Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

• US 20050244858A1
• Filed: 03/15/2005
• Published: 11/03/2005
• Est. Priority Date: 03/15/2004
• Status: Active Grant

• Current Assignee: City of Hope, Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: City of Hope, Integrated DNA Technologies (Danaher Corporation)
• Inventors: Behlke, Mark A., Kim, Dongho, Rossi, John J.
• Granted Patent: US 8,084,599 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 15/02 for disorders of the vagina
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/16 Otologicals
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/08 for leprosy
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 : specific for metastasis
• 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 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 5/14 : of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 : Antianaemics
• 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 2310/33 : of the base
• CPC Class Codes: C12N 2310/50 : Physical structure
• CPC Class Codes: C12N 2310/51 : in polymeric form, e.g. multimers, concatemers
• CPC Class Codes: C12N 2320/30 : Special therapeutic applications
• CPC Class Codes: C12N 2320/50 : Methods for regulating/modulating their activity
• CPC Class Codes: C12N 2320/51 : modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 : chemically synthesised

Abstract

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

Claims

1. A precursor RNAi molecule capable of reducing the expression of a target gene following cleavage by Dicer comprising:

a first oligonucleotide strand that is 22-30 nucleotides in length; and

a second oligonucleotide strand that is 22-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of the target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions,

wherein at least one of said first and second oligonucleotide strands is modified on the 3′

end to direct Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.

2. The precursor RNAi molecule of claim 1, wherein the nucleotide sequence of the second oligonucleotide strand sufficiently complementary to the RNA of the target gene comprises at least 19.

3. The precursor RNAi molecule of claim 1, wherein the first and second oligonucleotide strands have the same length.

4. The precursor RNAi molecule of claim 1, wherein the first and second oligonucleotide strands have different lengths.

5. The precursor RNAi molecule of claim 4, wherein the second oligonucleotide strand is longer than the first nucleotide strand and has a 3′

overhang when the first and second oligonucleotide strands are annealed.

6. The precursor RNAi molecule of claim 5, wherein the overhang is 1-3 nucleotides.

7. The precursor RNAi molecule of claim 5, wherein the overhang is 2 nucleotides.

8. The precursor RNAi molecule of claim 4, wherein first oligonucleotide strand is longer than the second nucleotide strand and has a 3′

overhang when the first and second oligonucleotide strands are annealed.

9. The precursor RNAi molecule of claim 8, wherein the overhang is 1-3 nucleotides.

10. The precursor RNAi molecule of claim 8, wherein the overhang is 2 nucleotides.

11. The precursor RNAi molecule of claim 1, wherein the first oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

12. The precursor RNAi molecule of claim 11, wherein the modification is the substitution of a ribonucleotide with a nucleotide selected from the group consisting of deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC), 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T), a monophosphate nucleotide of 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and a monophosphate nucleotide of 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

13. The precursor RNAi molecule of claim 11, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

14. The precursor RNAi molecule of claim 11, wherein the modification is 2 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

15. The precursor RNAi molecule of claim 11 wherein the modification is the addition of a sterically hindered molecule to the 3′

end of the first oligonucleotide strand.

16. The precursor RNAi molecule of claim 1, wherein the second oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

17. The precursor RNAi molecule of claim 16, wherein the modification is the substitution of a ribonucleotide with a nucleotide selected from the group consisting of deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC), 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T), a monophosphate nucleotide of 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and a monophosphate nucleotide of 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

18. The precursor RNAi molecule of claim 16, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the second oligonucleotide strand.

19. The precursor RNAi molecule of claim 16, wherein the modification is 2 deoxynucleotides substituted for ribonucleotides at the 3′

end of the second oligonucleotide strand.

20. The precursor RNAi molecule of claim 16, wherein the modification is the addition of a sterically hindered molecule to the 3′

end of the second oligonucleotide strand.

21. The precursor RNAi molecule of claim 5, wherein the first oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

22. The precursor RNAi molecule of claim 21, wherein the modification is the substitution of a ribonucleotide with a nucleotide is selected from the group consisting of deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC), 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T), a monophosphate nucleotide of 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and a monophosphate nucleotide of 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

23. The precursor RNAi molecule of claim 21, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

24. The precursor RNAi molecule of claim 21, wherein the modification is 2 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

25. The precursor RNAi molecule of claim 21 wherein the modification is the addition of a sterically hindered molecule to the 3′

end of the first oligonucleotide strand.

26. The precursor RNAi molecule of claim 8, wherein the second oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

27. The precursor RNAi molecule of claim 26, wherein the modification is the substitution of a ribonucleotide with a nucleotide selected from the group consisting of deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC), 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T), a monophosphate nucleotide of 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and a monophosphate nucleotide of 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

28. The precursor RNAi of molecule claim 26, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the second oligonucleotide strand.

29. The precursor RNAi molecule of claim 26, wherein the modification is 2 deoxynucleotides substituted for ribonucleotides at the 3′

end of the second oligonucleotide strand.

30. The precursor RNAi molecule of claim 26 wherein the modification is the addition of a sterically hindered molecule to the 3′

end of the first oligonucleotide strand.

31. The precursor RNAi molecule of claim 1, wherein the first and second oligonucleotide strands are separate strands that anneal under biological conditions.

32. The precursor RNAi molecule of claim 1, wherein the first and second oligonucleotide strands are linked by a linker wherein the strands anneal under biological conditions.

33. The precursor RNAi molecule of claim 32, wherein the linker is a chemical linker.

34. The precursor RNAi molecule of claim 32, wherein the linker is an oligonucleotide.

35. The precursor RNAi molecule of claim 1, wherein a ribonucleotide of the first or second strand is modified.

36. The precursor RNAi molecule of claim 35, wherein a ribonucleotide is substituted by a locked nucleic acid (LNA).

37. The precursor RNAi molecule of claim 36, wherein the LNA is in the 5′

region of the first strand.

38. The precursor RNAi molecule of claim 1, wherein the first and second oligonucleotide strands are fully complementary.

75. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 1 into the cell in an amount sufficient to reduce expression of the target gene.

76. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 5 into the cell in an amount sufficient to reduce expression of the target gene.

77. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 8 into the cell in an amount sufficient to reduce expression of the target gene.

78. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 11 into the cell in an amount sufficient to reduce expression of the target gene.

79. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 16 into the cell in an amount sufficient to reduce expression of the target gene.

80. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 21 into the cell in an amount sufficient to reduce expression of the target gene.

81. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 26 into the cell in an amount sufficient to reduce expression of the target gene.

88. A method for preparing the precursor RNAi molecule of claim 1 comprising selecting a target sequence of an RNA of a target gene, wherein the target sequence comprises at least 19 nucleotides;

synthesizing a second oligonucleotide strand comprising 22-30 nucleotides having a nucleotide sequence that is sufficiently complementary to the selected target seqeucne to direct target-specific RNAi; and

synthesizing a first oligonucleotide strand comprising 22-30 nucleotides that is capable of annealing to the second oligonucleotide strand under biological conditions,

wherein one of said first and second oligonucleotide strands is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.

89. The method of claim 88, wherein the first and second oligonucleotide strands have different lengths.

90. The precursor RNAi molecule of claim 88, wherein the second oligonucleotide strand is longer than the first nucleotide strand and has a 3′

overhang when the first and second oligonucleotide strands are annealed.

91. The method of claim 88, wherein the first oligonucleotide strand is longer than the second ucleotide strand and has a 3′

overhang when the first and second oligonucleotide strands are annealed.

92. The method of claim 88, wherein the first oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

93. The method of claim 92, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first strand.

94. The method of claim 88, wherein the second oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

95. The method of claim 94, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the second strand.

96. The method of claim 88, wherein a ribo nucleotide of the first or second strand is modified.

97. The precursor RNAi molecule of claim 96, wherein the modified nucleotide is a locked nucleic acid (LNA).

98. A method for preparing the precursor RNAi molecule of claim 1 comprising selecting a target sequence of an RNA of a target gene, wherein the target sequence comprises at least 19 nucleotides;

synthesizing a second oligonucleotide strand comprising 22-30 nucleotides having a nucleotide sequence that is sufficiently complementary to the selected target sequence to direct target-specific RNAi; and

synthesizing a first oligonucleotide strand comprising 22-30 nucleotides that is capable of annealing to the second oligonucleotide strand under biological conditions,

wherein one of said first and second oligonucleotide strands is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule and wherein one of said first and second oligonucleotide strands has a 3′

overhang that enhances Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.

99. The method of claim 98, wherein the first oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

100. The method of claim 99, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first strand.

101. The method of claim 98, wherein the second oligonucleotide strand is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule.

102. The method of claim 101, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the second strand.

103. The method of claim 98, wherein a ribo nucleotide of the first or second strand is modified.

108. A method for reducing expression of a target gene in a cell comprising identifying a target gene for attenuation;

synthesizing a precursor RNAi molecule of claim 1 for said target gene; and

introducing the precursor RNAi molecule into the cell in an amount sufficient to reduce expression of the target gene.

39. A precursor RNAi molecule capable of reducing the expression of a target gene following cleavage by Dicer comprising:

a first oligonucleotide strand that is 22-30 nucleotides in length; and

a second oligonucleotide strand that is 22-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of the target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions,

wherein the first and second oligonucleotide strands have a different length and wherein one of said first and second oligonucleotide strands has a 3′

overhang that enhances Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.

40. The precursor RNAi molecule of claim 39, wherein the nucleotide sequence of the second oligonucleotide strand sufficiently complementary to the RNA of the target gene comprises at least 19.

41. The precursor RNAi molecule of claim 39, wherein the second oligonucleotide strand is longer than the first nucleotide strand and has a 3′

overhang when the first and second oligonucleotide strands are annealed.

42. The precursor RNAi molecule of claim 41, wherein the overhang is 1-3 nucleotides.

43. The precursor RNAi molecule of claim 41, wherein the overhang is 2 nucleotides.

44. The precursor RNAi molecule of claim 41, wherein the first oligonucleotide strand is modified on the 3′

end to direct Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.

45. The precursor RNAi molecule of claim 44, wherein the modification is the substitution of a ribonucleotide with a nucleotide selected from the group consisting of deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymiidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC), 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T), a monophosphate nucleotide of 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and a monophosphate nucleotide of 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

46. The precursor RNAi molecule of claim 44, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

47. The precursor RNAi molecule of claim 44, wherein the modification is 2 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

48. The precursor RNAi molecule of claim 44 wherein the modification is the addition of a sterically hindered molecule to the 3′

end of the first oligonucleotide strand.

49. The precursor RNAi molecule of claim 39, wherein the first oligonucleotide strand is longer than the second nucleotide strand and has a 3′

overhang when the first and second oligonucleotide strands are annealed.

50. The precursor RNAi molecule of claim 49, wherein the overhang is 1-3 nucleotides.

51. The precursor RNAi molecule of claim 49, wherein the overhang is 2 nucleotides.

52. The precursor RNAi molecule of claim 49, wherein the second oligonucleotide strand is modified on the 3′

end to direct Dicer processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.

53. The precursor RNAi molecule of claim 49, wherein the modification is the substitution of a ribonucleotide with a nucleotide selected from the group consisting of deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC), 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T), a monophosphate nucleotide of 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and a monophosphate nucleotide of 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

54. The precursor RNAi molecule of claim 49, wherein the modification is 1-3 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

55. The precursor RNAi molecule of claim 49, wherein the modification is 2 deoxynucleotides substituted for ribonucleotides at the 3′

end of the first oligonucleotide strand.

56. The precursor RNAi molecule of claim 49 wherein the modification is the addition of a sterically hindered molecule to the 3′

end of the first oligonucleotide strand.

57. The precursor RNAi molecule of claim 39, wherein the first and second oligonucleotide strands are linked by a linker wherein the strands anneal under biological conditions.

58. The precursor RNAi molecule of claim 57, wherein the linker is a chemical linker.

59. The precursor RNAi molecule of claim 57, wherein the linker is a n oligonucleotide.

60. The precursor RNAi molecule of claim 39, wherein a ribonucleotide of the first or second strand is modified.

61. The precursor RNAi molecule of claim 39, wherein the first and second oligonucleotide strands are fully complementary.

82. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 39 into the cell in an amount sufficient to reduce expression of the target gene.

83. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 41 into the cell in an amount sufficient to reduce expression of the target gene.

84. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 44 into the cell in an amount sufficient to reduce expression of the target gene.

85. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 49 into the cell in an amount sufficient to reduce expression of the target gene.

86. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 55 into the cell in an amount sufficient to reduce expression of the target gene.

109. A method for reducing expression of a target gene in a cell comprising identifying a target gene for attenuation;

synthesizing a precursor RNAi molecule of claim 39 for said target gene; and

introducing the precursor RNAi molecule into the cell in an amount sufficient to reduce expression of the target gene.

62. A precursor RNAi molecule capable of reducing the expression of a target gene following cleavage by Dicer comprising:

a first oligonucleotide strand that is 25-30 nucleotides in length; and

a second oligonucleotide strand that is 25-30 nucleotides in length and comprises a nucleotide sequence that is sufficiently complementary to a sequence of an RNA of the target gene to direct target-specific RNAi and wherein the second oligonucleotide strand anneals to the first oligonucleotide strand under biological conditions,

wherein said precursor RNAi is a substrate for Dicer.

63. The precursor RNAi molecule of claim 62, wherein the second oligonucleotide is 1-3 nucleotides longer than the first strand.

64. The precursor RNAi molecule of claim 62, wherein the second oligonucleotide is 1-3 nucleotides longer than the first strand.

65. The precursor RNAi molecule of claim 62, wherein the first and second oligonucleotide strands have the same length.

66. The precursor RNAi molecule of claim 65, wherein the both ends of the molecule are blunt ends.

67. The precursor RNAi molecule of claim 65, wherein one end of the molecule is a blunt end.

68. The precursor RNAi molecule of claim 65, wherein the length is 27 nucleotides.

69. The precursor RNAi molecule of claim 62, wherein the nucleotide sequence of the second oligonucleotide strand sufficiently complementary to the RNA of the target gene comprises at least 19.

70. The precursor RNAi molecule of claim 62, wherein the first and second oligonucleotide strands are fully complementary.

71. The precursor RNAi molecule of claim 62, wherein a nucleotide of the first or second strand is modified.

72. The precursor RNAi molecule of claim 62, wherein the first oligonucleotide strand has a 3′

overhang when annealed to the second oligonucleotide strand.

73. The precursor RNAi molecule of claim 62, wherein the second oligonucleotide strand has a 3′

overhang when annealed to the first oligonucleotide strand.

74. The precursor RNAi molecule of claim 62, wherein the first oligonucleotide strand has a 3′

overhang when annealed to the second oligonucleotide strand and the second oligonucleotide strand has a 3′

overhang when annealed to the first oligonucleotide strand.

87. A method for reducing expression of a target gene in a cell comprising:

introducing the precursor RNAi molecule of claim 62 into the cell in an amount sufficient to reduce expression of the target gene.

104. A method for preparing the precursor RNAi molecule of claim 62 comprising selecting a target sequence of an RNA of a target gene, wherein the target sequence comprises at least 19 nucleotides;

synthesizing a second oligonucleotide strand comprising 25-30 nucleotides having a nucleotide sequence that is sufficiently complementary to the selected target seqeucne to direct target-specific RNAi; and

synthesizing a first oligonucleotide strand comprising 25-30 nucleotides that is capable of annealing to the second oligonucleotide strand under biological conditions,

wherein one of said first and second oligonucleotide strands is modified on the 3′

end to direct Dicer binding and processing of the precursor RNAi molecule and wherein said precursor RNAi is a substrate for Dicer.

105. The method of claim 104, wherein the first and second oligonucleotide strands have the same length.

106. The method of claim 105, wherein the length is 27 nucleotides.

107. The method of claim 104, wherein a nucleotide of the first or second strand is modified.

110. A method for reducing expression of a target gene in a cell comprising identifying a target gene for attenuation;

synthesizing a precursor RNAi molecule of claim 62 for said target gene; and

introducing the precursor RNAi molecule into the cell in an amount sufficient to reduce expression of the target gene.