In vivo production of small interfering RNAS that mediate gene silencing

• US 7,691,995 B2
• Filed: 07/12/2002
• Issued: 04/06/2010
• Est. Priority Date: 07/12/2001
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Grishok, Alla, Mello, Craig C., McLachlan, Juanita, Zamore, Phillip D., Hutvagner, Gyorgy
• Primary Examiner(s): WHITEMAN, BRIAN A
• Application Number:
  US10/195,034
• Publication Number:
  US 20060009402A1
• Time in Patent Office: 2,825 Days
• Field of Search: 536/24.5
• US Class Current: 536/24.5
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12N 2330/51 : Specially adapted vectors
• CPC Class Codes: C12N 2517/02 : Cells from transgenic animals
• CPC Class Codes: C12N 2799/021 : for the expression of a heterologous nucleic acid
• CPC Class Codes: C12N 2830/008 : cell type or tissue specific enhancer/promoter combination

Abstract

The invention provides engineered RNA precursors that when expressed in a cell are processed by the cell to produce targeted small interfering RNAs (siRNAs) that selectively silence targeted genes (by cleaving specific mRNAs) using the cell'"'"'s own RNA interference (RNAi) pathway. By introducing nucleic acid molecules that encode these engineered RNA precursors into cells in vivo with appropriate regulatory sequences, expression of the engineered RNA precursors can be selectively controlled both temporally and spatially, i.e., at particular times and/or in particular tissues, organs, or cells.

Claims

1. An isolated nucleic acid molecule capable of inducing RNA interference (RNAi) in a mammalian cell in vivo, comprising a regulatory sequence operably linked to a nucleic acid sequence that encodes an engineered ribonucleic acid (RNA) precursor, wherein the precursor comprises a stem from a naturally-occurring stRNA or miRNA precursor, the stem comprising a first stem portion of about 18 to about 40 nucleotides and a second stem portion of about 18 to about 40 nucleotides, wherein the first stem portion comprises an stRNA or miRNA sequence within additional stem sequences of the first stem portion, and wherein the second stem portion comprises a sequence complementary to the stRNA or miRNA sequence within additional stem sequences of the second stem portion, wherein:

(i) the stRNA or miRNA sequence within the first stem portion and the sequence within the second stem portion that is complementary to the stRNA or miRNA sequence are replaced with an siRNA antisense strand sequence that is complementary to a sequence of a messenger RNA (mRNA) of a target gene in the mammalian cell and an siRNA sense strand sequence that is complementary to the siRNA antisense strand sequence;

(ii) the second stem portion is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem;

(iii) a loop portion connects the two stem portions; and

(iv) the engineered RNA precursor is processed by the cell to generate an siRNA that mediates cleavage of the mRNA.

2. The nucleic acid molecule of claim 1, wherein the siRNA antisense strand sequence is fully complementary to the mRNA sequence.

3. The nucleic acid molecule of claim 1, wherein the siRNA sense strand sequence is fully complementary to the siRNA antisense strand sequence.

4. The nucleic acid molecule of claim 1, wherein the first stem portion is located at the 5′

end of the engineered RNA precursor.

5. The nucleic acid molecule of claim 1, wherein the first stem portion is located at the 3′

end of the engineered RNA precursor.

6. The nucleic acid molecule of claim 1, wherein the loop portion comprises at least 4 nucleotides.

7. The nucleic acid molecule of claim 1, wherein the loop portion comprises at least 7 nucleotides.

8. The nucleic acid molecule of claim 1, wherein the loop portion comprises 11 nucleotides.

9. The nucleic acid molecule of claim 1, wherein the sequence of the mRNA to which the siRNA antisense strand sequence is complementary, is located from 100 to 300 nucleotides 3′

of the start of translation of the mRNA of the target gene.

10. The nucleic acid molecule of claim 1, wherein the sequence of the mRNA to which the siRNA antisense strand sequence is complementary, is located in a 5′

untranslated region (UTR) or a 3′

UTR of the mRNA of the target gene.

11. The nucleic acid molecule of claim 1, wherein the first and second stem portions each comprise the same number of nucleotides.

12. The nucleic acid molecule of claim 1, wherein one of the first and second stem portions comprises 1 to 4 more nucleotides than the other stem portion.

13. The nucleic acid molecule of claim 1, wherein the regulatory sequence comprises a Pol III or Pol II promoter.

14. The nucleic acid molecule of claim 13, wherein the Pol III promoter is a U6 or H1-RNA promoter.

15. The nucleic acid molecule of claim 1, wherein the regulatory sequence is constitutive or inducible.

16. The nucleic acid molecule of claim 1, wherein the sequence of the engineered RNA precursor comprises a sequence selected from the group consisting of the sequence set forth in SEQ ID NO:

2, 3, 4, 5, 8, and 9.

17. The nucleic acid molecule of claim 1, comprising a sequence selected from the group consisting of the sequence set forth in SEQ ID NO:

10, 11, 17, 18, 20, 21 or a complement thereof.

18. The nucleic acid molecule of claim 1, wherein the first stem portion or the second stem portion further comprises an overhang of 1, 2, 3, or 4 nucleotides.

19. The nucleic acid molecule of claim 18, wherein the overhang comprises two uracils.

20. The nucleic acid molecule of claim 1, wherein the first stem portion is 18 to 40 nucleotides in length and the second stem portion is 18 to 40 nucleotides in length.

21. The nucleic acid molecule of claim 1, wherein the first stem portion is 22 to 28 nucleotides in length and the second stem portion is 22 to 28 nucleotides in length.

22. The nucleic acid molecule of claim 1, wherein the first stem portion is 18 to 30 nucleotides in length and the second stem portion is 18 to 30 nucleotides in length.

23. The nucleic acid molecule of claim 1, wherein the regulatory sequence is a mammalian regulatory sequence.

24. The nucleic acid molecule of claim 23, wherein the mammalian regulatory sequence is a tissue-specific regulatory sequence.

25. The nucleic acid molecule of claim 1, wherein the engineered RNA precursor is processed by Dicer.

26. The nucleic acid molecule of claim 1, wherein the loop portion of the engineered RNA precursor is at least 4 nucleotides in length and comprises a sequence from a loop of the naturally-occurring stRNA or miRNA precursor.

27. The nucleic acid molecule of claim 26, wherein the number of nucleotides in the loop portion of the engineered RNA precursor differs from the number of nucleotides in the loop of the naturally-occurring stRNA or miRNA precursor.

28. The nucleic acid molecule of claim 26, wherein the sequence of the loop portion of the engineered RNA precursor is the same as the sequence from the loop of the naturally-occurring stRNA or miRNA precursor.

29. The nucleic acid molecule of claim 1, wherein the siRNA sequences are about 21 to about 25 nucleotides in length.

30. The nucleic acid molecule of claim 1, wherein the siRNA sequences are 21 or 22 nucleotides in length.

31. The nucleic acid molecule of claim 1, wherein the cell is a human cell.

32. The nucleic acid molecule of claim 1, wherein the target gene is a human gene.

33. The nucleic acid molecule of claim 1, wherein the target gene is a mutant human gene.

34. The nucleic acid molecule of claim 1, wherein the target gene is a viral gene.

35. A vector comprising the nucleic acid molecule of claim 1.

36. The vector of claim 35, wherein the vector is a plasmid or a viral vector.

37. The vector of claim 36, wherein the viral vector is a retroviral vector.

38. A host cell containing the nucleic acid molecule of claim 1.

39. The host cell of claim 38, wherein the cell is a mammalian cell.

40. A transgene comprising the nucleic acid of claim 1.

41. An engineered RNA precursor capable of inducing RNA interference (RNAi) in a mammalian cell in vivo, comprising a stem from a naturally-occurring stRNA or miRNA precursor, the stem comprising a first stem portion of about 18 to about 40 nucleotides and a second stem portion of about 18 to about 40 nucleotides, wherein the first stem portion comprises an stRNA or miRNA sequence within additional stem sequences of the first stem portion, and wherein the second stem portion comprises a sequence complementary to the stRNA or miRNA sequence within additional stem sequences of the second stem portion, wherein:

(i) the stRNA or miRNA sequence within the first stem portion and the sequence within the second stem portion that is complementary to the stRNA or miRNA sequence are replaced with an siRNA antisense strand sequence that is complementary to a sequence of a messenger RNA (mRNA) of a target gene in the mammalian cell and an siRNA sense strand sequence that is complementary to the siRNA antisense strand sequence;

(ii) the second stem portion is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem;

(iii) a loop portion connects the two stem portions; and

(iv) the engineered RNA precursor is processed by the cell to generate an siRNA that mediates cleavage of the mRNA.

42. The engineered RNA precursor of claim 41, wherein the siRNA antisense strand sequence is fully complementary to the mRNA sequence.

43. The engineered RNA precursor of claim 41, wherein the siRNA sense strand sequence is fully complementary to the siRNA antisense strand sequence.

44. The engineered RNA precursor of claim 41, wherein the first stem portion is located at the 5′

end of the engineered RNA precursor.

45. The engineered RNA precursor of claim 41, wherein the first stem portion is located at the 3′

end of the engineered RNA precursor.

46. The engineered RNA precursor of claim 41, wherein the loop portion comprises at least 4 nucleotides.

47. The engineered RNA precursor of claim 41, wherein the loop portion comprises at least 7 nucleotides.

48. The engineered RNA precursor of claim 41, wherein the loop portion comprises 11 nucleotides.

49. The engineered RNA precursor of claim 41, wherein the sequence of the mRNA to which the siRNA antisense strand sequence is complementary, is located in a 5′

untranslated region (UTR) or a 3′

UTR of the mRNA of the target gene.

50. The engineered RNA precursor of claim 41, wherein the first and second stem portions each comprise the same number of nucleotides.

51. The engineered RNA precursor of claim 41, wherein one of the first and second stem portions comprises 1 to 4 more nucleotides than the other stem portion.

52. The engineered RNA precursor of claim 41, comprising a sequence selected from the group consisting of the sequence set forth in SEQ ID NO:

2, 3, 4, 5, 8, and 9.

53. The engineered RNA precursor of claim 41, wherein the target gene is a human gene.

54. The engineered RNA precursor of claim 41, wherein the target gene is a mutant human gene.

55. The engineered RNA precursor of claim 41, wherein the target gene is a viral gene.

56. The engineered RNA precursor of claim 41, wherein the first stem portion or the second stem portion further comprises an overhang of 1, 2, 3, or 4 nucleotides.

57. The engineered RNA precursor of claim 56, wherein the overhang comprises two uracils.

58. The engineered RNA precursor of claim 41, wherein the first stem portion is 18 to 40 nucleotides in length and the second stem portion is 18 to 40 nucleotides in length.

59. The engineered RNA precursor of claim 41, wherein the first stem portion is 22 to 28 nucleotides in length and the second stem portion is 22 to 28 nucleotides in length.

60. The engineered RNA precursor of claim 41, wherein the first stem portion is 18 to 30 nucleotides in length and the second stem portion is 18 to 30 nucleotides in length.

61. The engineered RNA precursor of claim 41, wherein the engineered RNA precursor is processed by Dicer.

62. The engineered RNA precursor of claim 41, wherein the loop portion of the engineered RNA precursor is at least 4 nucleotides in length and comprises a sequence from a loop of the naturally-occurring stRNA or miRNA precursor.

63. The engineered RNA precursor of claim 62, wherein the number of nucleotides in the loop portion of the engineered RNA precursor differs from the number of nucleotides in the loop of the naturally-occurring stRNA or miRNA precursor.

64. The engineered RNA precursor of claim 62, wherein the sequence of the loop portion of the engineered RNA precursor is the same as the sequence from the loop of the naturally-occurring stRNA or miRNA precursor.

65. The engineered RNA precursor of claim 41, wherein the siRNA sequences are about 21 to about 25 nucleotides in length.

66. The engineered RNA precursor of claim 41, wherein the siRNA sequences are 21 or 22 nucleotides in length.

67. The engineered RNA precursor of claim 41, wherein the sequence of the mRNA to which the siRNA antisense strand sequence is complementary, is located from 100 to 300 nucleotides 3′

of the start of translation of the mRNA of the target gene.

68. The engineered RNA precursor of claim 41, wherein the cell is a human cell.