High potency siRNAS for reducing the expression of target genes

• US 20030166282A1
• Filed: 01/31/2003
• Published: 09/04/2003
• Est. Priority Date: 02/01/2002
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Ford, Lance, Brown, David, Jarvis, Rich, Pallotta, Vince, Pasloske, Brittan
• Application Number:
  US10/355,820
• Publication Number:
  US 20030166282A1
• Time in Patent Office: Days
• US Class Current: 435/455
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• 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 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/334 Modified C
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/336 Modified G
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity
• CPC Class Codes: C12Y 102/01012 Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)

Abstract

The present invention provides improved methods of attenuating gene expression through the phenomenon of RNA interference. The invention provides methods of synthesis of double stranded RNAs (dsRNAs) of increased potency for use as small interfering RNA (siRNA). Surprisingly and unexpectedly, siRNAs made by the methods of the invention are significantly more potent than previously available siRNAs.

Claims

1. A method for making siRNA of increased potency comprising:

(a) obtaining nucleotides;

(b) incorporating the nucleotides into siRNA such that an RNA duplex of from 15 to 30 contiguous nucleotides is formed, wherein the siRNA has a sequence that is substantially identical to at least a portion of a selected target gene.

2. The method of claim 1, wherein the siRNA is further defined as having reduced duplex stability.

3. The method of claim 1, further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA.

4. The method of claim 3, wherein the modified nucleotide analog is selected from the group consisting of aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine, 5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine, alpha-S uridine, 4-thio uridine, 2-thio-cytidine, 2′

NH₂ uridine, 2′

NH₂ cytidine, and 2′

F uridine.

5. The method of claim 3, wherein the siRNA is further defined as having reduced duplex stability.

6. The method of claim 1, wherein incorporating the nucleotides into siRNA is further defined as comprising enzymatic synthesis.

7. The method of claim 6, further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA.

8. The method of claim 6, wherein the method of enzymatic incorporation comprises:

(a) obtaining a first polynucleotide template comprising a first promoter operatively linked to a first target sequence that has 5′ and

3′

ends and that is substantially identical to at least a portion of the target gene;

(b) obtaining a second polynucleotide template comprising a second promoter operatively linked to a second target sequence that has 5′ and

3′

ends and that is substantially the reverse complement of the first target sequence of the first template;

(c) enzymatically incorporating nucleotides into RNA by contacting the first template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the first template to form a first RNA product;

(e) enzymatically incorporating nucleotides into RNA by contacting the second template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the second template to form a second RNA product; and

(f) annealing the first and second RNA products to form a siRNA product.

9. The method of claim 8, wherein the first template further comprises an overhang encoding sequence joined to the 3′

end of the first target sequence and the second template further comprises an overhang encoding sequence joined to the 3′

end of the second target sequence.

10. The method of claim 9, wherein the first and second overhang sequences each comprise TT.

11. The method of claim 9, wherein the first two nucleotides of the 5′

end of the first target sequence are GG and the last two nucleotides of the 3′

end of the first target sequence are CC.

12. The method of claim 9, wherein the first two nucleotides of the 5′

end of the first target sequence are GA and the last two nucleotides of the 3′

end of the first target sequence are TC.

13. The method of claim 8, wherein the first target sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

14. The method of claim 8, wherein the first promoter is a T7, T3, or SP6 promoter.

15. The method of claim 8, wherein the second promoter is a T7, T3, or SP6 promoter.

16. The method of claim 8, wherein the first promoter and the second promoter are the same promoter.

17. The method of claim 8, wherein the first promoter and the second promoter are different promoters.

18. The method of claim 8, wherein at least one nucleotide is a modified nucleotide analog.

19. The method of claim 8, wherein the first template further comprises a first leader sequence of about 10 nucleotides positioned between the first promoter and the first target sequence.

20. The method of claim 19, wherein the second template further comprises a second leader sequence of about 10 nucleotides positioned between the second promoter and the second target sequence.

21. The method of claim 20, wherein the second leader sequence is substantially non-complementary to any portion of either strand of the siRNA.

22. The method of claim 20, wherein the first and second leader sequences each comprise SEQ ID NO:

1.

23. The method of claim 20, wherein the first and second leader sequences each comprise SEQ ID NO:

2.

24. The method of claim 20, wherein the first leader sequence comprises SEQ ID NO:

1 and the second leader sequence comprises SEQ ID NO;

2.

25. The method of claim 8 further comprising the step of contacting the siRNA product with a nuclease.

26. The method of claim 25, wherein the nuclease is RNase T1, RNase A, RNase Sa, RNase Sa2, or RNase Sa3.

27. The method of of claim 8, wherein at least one step is performed in at least one container.

28. The method of claim 27, wherein the transcription of the first template and the transcription of the second template are performed in the same container.

29. The method of claim 27, wherein the transcription of the first template and the transcription of the second template are performed in different containers.

30. The method of claim 27, wherein the annealing is performed in the same container as at least one of the transcription steps.

31. The method of claim 6, wherein the method of enzymatic incorporation comprises:

(a) obtaining a polynucleotide template comprising a promoter operatively linked to a first target sequence, a loop sequence, and a second target sequence having 5′ and

3′

ends and that is substantially the reverse complement of the first target sequence; and

(b) enzymatically incorporating nucleotides into RNA by contacting the template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the template to form an siRNA product

32. The method of claim 31, wherein the promoter is a T7, T3, or SP6 promoter.

33. The method of claim 31, further comprising the step of annealing the siRNA product to form a stem and loop siRNA product.

34. The method of claim 31, wherein the template further comprises an overhang encoding sequence attached to the 3′

end of the second target sequence.

35. The method of claim 34, wherein the overhang encoding sequence comprises TT.

36. The method of claim 31, wherein the template further comprises a leader sequence of about 10 nucleotides positioned between the promoter and the first target sequence.

37. The method of claim 36, wherein the leader sequence comprises SEQ ID NO:

1.

38. The method of claim 36, wherein the leader sequence comprises SEQ ID NO:

2.

39. The method of claim 34, wherein the loop sequence is selected such that the loop is resistant to nuclease digestion.

40. The method of claim 39, wherein the loop sequence is AAGC.

41. The method of claim 39, further comprising the step of digesting the stem and loop siRNA product with a nuclease.

42. The method of claim 41, wherein the nuclease is RNase T1, RNase A, RNase Sa, RNase Sa2, or RNase Sa3.

43. The method of claim 1, further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA with a method comprising chemical synthesis.

44. The method of claim 43, wherein the modified nucleotide analog is selected from the group consisting of aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine, 5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine, alpha-S uridine, 4-thio uridine, 2-thio-cytidine, 2′

NH₂ uridine, 2′

NH₂ cytidine, and 2′

F uridine.

45. The method of claim 43, wherein the siRNA is further defined as having reduced duplex stability.

46. A method for attenuating the expression of a target gene in a cell comprising:

(a) obtaining a siRNA of increased potency;

(b) introducing the siRNA of increased potency into the cell in an amount sufficient to attenuate expression of the target gene.

47. The method of claim 46, wherein the cell is comprised within a tissue.

48. The method of claim 46, wherein the cell is comprised within an organism.

49. The method of claim 48, wherein the organism is a plant, animal, protozoan, virus, bacterium, or fungus.

50. The method of claim 49, wherein the organism is an animal.

51. The method of claim 50, wherein the animal is a vertebrate.

52. The method of claim 51, wherein the vertebrate is a fish.

53. The method of claim 51, wherein the animal is a mammal.

54. The method of claim 52, wherein the mammal is a mouse, a rat, or a primate.

55. The method of claim 54, wherein the primate is a human.

56. The method of claim 46, wherein obtaining siRNA of increased potency comprises practicing a method of making siRNA of increased potency comprising:

(a) obtaining nucleotides;

(b) incorporating the nucleotides into siRNA such that an RNA duplex of from 15 to 30 contiguous nucleotides is formed, wherein the siRNA has a sequence that is substantially identical to at least a portion of a selected target gene.

57. The method of claim 56, wherein the siRNA is further defined as having reduced duplex stability.

58. The method of claim 56, further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA.

59. The method of claim 58, wherein the modified nucleotide analog is selected from the group consisting of aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine, 5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine, alpha-S uridine, 4-thio uridine, 2-thio-cytidine, 2′

NH₂ uridine, 2′

NH₂ cytidine, and 2′

F uridine.

60. The method of claim 56, wherein incorporating the nucleotides into siRNA is further defined as comprising enzymatic synthesis.

61. The method of claim 60, further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA.

62. The method of claim 60, wherein the method of enzymatic incorporation comprises:

(a) obtaining a first polynucleotide template comprising a first promoter operatively linked to a first target sequence that has 5′ and

3′

ends and that is substantially identical to at least a portion of the target gene;

(b) obtaining a second polynucleotide template comprising a second promoter operatively linked to a second target sequence that has 5′ and

3′

ends and that is substantially the reverse complement of the first target sequence of the first template;

(c) enzymatically incorporating nucleotides into RNA by contacting the first template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the first template to form a first RNA product;

(e) enzymatically incorporating nucleotides into RNA by contacting the second template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the second template to form a second RNA product; and

(f) annealing the first and second RNA products to form a siRNA product.

63. The method of claim 62, wherein the first template further comprises an overhang encoding sequence joined to the 3′

end of the first target sequence and the second template further comprises an overhang encoding sequence joined to the 3′

end of the second target sequence.

64. The method of claim 63, wherein the first and second overhang encoding sequences each comprise TT.

65. The method of claim 63, wherein the first two nucleotides of the 5′

end of the first target sequence are GG and the last two nucleotides of the 3′

end of the first target sequence are CC.

66. The method of claim 63, wherein the first two nucleotides of the 5′

end of the first target sequence are GA and the last two nucleotides of the 3′

end of the first target sequence are TC.

67. The method of claim 62, wherein the first target sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

68. The method of claim 62, wherein the first promoter is a T7, T3, or SP6 promoter.

69. The method of claim 62, wherein the second promoter is a T7, T3, or SP6 promoter.

70. The method of claim 62, wherein the first promoter and the second promoter are the same promoter.

71. The method of claim 62, wherein the first promoter and the second promoter are different promoters.

72. The method of claim 62, wherein at least one nucleotide is a modified nucleotide analog.

73. The method of claim 62, wherein the first template further comprises a first leader sequence of about 10 nucleotides positioned between the first promoter and the first target sequence.

74. The method of claim 73, wherein the second template further comprises a second leader sequence of about 10 nucleotides positioned between the second promoter and the second target sequence.

75. The method of claim 74, wherein the second leader sequence is substantially non-complementary to the first leader sequence.

76. The method of claim 74, wherein the first and second leader sequences each comprise SEQ ID NO:

1.

77. The method of claim 74, wherein the first and second leader sequences each comprise SEQ ID NO:

2.

78. The method of claim 74, wherein the first leader sequence comprises SEQ ID NO:

1 and the second leader sequence comprises SEQ ID NO;

2.

79. The method of claim 62 further comprising the step of contacting the siRNA product with a nuclease.

80. The method of claim 79, wherein the nuclease is RNase T1, RNase A, RNase Sa, RNase Sa2, or RNase Sa3.

81. The method of claim 62, wherein at least one step is performed in at least one container.

82. The method of claim 81, wherein the transcription of the first template and the transcription of the second template are performed in the same container.

83. The method of claim 81, wherein the transcription of the first template and the transcription of the second template are performed in different containers.

84. The method of claim 81, wherein the annealing is performed in the same container as at least one of the transcription steps.

85. The method of claim 60, wherein the method of enzymatic incorporation comprises:

(a) obtaining a polynucleotide template comprising a promoter operatively linked to a first target sequence, a loop sequence, and a second target sequence having 5′ and

3′

ends and that is substantially the reverse complement of the first target sequence; and

(b) enzymatically incorporating nucleotides into RNA by contacting the template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the template to form an siRNA product

86. The method of claim 85, wherein the promoter is a T7, T3, or SP6 promoter.

87. The method of claim 85, further comprising the step of annealing the siRNA product to form a stem and loop siRNA product.

88. The method of claim 85, wherein the template further comprises an overhang encoding sequence attached to the 3′

end of the second target sequence.

89. The method of claim 88, wherein the overhang encoding sequence comprises TT.

90. The method of claim 85, wherein the template further comprises a leader sequence of about 10 nucleotides positioned between the promoter and the first target sequence.

91. The method of claim 90, wherein the leader sequence comprises SEQ ID NO:

1.

92. The method of claim 90, wherein the leader sequence comprises SEQ ID NO:

2.

93. The method of claim 88, wherein the loop sequence is selected such that the loop is resistant to nuclease digestion.

94. The method of claim 93, wherein the loop sequence is AAGC.

95. The method of claim 93, further comprising the step of digesting the stem and loop siRNA product with a nuclease.

96. The method of claim 95, wherein the nuclease is RNase T1, RNase A, RNase Sa, RNase Sa2, or RNase Sa3.

97. The method of claim 46, further defined as comprising obtaining at least one modified nucleotide analog and incorporating the at least one modified nucleotide analog into the siRNA and incorporating the nucleotides into siRNA with a method comprising chemical synthesis.

98. The method of claim 97, wherein the siRNA is further defined as having reduced duplex stability.

99. The method of claim 97, wherein the modified nucleotide analog is selected from the group consisting of aminoallyl UTP, pseudo-UTP, 5-I-UTP, 5-I-CTP, 5-Br-UTP, alpha-S ATP, alpha-S CTP, alpha-S GTP, alpha-S UTP, 4-thio UTP, 2-thio-CTP, 2′

NH₂ UTP, 2′

NH₂ CTP, and 2′

F UTP.

100. A siRNA of increased potency comprising a duplex structure of from 15 to 30 nucleotides that has a nucleotide sequence substantially identical to at least a portion of a target gene.

101. The siRNA of claim 100, wherein the duplex structure is of reduced stability.

102. The siRNA of claim 100, further defined as being produced by a method comprising enzymatic synthesis.

103. The siRNA of claim 100, further defined as comprising at least one nucleotide that is a modified nucleotide analog.

104. The siRNA of claim 103, wherein the modified nucleotide analog is selected from the group consisting of aminoallyl UTP, pseudo-UTP, 5-I-UTP, 5-I-CTP, 5-Br-UTP, alpha-S ATP, alpha-S CTP, alpha-S GTP, alpha-S UTP, 4-thio UTP, 2-thio-CTP, 2′

NH₂ UTP, 2′

NH₂ CTP, and 2′

F UTP.

105. The siRNA of claim 104, further defined as being produced by a method comprising chemical synthesis.

106. The siRNA of claim 104, further defined as being produced by a method comprising enzymatic synthesis.

107. The siRNA of claim 100, wherein the duplex structure is 15 nucleotides in length.

108. The siRNA of claim 100, wherein the duplex structure is 16 nucleotides in length.

109. The siRNA of claim 100, wherein the duplex structure is 17 nucleotides in length.

110. The siRNA of claim 100, wherein the duplex structure is 18 nucleotides in length.

111. The siRNA of claim 100, wherein the duplex structure is 19 nucleotides in length.

112. The siRNA of claim 100, wherein the duplex structure is 20 nucleotides in length.

113. The siRNA of claim 100, wherein the duplex structure is 21 nucleotides in length.

114. The siRNA of claim 100, wherein the duplex structure is 22 nucleotides in length.

115. The siRNA of claim 100, wherein the duplex structure is 23 nucleotides in length.

116. The siRNA of claim 100, wherein the duplex structure is 24 nucleotides in length.

117. The siRNA of claim 100, wherein the duplex structure is 25 nucleotides in length.

118. The siRNA of claim 100, wherein the duplex structure is 26 nucleotides in length.

119. The siRNA of claim 100, wherein the duplex structure is 27 nucleotides in length.

120. The siRNA of claim 100, wherein the duplex structure is 28 nucleotides in length.

121. The siRNA of claim 100, wherein the duplex structure is 29 nucleotides in length.

122. The siRNA of claim 100, wherein the duplex structure is 30 nucleotides in length.

123. The siRNA of claim 100, wherein the duplex structure is of reduced stability.

124. A cell comprising a target gene whose expression is attenuated by a method comprising:

(a) obtaining siRNA of increased potency comprising a duplex structure of from 15 to 30 nucleotides that has a nucleotide sequence substantially identical to at least a portion of a target gene; and

(b) introducing the siRNA of increased potency into the cell in an amount sufficient to attenuate expression of the target gene.

125. A kit for making siRNA of increased potency comprising nucleotides.

126. The kit of claim 125, further comprising at least one polynucleotide template.

127. The kit of claim 126, further comprising at least one component for enzymatic synthesis of siRNA of increased potency

128. The kit of claim 127, further comprising at least one component for isolation and purification of siRNA of increased potency.

129. A kit for attenuating expression of a target gene in a cell comprising at least one siRNA of increased potency.