Modified siRNA molecules and uses thereof

• US 20070135372A1
• Filed: 11/02/2006
• Published: 06/14/2007
• Est. Priority Date: 11/02/2005
• Status: Active Grant

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Judge, Adam, MacLachlan, Ian
• Granted Patent: US 8,101,741 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• 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 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• 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 2310/351 Conjugate
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

The present invention provides chemically modified siRNA molecules and methods of using such siRNA molecules to silence target gene expression. Advantageously, the modified siRNA of the present invention is less immunostimulatory than its corresponding unmodified siRNA sequence and retains RNAi activity against the target sequence. The present invention also provides nucleic acid-lipid particles comprising a modified siRNA, a cationic lipid, and a non-cationic lipid, which can further comprise a conjugated lipid that inhibits aggregation of particles. The present invention further provides methods of silencing gene expression by administering a modified siRNA to a mammalian subject. Methods for identifying and/or modifying an siRNA having immunostimulatory properties are also provided.

Claims

1. A modified siRNA comprising a double-stranded region of about 15 to about 60 nucleotides in length, wherein less than about 20% of the nucleotides in the double-stranded region comprise modified nucleotides,

wherein the modified siRNA is less immunostimulatory than a corresponding unmodified siRNA sequence, and

wherein the modified siRNA is capable of silencing expression of a target sequence.

2. The modified siRNA of claim 1, wherein the modified siRNA comprises modified nucleotides selected from the group consisting of 2′

-O-methyl (2′

OMe) nucleotides, 2′

-deoxy-2′

-fluoro (2′

F) nucleotides, 2′

-deoxy nucleotides, 2′

-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof.

3. The modified siRNA of claim 1, wherein the modified siRNA comprises 2′

OMe nucleotides.

4. The modified siRNA of claim 1, wherein the modified siRNA comprises 2′

OMe nucleotides selected from the group consisting of 2′

OMe-guanosine nucleotides, 2′

OMe-uridine nucleotides, 2′

OMe-adenosine nucleotides, and mixtures thereof.

5. The modified siRNA of claim 1, wherein the modified siRNA does not comprise 2′

OMe-cytosine nucleotides.

6. The modified siRNA of claim 1, wherein the modified siRNA comprises a double-stranded region of about 15 to about 30 nucleotides in length.

7. The modified siRNA of claim 1, wherein the modified siRNA comprises modified nucleotides in one strand of the modified siRNA.

8. The modified siRNA of claim 1, wherein the modified siRNA comprises modified nucleotides in the sense or antisense strand of the modified siRNA.

9. The modified siRNA of claim 7 or 8, wherein the modified siRNA comprises 2′

OMe-guanosine nucleotides, 2′

OMe-uridine nucleotides, or mixtures thereof.

10. The modified siRNA of claim 1, wherein the modified siRNA comprises modified nucleotides in both strands of the modified siRNA.

11. The modified siRNA of claim 10, wherein the modified siRNA comprises 2′

OMe-guanosine nucleotides, 2′

OMe-uridine nucleotides, or mixtures thereof.

12. The modified siRNA of claim 1, wherein less than about 15% of the nucleotides in the double-stranded region comprise modified nucleotides.

13. The modified siRNA of claim 1, wherein less than about 10% of the nucleotides in the double-stranded region comprise modified nucleotides.

14. The modified siRNA of claim 1, wherein less than about 5% of the nucleotides in the double-stranded region comprise modified nucleotides.

15. The modified siRNA of claim 1, wherein from about 1% to about 20% of the nucleotides in the double-stranded region comprise modified nucleotides.

16. The modified siRNA of claim 1, wherein from about 5% to about 20% of the nucleotides in the double-stranded region comprise modified nucleotides.

17. The modified siRNA of claim 1, wherein from about 10% to about 20% of the nucleotides in the double-stranded region comprise modified nucleotides.

18. The modified siRNA of claim 1, wherein the modified siRNA is at least about 80% less immunostimulatory than the corresponding unmodified siRNA sequence.

19. The modified siRNA of claim 1, wherein the modified siRNA has an IC₅₀ that is less than or equal to ten-fold that of the corresponding unmodified siRNA sequence.

20. The modified siRNA of claim 1, wherein the modified siRNA has an IC₅₀ that is less than or equal to three-fold that of the corresponding unmodified siRNA sequence.

21. The modified siRNA of claim 1, wherein the modified siRNA has an IC₅₀ that is less than or equal to two-fold that of the corresponding unmodified siRNA sequence.

22. The modified siRNA of claim 1, wherein the modified siRNA does not comprise phosphate backbone modifications.

23. The modified siRNA of claim 1, wherein the modified siRNA does not comprise 2′

-deoxy nucleotides in the double-stranded region.

24. The modified siRNA of claim 1, wherein the modified siRNA comprises 3′

overhangs in one strand of the modified siRNA.

25. The modified siRNA of claim 1, wherein the modified siRNA comprises 3′

overhangs in both strands of the modified siRNA.

26. The modified siRNA of claim 1, wherein the modified siRNA does not comprise 3′

overhangs.

27. The modified siRNA of claim 1, wherein the corresponding unmodified siRNA sequence comprises at least one 5′

-GU-3′

motif.

28. The modified siRNA of claim 1, wherein the corresponding unmodified siRNA sequence comprises at least one 5′

-GU-3′

motif in the sense strand.

29. The modified siRNA of claim 1, wherein the corresponding unmodified siRNA sequence comprises at least one 5′

-GU-3′

motif in the antisense strand.

30. The modified siRNA of claim 1, wherein the modified siRNA comprises a hairpin loop structure.

31. The modified siRNA of claim 1, further comprising a carrier system.

32. The modified siRNA of claim 31, wherein the carrier system is selected from the group consisting of a nucleic acid-lipid particle, a liposome, a micelle, a virosome, a nucleic acid complex, and mixtures thereof.

33. The modified siRNA of claim 32, wherein the carrier system is a nucleic acid-lipid particle.

34. The modified siRNA of claim 32, wherein the nucleic acid complex comprises the modified siRNA complexed with a cationic lipid, a cationic polymer, a cyclodextrin, or mixtures thereof.

35. The modified siRNA of claim 34, wherein the modified siRNA is complexed with a cationic polymer, wherein the cationic polymer is polyethylenimine (PEI).

36. A pharmaceutical composition comprising a modified siRNA of claim 1 and a pharmaceutically acceptable carrier.

59. A nucleic acid-lipid particle comprising:

a modified siRNA of claim 1 or 37;

a cationic lipid; and

a non-cationic lipid.

60. The nucleic acid-lipid particle of claim 59, wherein the cationic lipid is a member selected from the group consisting of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLendMA), and a mixture thereof.

61. The nucleic acid-lipid particle of claim 59, wherein the cationic lipid is DLinDMA.

62. The nucleic acid-lipid particle of claim 59, wherein the non-cationic lipid is an anionic lipid.

63. The nucleic acid-lipid particle of claim 59, wherein the non-cationic lipid is a neutral lipid.

64. The nucleic acid-lipid particle of claim 59, wherein the non-cationic lipid is a member selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylcholine (DPPC), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), cholesterol, and a mixture thereof.

65. The nucleic acid-lipid particle of claim 59, wherein the non-cationic lipid is DSPC, DPPC, or DSPE.

66. The nucleic acid-lipid particle of claim 59, further comprising a conjugated lipid that inhibits aggregation of particles.

67. The nucleic acid-lipid particle of claim 66, wherein the conjugated lipid that inhibits aggregation of particles is a member selected from the group consisting of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and a mixture thereof.

68. The nucleic acid-lipid particle of claim 67, wherein the PEG-lipid is a member selected from the group consisting of a PEG-diacylglycerol, a PEG dialkyloxypropyl, a PEG-phospholipid, a PEG-ceramide, and a mixture thereof.

69. The nucleic acid-lipid particle of claim 67, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-dialkyloxypropyl (PEG-DAA) conjugate.

70. The nucleic acid-lipid particle of claim 69, wherein the PEG-DAA conjugate is a member selected from the group consisting of a PEG-dilauryloxypropyl (C₁₂), a PEG-dimyristyloxypropyl (C₁₄), a PEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈).

71. The nucleic acid-lipid particle of claim 69, wherein the PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C₁₄).

72. The nucleic acid-lipid particle of claim 59, wherein the cationic lipid comprises from about 20 mol % to about 50 mol % of the total lipid present in the particle.

73. The nucleic acid-lipid particle of claim 59, wherein the cationic lipid comprises about 40 mol % of the total lipid present in the particle.

74. The nucleic acid-lipid particle of claim 59, wherein the non-cationic lipid comprises from about 5 mol % to about 90 mol % of the total lipid present in the particle.

75. The nucleic acid-lipid particle of claim 59, wherein the non-cationic lipid comprises about 20 mol % of the total lipid present in the particle.

76. The nucleic acid-lipid particle of claim 69, wherein the PEG-DAA conjugate comprises from 0 mol % to about 20 mol % of the total lipid present in the particle.

77. The nucleic acid-lipid particle of claim 69, wherein the PEG-DAA conjugate comprises about 2 mol % of the total lipid present in the particle.

78. The nucleic acid-lipid particle of claim 59, further comprising cholesterol.

79. The nucleic acid-lipid particle of claim 78, wherein the cholesterol comprises from about 10 mol % to about 60 mol % of the total lipid present in the particle.

80. The nucleic acid-lipid particle of claim 78, wherein the cholesterol comprises about 48 mol % of the total lipid present in the particle.

81. The nucleic acid-lipid particle of claim 59, wherein the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after exposure of the particle to a nuclease at 37°

C. for 20 minutes.

82. The nucleic acid-lipid particle of claim 59, wherein the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after incubation of the particle in serum at 37°

C. for 30 minutes.

83. The nucleic acid-lipid particle of claim 59, wherein the nucleic acid is fully encapsulated in the nucleic acid-lipid particle.

84. The nucleic acid-lipid particle of claim 59, wherein the particle has a nucleic acid:

lipid mass ratio of from about 0.01 to about 0.2.

85. The nucleic acid-lipid particle of claim 59, wherein the particle has a nucleic acid:

lipid mass ratio of from about 0.02 to about 0.1.

86. The nucleic acid-lipid particle of claim 59, wherein the particle has a nucleic acid:

lipid mass ratio of about 0.08.

87. The nucleic acid-lipid particle of claim 59, wherein the particle has a median diameter of from about 50 nm to about 150 nm.

88. The nucleic acid-lipid particle of claim 59, wherein the particle has a median diameter of from about 70 nm to about 90 nm.

89. A pharmaceutical composition comprising a nucleic acid-lipid particle of claim 59 and a pharmaceutically acceptable carrier.

90. A method for introducing an siRNA that silences expression of a target sequence into a cell, the method comprising:

contacting the cell with a modified siRNA of claim 1 or 37.

91. The method of claim 90, wherein the modified siRNA is in a carrier system.

92. The method of claim 91, wherein the carrier system is selected from the group consisting of a nucleic acid-lipid particle, a liposome, a micelle, a virosome, a nucleic acid complex, and a mixture thereof.

93. The method of claim 92, wherein the nucleic acid complex comprises the modified siRNA complexed with a cationic lipid, a cationic polymer, a cyclodextrin, or a mixture thereof.

94. The method of claim 93, wherein the modified siRNA is complexed with a cationic polymer, wherein the cationic polymer is polyethylenimine (PEI).

95. The method of claim 91, wherein the carrier system is a nucleic acid-lipid particle comprising:

the modified siRNA;

a cationic lipid; and

a non-cationic lipid.

96. The method of claim 95, wherein the nucleic acid-lipid particle further comprises a conjugated lipid that prevents aggregation of particles.

97. The method of claim 95, wherein the presence of the nucleic acid-lipid particle is detectable at least 1 hour after administration of said particle.

98. The method of claim 95, wherein more than 10% of a plurality of the particles are present in the plasma of a mammal about 1 hour after administration.

99. The method of claim 95, wherein an effect of the modified siRNA at a site distal to the site of administration is detectable for at least 72 hours after administration of the nucleic acid-lipid particle.

100. The method of claim 90, wherein the cell is in a mammal.

101. The method of claim 100, wherein the mammal is a human.

102. A method for in vivo delivery of an siRNA that silences expression of a target sequence, the method comprising:

administering to a mammalian subject a modified siRNA of claim 1 or 37.

103. The method of claim 102, wherein the modified siRNA is in a carrier system.

104. The method of claim 103, wherein the carrier system is selected from the group consisting of a nucleic acid-lipid particle, a liposome, a micelle, a virosome, a nucleic acid complex, and a mixture thereof.

105. The method of claim 104, wherein the nucleic acid complex comprises the modified siRNA complexed with a cationic lipid, a cationic polymer, a cyclodextrin, or a mixture thereof.

106. The method of claim 105, wherein the modified siRNA is complexed with a cationic polymer, wherein the cationic polymer is polyethylenimine (PEI).

107. The method of claim 103, wherein the carrier system is a nucleic acid-lipid particle comprising:

the modified siRNA;

a cationic lipid; and

a non-cationic lipid.

108. The method of claim 107, wherein the nucleic acid-lipid particle further comprises a conjugated lipid that prevents aggregation of particles.

109. The method of claim 102, wherein the administration is selected from the group consisting of oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal.

110. The method of claim 102, wherein the mammalian subject is a human.

37. A modified siRNA comprising a double-stranded region of about 15 to about 60 nucleotides in length, wherein at least two of the nucleotides in the double-stranded region comprise modified nucleotides,

wherein the modified nucleotides are selected from the group consisting of modified guanosine nucleotides, modified uridine nucleotides, and mixtures thereof,

wherein the modified siRNA is less immunostimulatory than a corresponding unmodified siRNA sequence, and

wherein the modified siRNA is capable of silencing expression of a target sequence.

38. The modified siRNA of claim 37, wherein the modified siRNA comprises modified nucleotides selected from the group consisting of 2′

OMe-guanosine nucleotides, 2′

OMe-uridine nucleotides, and mixtures thereof.

39. The modified siRNA of claim 37, wherein the modified siRNA comprises a double-stranded region of about 15 to about 30 nucleotides in length.

40. The modified siRNA of claim 37, wherein the modified siRNA comprises modified nucleotides in one strand of the modified siRNA.

41. The modified siRNA of claim 37, wherein the modified siRNA comprises modified nucleotides in the sense or antisense strand of the modified siRNA.

42. The modified siRNA of claim 37, wherein the modified siRNA comprises modified nucleotides in both strands of the modified siRNA.

43. The modified siRNA of claim 37, wherein less than about 30% of the nucleotides in the double-stranded region comprise modified nucleotides.

44. The modified siRNA of claim 37, wherein less than about 25% of the nucleotides in the double-stranded region comprise modified nucleotides.

45. The modified siRNA of claim 37, wherein less than about 20% of the nucleotides in the double-stranded region comprise modified nucleotides.

46. The modified siRNA of claim 37, wherein the modified siRNA is at least about 80% less immunostimulatory than the corresponding unmodified siRNA sequence.

47. The modified siRNA of claim 37, wherein the modified siRNA wherein the modified siRNA has an IC₅₀ that is less than or equal to ten-fold that of the corresponding unmodified siRNA sequence.

48. The modified siRNA of claim 37, wherein the modified siRNA wherein the modified siRNA has an IC₅₀ that is less than or equal to three-fold that of the corresponding unmodified siRNA sequence.

49. The modified siRNA of claim 37, wherein the modified siRNA wherein the modified siRNA has an IC₅₀ that is less than or equal to two-fold that of the corresponding unmodified siRNA sequence.

50. The modified siRNA of claim 37, wherein the modified siRNA does not comprise phosphate backbone modifications.

51. The modified siRNA of claim 37, wherein the modified siRNA does not comprise 2′

-deoxy nucleotides in the double-stranded region.

52. The modified siRNA of claim 37, wherein the modified siRNA comprises 3′

overhangs in one strand of the modified siRNA.

53. The modified siRNA of claim 37, wherein the modified siRNA comprises 3′

overhangs in both strands of the modified siRNA.

54. The modified siRNA of claim 37, wherein the modified siRNA does not comprise 3′

overhangs.

55. The modified siRNA of claim 37, wherein the corresponding unmodified siRNA sequence comprises at least one 5′

-GU-3′

motif.

56. The modified siRNA of claim 37, wherein the modified siRNA comprises a hairpin loop structure.

57. The modified siRNA of claim 37, further comprising a carrier system.

58. A pharmaceutical composition comprising a modified siRNA of claim 37 and a pharmaceutically acceptable carrier.

111. A method for modifying an siRNA having immunostimulatory properties, the method comprising:

(a) providing an unmodified siRNA sequence capable of silencing expression of a target sequence and comprising a double-stranded sequence of about 15 to about 60 nucleotides in length; and

(b) modifying the siRNA by substituting at least one nucleotide in the sense or antisense strand with a modified nucleotide, thereby generating a modified siRNA that is less immunostimulatory than the unmodified siRNA sequence and is capable of silencing expression of the target sequence.

112. The method of claim 111, wherein the modified nucleotide is selected from the group consisting of a 2′

-O-methyl (2′

OMe) nucleotide, 2′

-deoxy-2′

-fluoro (2′

F) nucleotide, 2′

-deoxy nucleotide, 2′

-O-(2-methoxyethyl) (MOE) nucleotide, locked nucleic acid (LNA) nucleotide, and mixtures thereof.

113. The method of claim 111, wherein the modified nucleotide is a 2′

OMe nucleotide.

114. The method of claim 111, wherein the modified nucleotide is selected from the group consisting of a 2′

OMe-guanosine nucleotide, 2′

OMe-uridine nucleotide, 2′

OMe-adenosine nucleotide, and mixtures thereof.

115. The method of claim 111, wherein the modified nucleotide is not a 2′

OMe-cytosine nucleotide.

116. The method of claim 111, wherein the unmodified siRNA sequence comprises a 5′

-GU-3′

motif.

117. The method of claim 116, wherein the sense strand of the unmodified siRNA sequence comprises a 5′

-GU-3′

motif.

118. The method of claim 116, wherein the antisense strand of the unmodified siRNA sequence comprises a 5′

-GU-3′

motif.

119. The method of claim 116, wherein at least one nucleotide in the 5′

-GU-3′

motif is substituted with a modified nucleotide.

120. The method of claim 116, wherein both nucleotides in the 5′

-GU-3′

motif are substituted with modified nucleotides.

121. The method of claim 111, further comprising:

(c) confirming that the modified siRNA is less immunostimulatory by contacting the modified siRNA with a mammalian responder cell under conditions suitable for the responder cell to produce a detectable immune response.

122. A method for identifying and modifying an siRNA having immunostimulatory properties, the method comprising:

(a) contacting an unmodified siRNA sequence with a mammalian responder cell under conditions suitable for the responder cell to produce a detectable immune response;

(b) identifying the unmodified siRNA sequence as an immunostimulatory siRNA by the presence of a detectable immune response in the responder cell; and

(c) modifying the immunostimulatory siRNA by substituting at least one nucleotide with a modified nucleotide, thereby generating a modified siRNA sequence that is less immunostimulatory than the unmodified siRNA sequence.

123. The method of claim 122, wherein the modified nucleotide is selected from the group consisting of a 2′

-O-methyl (2′

OMe) nucleotide, 2′

-deoxy-2′

-fluoro (2′

F) nucleotide, 2′

-deoxy nucleotide, 2′

-O-(2-methoxyethyl) (MOE) nucleotide, locked nucleic acid (LNA) nucleotide, and mixtures thereof.

124. The method of claim 122, wherein the modified nucleotide is a 2′

OMe nucleotide.

125. The method of claim 122, wherein the modified nucleotide is selected from the group consisting of a 2′

OMe-guanosine nucleotide, 2′

OMe-uridine nucleotide, 2′

OMe-adenosine nucleotide, and mixtures thereof.

126. The method of claim 122, wherein the modified nucleotide is not a 2′

OMe-cytosine nucleotide.

127. The method of claim 122, wherein the unmodified siRNA sequence comprises a 5′

-GU-3′

motif.

128. The method of claim 122, wherein the mammalian responder cell is a peripheral blood mononuclear cell.

129. The method of claim 122, wherein the detectable immune response comprises production of a cytokine or growth factor selected from the group consisting of TNF-α

, IFN-α

, IFN-β

, IFN-γ

, IL-6, IL-12, and combinations thereof.