid formulation.

6. The method of claim 1 , wherein the contacting step comprises injection of the RNA molecule.

7. The method of claim 1 , wherein the cell or the organism is a mammalian cell or a mammalian organism.

8. The method of claim 1 , wherein the cell or the organism is a human cell or a human organism.

9. The method of claim 1 , wherein the cell or the organism is a plant cell or a plant organism.

10. The method of claim 1 , wherein the cell is chosen from an embryonic cell, a pluripotent stem cell, a tumor cell, or a virus-infected cell.

11. The method of claim 1 , wherein said target-specific RNA interference modulates the function of a gene in the cell or the organism.

12. The method of claim 1 , wherein the RNA molecule modulates the function of a gene associated with a pathological condition.

13. The method of claim 1 , wherein the RNA molecule is synthesized by chemical synthesis.

14. The method of claim 1 , wherein the RNA molecule is not prepared by enzymatic transcription.

15. The method of claim 1 , wherein the at least one nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.

16. The method of claim 1 , wherein the at least one nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′—OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is C 1 -C 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

17. The method of claim 1 , wherein the at least one nucleotide analogue is a backbone-modified ribonucleotide comprising a phosphorothioate group.

18. The method of claim 1 , wherein the nucleotide analogue comprises a modified nucleobase or a non-naturally-occurring nucleobase.

19. The method of claim 1 , wherein the RNA molecule comprises a single double stranded region.

20. The method of claim 1 , wherein the 3′-overhang is stabilized against degradation.

21. The method of claim 1 , wherein the 3′-overhang is 1-5 nucleotides in length.

22. The method of claim 1 , wherein the 3′-overhang is 1-3 nucleotides in length.

23. The method of claim 1 , wherein the 3′-overhang is 2 nucleotides in length.

24. The method of claim 1 , wherein the RNA molecule comprises a blunt end.

25. The method of claim 1 , wherein each strand is 19-25 nucleotides in length.

26. The method of claim 1 , wherein the target mRNA is encoded by a pathogen-associated gene, a viral gene, a tumor-associated gene, or an autoimmune disease-associated gene.

27. The method of claim 1 , wherein the sense strand is at least 85% identical in the double-stranded portion of the RNA molecule to the target mRNA.

28. The method of claim 1 , wherein the sense strand is 100% identical in the double-stranded portion of the RNA molecule to the target mRNA.

29. A method of mediating target-specific RNA interference in a cell or an organism comprising contacting said cell or organism with an isolated double-stranded RNA molecule, said contacting step comprising administration of the RNA molecule under conditions wherein target-specific RNA interference can occur, wherein said RNA molecule comprises a double-stranded region consisting of 19-52 base pairs and a 3′-overhang, wherein the RNA molecule is non-enzymatically processed, and is capable of target-specific RNA interference, and wherein said RNA molecule comprises at least one nucleotide analogue.

30. The method of claim 29 , wherein the contacting step occurs in a cell.

31. The method of claim 29 , wherein the contacting step occurs in an organism.

32. The method of claim 29 , wherein the RNA molecule is administered using a carrier-mediated delivery.

33. The method of claim 32 , wherein said carrier-mediated delivery comprises a liposomal carrier or a lipid formulation.

34. The method of claim 29 , wherein the contacting step comprises injection of the RNA molecule.

35. The method of claim 29 , wherein the cell or the organism is a mammalian cell or a mammalian organism.

36. The method of claim 29 , wherein the cell or the organism is a human cell or a human organism.

37. The method of claim 29 , wherein the cell or the organism is a plant cell or a plant organism.

38. The method of claim 29 , wherein the cell is chosen from an embryonic cell, a pluripotent stem cell, a tumor cell or a virus-infected cell.

39. The method of claim 29 , wherein said target-specific RNA interference modulates the function of a gene in the cell or the organism.

40. The method of claim 29 , wherein the RNA molecule modulates the function of a gene associated with a pathological condition.

41. The method of claim 29 , wherein the RNA molecule is synthesized by chemical synthesis.

42. The method of claim 29 , wherein the RNA molecule is not prepared by enzymatic transcription.

43. The method of claim 29 , wherein the at least one nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.

44. The method of claim 29 , wherein the at least one nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′—OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is C 1 -C 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

45. The method of claim 29 , wherein the at least one nucleotide analogue is a backbone-modified ribonucleotide comprising a phosphorothioate group.

46. The method of claim 29 , wherein the nucleotide analogue comprises a modified nucleobase or a non-naturally-occurring nucleobase.

47. The method of claim 29 , wherein the RNA molecule comprises a single double stranded region.

48. The method of claim 29 , wherein the 3′-overhang is stabilized against degradation.

49. The method of claim 29 , wherein the 3′-overhang is 1-5 nucleotides in length.

50. The method of claim 29 ,

Assignees

• Max Planck Gesellschaft
• Massachusetts Inst Technology
• Whitehead Inst Biomedical Res
• Univ Massachusetts

Inventors

• Tuschl Thomas
• Elbashir Sayda Mahgoub
• Lendeckel Winfried

Classifications

• A61K9/0019

  Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00) · CPC title

• A61K38/00

  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 title

• C12N15/11

  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 title

• C12N2310/53

  partially self-complementary or closed · CPC title

• C12N2310/321

  2'-O-R Modification · CPC title