RNA interference mediating small RNA molecules

• US 8,362,231 B2
• Filed: 01/06/2010
• Issued: 01/29/2013
• Est. Priority Date: 12/01/2000
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology, Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e), University Of Massachusetts, Whitehead Institute for Biomedical Research
• Original Assignee: Massachusetts Institute of Technology, Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e), University Of Massachusetts, Whitehead Institute for Biomedical Research
• Inventors: Tuschl, Thomas, Elbashir, Sayda Mahgoub, Lendeckel, Winfried
• Primary Examiner(s): Bowman, Amy
• Application Number:
  US12/683,081
• Publication Number:
  US 20110070162A1
• Time in Patent Office: 1,119 Days
• Field of Search: None
• US Class Current: 536/24.5
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• 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: A61K 9/0019 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 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 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• 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 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2330/30 chemically synthesised

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.

Claims

1. An isolated, synthetic double-stranded RNA molecule, wherein:

(i) each RNA strand of said double-stranded RNA molecule independently consists of 19-23 nucleotides in length;

(ii) at least one RNA strand forms a single-stranded 3′

-overhang from 1 to 3 nucleotides;

(iii) the 3′

-overhang of said double-stranded RNA molecule comprises at least one nucleotide analogue,

wherein said RNA molecule is capable of target-specific RNA interference.

2. The double-stranded RNA molecule of claim 1, wherein one RNA strand forms the single-stranded 3′

-overhang from 1 to 3 nucleotides in length.

3. The double-stranded RNA molecule of claim 1, wherein each of the RNA strands forms a single-stranded 3′

-overhang from 1-3 nucleotides in length.

4. The double-stranded RNA molecule of claim 1, wherein each strand consists of 21-23 nucleotide in length.

5. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang is 2 nucleotides in length.

6. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang has been stabilized against degradation.

7. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a nucleotide lacking a 2′

-hydroxyl in a sugar.

8. The double-stranded RNA molecule of claim 1, wherein the nucleotide analogue of the 3′

-overhang is selected from a sugar- or a backbone modified ribonucleotide, or a combination thereof.

9. The double-stranded RNA molecule according to claim 1, wherein the nucleotide analogue of the 3′

-overhang is a sugar-modified ribonucleotide, wherein the 2′

-OH group is replaced by a group selected from H, OR, R, halo, SH, SR′

, NH₂, NHR, N(R)₂ or CN, wherein R is C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl and halo is F, Cl, Br or I.

10. The double-stranded RNA molecule of claim 1, wherein the nucleotide analogue of the 3′

-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group.

11. The double-stranded RNA molecule of claim 1, which has a sequence having an identity of at least 70 percent in the double-stranded portion of the RNA molecule to a predetermined mRNA target molecule.

12. The double-stranded RNA molecule of claim 11, wherein the identity is at least 85 percent in the double-stranded portion of the RNA molecule.

13. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 1 and a pharmaceutical carrier.

14. The composition of claim 13 for diagnostic applications.

15. The composition of claim 13 for therapeutic applications.

16. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a 2′

-deoxythymidine.

17. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang of the double-stranded RNA molecule is chosen from NN/UG, NN/UU, NN/TdG or NN/TT, wherein N is any nucleotide.

18. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang of the double-stranded RNA molecule is TT.

19. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a purine nucleotide.

20. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a modified pyrimidine nucleotide.

21. The double-stranded RNA molecule of claim 1, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a nucleobase-modified ribonucleotide chosen from a non-naturally-occurring nucleobase modified at the 5-position;

an adenosine or a guanosine modified at the 8-position;

or an O- or an N-alkylated nucleotide.

22. The double-stranded RNA molecule of claim 4, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a 2′

-deoxythymidine or a nucleotide lacking a 2′

-hydroxyl in a sugar.

23. The double-stranded RNA molecule of claim 4, wherein the nucleotide analogue of the 3′

-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.

24. The double-stranded RNA molecule of claim 4, wherein the nucleotide analogue of the 3′

-overhang is a sugar-modified ribonucleotide, wherein the 2′

-OH group is replaced by a group selected from H, OR, R, halo, SH, SR′

, NH₂, NHR, N(R)₂ or CN, wherein R is C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl and halo is F, Cl, Br or I.

25. The double-stranded RNA molecule of claim 4, wherein the nucleotide analogue of the 3′

-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group.

26. The double-stranded RNA molecule of claim 4, wherein the 3′

-overhang of the double-stranded RNA molecule is TT.

27. The double-stranded RNA molecule of claim 1, wherein the double-stranded portion of the RNA molecule comprises at least one nucleotide analogue.

28. The double-stranded RNA molecule of claim 27, wherein the nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.

29. The double-stranded RNA molecule of claim 27, wherein the 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, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl and halo is F, Cl, Br or I.

30. The double-stranded RNA molecule of claim 27, wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group.

31. The double-stranded RNA molecule of claim 27, wherein the modified nucleotide analogue is located at the 5′

-end, the 3′

-end, or both, of the double stranded RNA molecule.

32. The double-stranded RNA molecule of claim 1, wherein each strand has a length from 20 to 22 nucleotides.

33. The double-stranded RNA molecule of claim 1, wherein each strand has a length of 21 or 22 nucleotides.

34. The double-stranded RNA molecule of claim 1, wherein the only single stranded regions in said double-stranded RNA molecule are the one or two 3′

-overhangs.

35. The double-stranded RNA molecule of claim 3, wherein said double-stranded RNA molecule has a double-stranded region of 16-22 nucleotides in length.

36. The double-stranded RNA molecule of claim 1, which consists of a single double-stranded region and a single-stranded region of 1 to 3 nucleotides at the 3′

end of at least one of the strands of said double-stranded RNA molecule.

37. The double-stranded RNA molecule of claim 1, wherein the RNA strands of said double-stranded RNA are not covalently linked to each other.

38. The double-stranded RNA molecule of claim 1, which comprises a terminal 3′

hydroxyl group.

39. The double-stranded RNA molecule of claim 1, which is chemically synthesized.

40. The double-stranded RNA molecule of claim 1, which cleaves a target mRNA at a site within the region spanned by the double-stranded RNA molecule.

41. The double-stranded RNA molecule of claim 40, which cleaves a target mRNA at a single site.

42. The double-stranded RNA molecule of claim 1, which has a sequence in the double-stranded portion of the RNA molecule identical to a predetermined mRNA target molecule.

43. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 22 and a pharmaceutical carrier.

44. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 23 and a pharmaceutical carrier.

45. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 24 and a pharmaceutical carrier.

46. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 25 and a pharmaceutical carrier.

47. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 26 and a pharmaceutical carrier.

48. An isolated, synthetic double-stranded RNA molecule, wherein:

(i) each RNA strand of said double-stranded RNA molecule independently consists of 19-25 nucleotides in length;

(ii) at least one RNA strand forms a single-stranded 3′

-overhang from 1 to 5 nucleotides;

(iii) the 3′

-overhang of said double-stranded RNA molecule comprises at least one nucleotide analogue,

wherein said RNA molecule is capable of target-specific RNA interference.

49. The double-stranded RNA molecule of claim 48, wherein one RNA strand forms the 3′

-overhang from 1 to 5 nucleotides in length.

50. The double-stranded RNA molecule of claim 48, wherein each of the RNA strands forms a 3′

-overhang from 1-5 nucleotides in length.

51. The double-stranded RNA molecule of claim 50, wherein the 3′

-overhang is 2 nucleotides in length.

52. The double-stranded RNA molecule of claim 48, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a nucleotide lacking a 2′

-hydroxyl in a sugar.

53. The double-stranded RNA molecule of claim 48, wherein the nucleotide analogue of the 3′

-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.

54. The double-stranded RNA molecule according to claim 48, wherein the nucleotide analogue of the 3′

-overhang is a sugar-modified ribonucleotide, wherein the 2′

-OH group is replaced by a group selected from H, OR, R, halo, SH, SR′

, NH₂, NHR, N(R)₂ or CN, wherein R is C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl and halo is F, Cl, Br or I.

55. The double-stranded RNA molecule of claim 48, wherein the nucleotide analogue of the 3′

-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group.

56. The double-stranded RNA molecule of claim 48, wherein the RNA strands of said double-stranded RNA are not covalently linked to each other.

57. The double-stranded RNA molecule of claim 48, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a 2′

-deoxythymidine.

58. The double-stranded RNA molecule of claim 48, wherein the 3′

-overhang of the double-stranded RNA molecule is TT.

59. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 48 and a pharmaceutical carrier.

60. An isolated, synthetic double-stranded RNA molecule, wherein:

(i) each RNA strand of said double-stranded RNA molecule independently consists of 19-23 nucleotides in length;

(ii) at least one RNA strand forms a single-stranded 3′

-overhang from 1 to 3 nucleotides;

(iii) the 3′

-overhang of said double-stranded RNA molecule has been stabilized against degradation,

wherein said RNA molecule is capable of target-specific RNA interference.

61. The double-stranded RNA molecule of claim 60, wherein one RNA strand forms the single-stranded 3′

-overhang from 1 to 3 nucleotides in length.

62. The double-stranded RNA molecule of claim 60, wherein each of the RNA strands forms a 3′

-overhang from 1-3 nucleotides in length.

63. The double-stranded RNA molecule of claim 62, wherein the 3′

-overhang has been stabilized against degradation by incorporating a nucleotide analogue.

64. The double-stranded RNA molecule of claim 63, wherein the 3′

-overhang is 2 nucleotides in length.

65. The double-stranded RNA molecule of claim 64, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a nucleotide lacking a 2′

-hydroxyl in a sugar.

66. The double-stranded RNA molecule of claim 63, wherein the nucleotide analogue of the 3′

-overhang is selected from a sugar- or a backbone modified ribonucleotide, or a combination thereof.

67. The double-stranded RNA molecule according to claim 63, wherein the nucleotide analogue of the 3′

-overhang is a sugar-modified ribonucleotide, wherein the 2′

-OH group is replaced by a group selected from H, OR, R, halo, SH, SR′

, NH₂, NHR, N(R)₂ or CN, wherein R is C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl and halo is F, Cl, Br or I.

68. The double-stranded RNA molecule of claim 63, wherein the nucleotide analogue of the 3′

-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group.

69. The double-stranded RNA molecule of claim 64, wherein the 3′

-overhang of the double-stranded RNA molecule comprises a 2′

-deoxythymidine.

70. The double-stranded RNA molecule of claim 64, wherein the 3′

-overhang of the double-stranded RNA molecule is TT.

71. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 60 and a pharmaceutical carrier.

72. An isolated, synthetic double-stranded RNA molecule, wherein:

(i) each strand independently consists of 21-23 nucleotides;

(ii) each strand forms a single-stranded two-nucleotide 3′

-overhang;

(iii) the only single-stranded regions in said RNA molecule are the single stranded two-nucleotide 3′

-overhangs; and

(iv) each of the two-nucleotide 3′

-overhangs comprises at least one nucleotide analogue to stabilize the 3′

-overhang against degradation,

wherein said RNA molecule is capable of target-specific RNA interference.

73. The double-stranded RNA molecule of claim 72, wherein each of the 3′

-overhangs of the double-stranded RNA molecule comprises a nucleotide lacking a 2′

-hydroxyl in a sugar.

74. The double-stranded RNA molecule of claim 72, wherein each of the 3′

-overhangs of the double-stranded RNA molecule comprises a 2′

-deoxythymidine.

75. The double-stranded RNA molecule of claim 72, wherein each of the two-nucleotide 3′

-overhangs of the double-stranded RNA molecule is TT.

76. The double-stranded RNA molecule of claim 72, wherein each of the two-nucleotide 3′

-overhangs has at least one backbone modified ribonucleotide containing a phosphorothioate group.

77. The double-stranded RNA molecule of claim 72, wherein each of the two-nucleotide 3′

-overhangs has two backbone-modified ribonucleotides containing a phosphorothioate group.

78. The double-stranded RNA molecule of claim 72, wherein the only modified nucleotides in the double-stranded RNA molecule are in the two-nucleotide 3′

-overhangs.

79. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 72 and a pharmaceutical carrier.