Chen, Jianzhu (Brookline, MA, US)
Eisen, Herman N. (Waban, MA, US)
Ge, Qing (Cambridge, MA, US)

Plaque It! Sponsored by: Flash of Genius

10/674159

12/02/2004

09/29/2003

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Massachusetts Institute of Technology

514/44

536/23.100

(IPC1-7): A61K048/00; C07H021/02

Choate, Hall & Stewart (Exchange Place, Boston, MA, 02109, US)

We claim:

1. A composition comprising: an siRNA or shRNA targeted to a target transcript, wherein the target transcript is an agent-specific transcript, which transcript is involved in infection by or replication of an infectious agent.

2. The composition of claim 1, wherein: the infectious agent is an agent whose genome comprises multiple independent nucleic acid molecules.

3. The composition of claim 2, wherein: the nucleic acid molecules are RNA.

4. The composition of claim 2, wherein: the RNA molecules are single-stranded.

5. The composition of claim 1, wherein: multiple variants of the infectious agent exist and wherein the agent is capable of undergoing genetic reassortment.

6. The composition of claim 1, wherein: multiple variants of the infectious agent exist and wherein the siRNA or shRNA comprises a duplex region whose antisense strand or antisense portion is perfectly complementary to a portion of a target mRNA, which portion is at least 10 nucleotides in length and is highly conserved among a plurality of variants.

7. The composition of claim 6, wherein: multiple variants of the infectious agent exist and wherein the siRNA or shRNA comprises a duplex region whose antisense strand or antisense portion is perfectly complementary to a portion of a target mRNA, which portion is at least 12 nucleotides in length and is highly conserved among a plurality of variants.

8. The composition of claim 6, wherein: multiple variants of the infectious agent exist and wherein the siRNA or shRNA comprises a duplex region whose antisense strand or antisense portion is perfectly complementary to a portion of a target mRNA, which portion is at least 15 nucleotides in length and is highly conserved among a plurality of variants.

9. The composition of claim 6, wherein: multiple variants of the infectious agent exist and wherein the siRNA or shRNA comprises a duplex region whose antisense strand or antisense portion is perfectly complementary to a portion of a target mRNA, which portion is at least 17 nucleotides in length and is highly conserved among a plurality of variants.

10. The composition of claim 6, wherein: multiple variants of the infectious agent exist and wherein the siRNA or shRNA comprises a duplex region whose antisense strand or antisense portion is perfectly complementary to a portion of a target mRNA, which portion is at least 19 nucleotides in length and is highly conserved among a plurality of variants.

11. The composition of claim 8, wherein: a portion is highly conserved among variants if it is identical among the different variants.

12. The composition of claim 8, wherein a portion is highly conserved among variants if it varies by at most one nucleotide between different variants.

13. The composition of claim 8, wherein: a portion is highly conserved among variants if it varies by at most two nucleotides between different variants.

14. The composition of claim wherein: the portion is highly conserved among at least 5 variants.

15. The composition of claim 8, wherein: the portion is highly conserved among at least 10 variants.

16. The composition of claim 8, wherein: the portion is highly conserved among at least 15 variants.

17. The composition of claim 8, wherein: the portion is highly conserved among at least 20 variants.

18. The composition of claim 1, wherein: the infectious agent infects respiratory epithelial cells.

19. The composition of claim 1, wherein: the infectious agent is an influenza virus.

20. The composition of claim 19, wherein: the influenza virus is an influenza A virus.

21. The composition of claim 19, wherein: the influenza virus is an influenza B virus.

22. The composition of claim 1, wherein: the infectious agent inhibits host cell mRNA translation.

23. The composition of claim 1, wherein: the infectious agent infects a host cell and the siRNA or shRNA is present at a level sufficient to inhibit production of the agent by the host cell by at least about 2 fold.

24. The composition of claim 1, wherein: the infectious agent infects a host cell and the siRNA or shRNA is present at a level sufficient to inhibit production of the agent by a host cell by at least about 5 fold.

25. The composition of claim 1, wherein: the infectious agent infects a host cell and the siRNA or shRNA is present at a level sufficient to inhibit production of the agent by a host cell by at least about 10 fold.

26. The composition of claim 1, wherein: the infectious agent infects a host cell and the siRNA or shRNA is present at a level sufficient to inhibit production of the agent by a host cell by at least about 50 fold.

27. The composition of claim 1, wherein: the infectious agent infects a host cell and the siRNA or shRNA is present at a level sufficient to inhibit production of the agent by a host cell by at least about 100 fold.

28. The composition of claim 1, wherein: the infectious agent infects a host cell and the siRNA or shRNA is present at a level sufficient to inhibit production of the agent by a host cell by at least about 200 fold.

29. The composition of claim 1, wherein: the target transcript encodes a subunit of a viral RNA polymerase.

30. The composition of claim 1, wherein: the target transcript encodes a hemagglutinin or a neuraminidase.

31. The composition of claim 1, wherein: the infectious agent is an influenza virus and the target transcript encodes a protein selected from the group consisting of hemagglutinin, neuraminidase, membrane protein 1, membrane protein 2, nonstructural protein 1, nonstructural protein 2, polymerase protein PB1, polymerase protein PB2, polymerase protein PA, polymerase protein NP.

32. The composition of claim 1, wherein: the siRNA or shRNA is present at a level sufficient to inhibit replication of the infectious agent.

33. The composition of claim 1, wherein: the siRNA or shRNA comprises a base-paired region at least 15 nucleotides long.

34. The composition of claim 1, wherein: the siRNA or shRNA comprises a base-paired region approximately 19 nucleotides long.

35. The composition of claim 1, wherein: the siRNA or shRNA comprises a base-paired region at least 15 nucleotides long and at least one single-stranded 3 prime overhang.

36. The composition of claim 1, wherein: the siRNA or shRNA comprises a portion that is perfectly complementary to a region of the target transcript, wherein the portion is at least 15 nucleotides in length.

37. The composition of claim 1, wherein: the siRNA or shRNA comprises a portion that is perfectly complementary to a portion of the target transcript, with the exception of at most one nucleotide, wherein the portion is at least 15 nucleotides in length.

38. The composition of claim 1, wherein: the siRNA or shRNA comprises a portion that is perfectly complementary with a portion of the target transcript with the exception at most two nucleotides, wherein the portion is at least 15 nucleotides in length.

39. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 10 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68.

40. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 12 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68.

41. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 15 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68.

42. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 17 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68.

43. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 19 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68.

44. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 10 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68, with the proviso that either one or two nucleotides among the 10 consecutive nucleotides may differ from that sequence.

45. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 12 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68, with the proviso that either one or two nucleotides among the 12 consecutive nucleotides may differ from that sequence.

46. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 15 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68, with the proviso that either one or two nucleotides among the 15 consecutive nucleotides may differ from that sequence.

47. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 17 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68, with the proviso that either one or two nucleotides among the 17 consecutive nucleotides may differ from that sequence.

48. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 19 consecutive nucleotides as set forth in nucleotides 3 through 21 of the sequence presented in any of SEQ ID NOS: 1 through 68, with the proviso that either one or two nucleotides among the 19 consecutive nucleotides may differ from that sequence.

49. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 10 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 2210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268.

50. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 12 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268.

51. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 15 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268.

52. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 17 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268.

53. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 19 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268.

54. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 10 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268, with the proviso that either one or two nucleotides among the 10 consecutive nucleotides may differ from that sequence.

55. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 12 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268, with the proviso that either one or two nucleotides among the 12 consecutive nucleotides may differ from that sequence.

56. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 15 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268, with the proviso that either one or two nucleotides among the 15 consecutive nucleotides may differ from that sequence.

57. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 17 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268, with the proviso that either one or two nucleotides among the 17 consecutive nucleotides may differ from that sequence.

58. The composition of claim 1, wherein: the siRNA or shRNA comprises a core duplex region, wherein the sequence of the sense strand or portion of the core duplex region comprises at least 19 consecutive nucleotides as set forth in nucleotides 1 through 19 of the sequence presented in any of SEQ ID NOS: 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, or 268, with the proviso that either one or two nucleotides among the 19 consecutive nucleotides may differ from that sequence.

59. The composition of claim 1, wherein the siRNA or shRNA comprises sense and antisense strands or portions whose sequences comprise sequences given by nucleotides 1-19 of SEQ ID NOS: 77 and 78 respectively, with, optionally, a 3′ overhang on one or both sequences.

60. The composition of claim 1, wherein the siRNA or shRNA comprises sense and antisense portions whose sequences comprise sequences given by nucleotides 1-19 of SEQ ID NOS: 71 and 72 respectively, with, optionally, a 3′ overhang on one or both sequences.

61. The composition of claim 1, wherein the siRNA or shRNA comprises sense and antisense portions whose sequences comprise sequences given by nucleotides 1-19 of SEQ ID NOS: 83 and 84 respectively, with, optionally, a 3′ overhang on one or both sequences.

62. The composition of claim 1, wherein the siRNA or shRNA comprises sense and antisense portions whose sequences comprise sequences given by nucleotides 1-19 of SEQ ID NOS: 89 and 90 respectively, with, optionally, a 3′ overhang on one or both sequences.

63. The composition of claim 1, wherein the siRNA or shRNA comprises sense and antisense portions whose sequences comprise sequences given by nucleotides 1-19 of SEQ ID NOS: 91 and 92 respectively, with, optionally, a 3′ overhang on one or both sequences.

64. The composition of claim 1, wherein the siRNA or shRNA comprises sense and antisense portions whose sequences comprise sequences given by nucleotides 1-19 of SEQ ID NOS: 93 and 94 respectively, with, optionally, a 3′ overhang on one or both sequences.

65. The composition of claim 1, wherein the siRNA or shRNA comprises sense and antisense portions whose sequences comprise sequences given by nucleotides 1-20 of SEQ ID NOS: 188 and 189 respectively, with, optionally, a 3′ overhang on one or both sequences.

66. The composition of claim 1, wherein the siRNA or shRNA comprises a duplex portion selected from the group consisting of duplex portions of: NP-1496, NP-1496a, PA-2087, PB1-2257, PB1-129, PB2-2240, M-37, or M-598 or a variant of any of the foregoing, which variant differs by at most one nucleotide from the corresponding siRNA.

67. The composition of claim 66, wherein the siRNA or shRNA duplex portion is identical to the duplex portion of NP-1496.

68. The composition of claim 66, wherein the siRNA or shRNA duplex portion is identical to the duplex portion of NP-1496a.

69. The composition of claim 1, wherein the sense strand or portion of the siRNA or shRNA has a sequence selected from the group consisting of: the first 19 nucleotides of SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 83, SEQ ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 99, and SEQ ID NO: 188, reading in a 5′ to 3′ direction.

70. An analog of the siRNA or shRNA of claim 1, wherein the analog differs from the siRNA or shRNA in that it contains at least one modification.

71. The analog of claim 70, wherein: the modification results in increased stability of the siRNA, enhances absorption of the siRNA, enhances cellular entry of the siRNA, or any combination of the foregoing.

72. The analog of claim 70, wherein: the modification modifies a base, a sugar, or an internucleoside linkage.

73. The analog of claim 70, wherein: the modification is not a nucleotide 2′ modification.

74. The analog of claim 70, wherein: the modification is a nucleotide 2′ modification.

75. An analog of the siRNA or shRNA of claim 1, wherein: the analog differs from the siRNA in that at least one ribonucleotide is replaced by a deoxyribonucleotide.

76. A composition comprising a plurality of single-stranded RNAs which, when hybridized to each other, form the composition of claim 1.

77. The composition of claim 76, wherein: the single-stranded RNAs range in length between approximately 21 and 23 nucleotides, inclusive.

78. A composition comprising a plurality of the siRNAs or shRNAs of claim 1.

79. The composition of claim 78, wherein at least some of the siRNAs or shRNAs are targeted to different influenza virus transcripts.

80. The composition of claim 78, wherein at least some of the siRNAs or shRNAs are targeted to different regions of the same influenza virus transcript.

81. The siRNA or shRNA of claim 1, wherein: presence of the siRNA or shRNA within a cell susceptible to influenza virus infection reduces the susceptibility of the cell to infection by at least two influenza strains.

82. The siRNA or shRNA of claim 1, wherein presence of the siRNA or shRNA within a subject susceptible to infection with influenza virus reduces the susceptibility of the subject to infection by at least two influenza strains.

83. A cell comprising the siRNA or shRNA of claim 1.

84. A vector that provides a template for synthesis of the siRNA or shRNA of claim 1.

85. The vector of claim 84, wherein the vector comprises a nucleic acid operably linked to expression signals active in a host cell so that, when the construct is introduced into the host cell, the siRNA or shRNA of claim 1 is produced inside the host cell

86. A vector comprising a nucleic acid operably linked to expression signals active in a host cell so that, when the construct is introduced into the host cell, an siRNA or shRNA is produced inside the host cell that is targeted to an transcript specific to an infectious agent, which transcript is involved in infection by or replication of the agent.

87. The vector of claim 86, wherein the infectious agent is a virus and wherein multiple variants of the virus exist and wherein the virus is capable of undergoing genetic reassortment or mixing.

88. A cell comprising the vector of claim 87.

89. A transgenic animal comprising the vector of claim 87.

90. The vector of claim 87, wherein the virus is one whose genome comprises multiple independent nucleic acid molecules.

91. The vector of claim 87, wherein the infectious agent is an influenza virus.

92. The vector of claim 91, wherein the vector provides a template for transcription of one or more strands of an siRNA or an shRNA that reduces susceptibility of the cell to infection by influenza virus or inhibits influenza virus production.

93. The vector of claim 91, wherein degradation of the target transcript delays, prevents, or inhibits one or more aspects of influenza virus infection or replication.

94. The vector of claim 92, wherein the siRNA or shRNA duplex portion is selected from the group consisting of duplex portions of: NP-1496, NP-1496a, PA-2087, PB1-2257, PB1-129, PB2-2240, M-37, and M-598, or a variant of any of the foregoing, wherein the variant differs by at most one nucleotide from the corresponding siRNA in either its sense portion, antisense portion, or both.

95. The vector of claim 94, wherein the siRNA or shRNA duplex portion is identical to the duplex portion of NP-1496.

96. The vector of claim 94, wherein the siRNA duplex portion is identical to the duplex portion of NP-1496a.

97. The vector of claim 94, wherein the sense strand or portion of the siRNA or shRNA has a sequence selected from the group consisting of: the first 19 nucleotides of any of SEQ ID NOS: 71, 75, 77, 83, 93, 95, 99, and 188, reading in a 5′ to 3′ direction.

98. The vector of claim 86, wherein: the nucleic acid is operably linked to a promoter for RNA polymerase III.

99. The vector of claim 98, wherein: the promoter is a U6 or H1 promoter.

100. The vector of claim 86, wherein: the vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors.

101. The vector of claim 86, wherein the vector is a lentiviral vector.

102. The vector of claim 86, wherein the vector is a DNA vector.

103. The vector of claim 86, wherein the vector is a virus.

104. The vector of claim 86, wherein the vector is a lentivirus.

105. A method of treating or preventing infection by an infectious agent, the method comprising steps of: administering to a subject prior to, simultaneously with, or after exposure of the subject to the infectious agent, a composition comprising the vector of claim 86 or the cell of claim 88.

106. The method of claim 105, wherein the infectious agent is a virus.

107. The method of claim 105, wherein the infectious agent infects respiratory epithelial cells.

108. The method of claim 105, wherein the infectious agent is an influenza virus.

109. The method of claim 105, wherein the composition is administered intravenously.

110. The method of claim 105, wherein the composition is administered intranasally.

111. The method of claim 105, wherein the composition is administered by inhalation.

112. A pharmaceutical composition comprising: the composition of claim 1; and a pharmaceutically acceptable carrier.

113. The pharmaceutical composition of claim 112, wherein: the composition is formulated as an aerosol.

114. The pharmaceutical composition of claim 112, wherein: the composition is formulated as a nasal spray.

115. The pharmaceutical composition of claim 112, wherein: the composition is formulated for intravenous administration.

116. The pharmaceutical composition of claim 112, wherein: the infectious agent is an influenza virus and wherein the composition further comprises a second anti-influenza agent.

117. The pharmaceutical composition of claim 116, wherein the second anti-influenza agent is approved by the United States Food and Drug Administration.

118. A method for identifying viral inhibitors, the method comprising steps of: providing a cell including a candidate siRNA or shRNA whose sequence includes a region of complementarity with at least one transcript produced during infection with a virus, which transcript is characterized in that its degradation delays, prevents, or inhibits one or more aspects of viral infection or replication; detecting infection by or replication of the virus in the cell; and identifying an siRNA or shRNA that inhibits viral infectivity or replication, which siRNA or shRNA is a viral inhibitor.

119. The method of claim 118, wherein: the virus is an influenza virus.

120. The method of claim 118, wherein: the cell is characterized in that in the absence of the siRNA or shRNA the cell produces at least one viral transcript.

121. The method of claim 118, further comprising the step of: transfecting the cell with a viral genome or infecting the cell with the virus.

122. A method of treating or preventing infection by a virus, the method comprising steps of: administering to a subject prior to, simultaneously with, or after exposure of the subject to the virus, a composition comprising an effective amount of an RNAi-inducing entity, wherein the RNAi-inducing entity is targeted to a transcript produced during infection by the virus, which transcript is characterized in that reduction in levels of the transcript delays, prevents, or inhibits one or more aspects of infection by or replication of the virus.

123. The method of claim 122, wherein: the virus infects respiratory epithelial cells.

124. The method of claim 122, wherein: the virus is an influenza virus.

125. The method of claim 122, wherein the composition is administered into the respiratory tract.

126. The method of claim 122, wherein the composition is administered by a conventional intravenous delivery method.

127. The method of claim 122, wherein in the absence of the RNAi-inducing entity the virus is able to undergo a complete life cycle leading to production of infectious virus, and wherein the presence of the siRNA or shRNA inhibits production of the virus.

128. The method of claim 122, wherein the RNAi-inducing entity comprises a duplex portion selected from the group consisting of: duplex portions of: NP-1496, NP-1496a, PA-2087, PB1-2257, PB1-129, PB2-2240, M-37, and M-598, or a variant of any of the foregoing, wherein the variant differs by at most one nucleotide from the corresponding siRNA in either its sense portion, antisense portion, or both.

129. The method of claim 128, wherein the duplex portion is identical to the duplex portion of NP-1496.

130. The vector of claim 128, wherein the duplex portion is identical to the duplex portion of NP-1496a.

131. A method for designing an siRNA or shRNA having a duplex portion, the method comprising steps of: identifying a portion of a target transcript, which portion is highly conserved among a plurality of variants of an infectious agent and comprises at least 15 consecutive nucleotides; and selecting the sequence of the portion as the sequence for the duplex portion of the siRNA or shRNA sense strand or portion.

132. The method of claim 131, further comprising: selecting a sequence complementary to the portion as the sequence for the duplex portion of the siRNA or shRNA antisense strand or portion.

133. The method of claim 132, further comprising: adding a 3′ overhang to either or both of the sense and antisense strands of the siRNA duplex.

134. The method of claim 131, wherein: the plurality of variants comprises at least 10 variants.

135. The method of claim 131, wherein: the plurality of variants comprises at least 15 variants.

136. The method of claim 131, wherein: the plurality of variants comprises at least 20 variants.

137. The method of claim 131, wherein: the portion comprises approximately 19 nucleotides.

138. The method of claim 131, wherein: a portion is considered highly conserved among a plurality of variants if it differs by at most one nucleotide between the variants.

139. The method of claim 131, wherein: the infectious agent is an influenza virus.

140. The method of claim 131, wherein: the infectious agent is capable of undergoing reassortment.

141. The method of claim 131, wherein: the variants include at least two variants, each of which naturally infects a host of a different species.

142. The method of claim 141, wherein: the species include at least two species selected from the group consisting of humans, swine, horse, and bird species.

143. The method of claim 131, wherein: the variants include at least two variants, each of which arose in a host of a different species.

144. The method of claim 143, wherein: the species include at least two species selected from the group consisting of humans, swine, horse, and bird species.

145. A composition comprising an siRNA or shRNA designed in accordance with the method of claim 131.

146. A method of reducing or lowering levels of a transcript, which transcript is a vRNA or cRNA, comprising administering an RNAi-inducing entity targeted to an mRNA transcript having a sequence at least a portion of which is complementary to or identical to the vRNA or cRNA transcript.

147. A method of inhibiting a first transcript comprising administering an RNAi-inducing entity targeted to a second transcript, wherein inhibition of the second transcript results in inhibition of the first transcript.

148. The method of claim 147, wherein the level of the first transcript is reduced relative to its level in the absence of the RNAi-inducing entity.

149. The method of claim 147, wherein the level of the second transcript is reduced relative to its level in the absence of the RNAi-inducing entity.

150. The method of claim 147, wherein the levels of the first and second transcript are reduced relative to their levels in the absence of the RNAi-inducing entity.

151. The method of claim 147, wherein the RNAi-inducing entity is not specifically targeted to the first transcript.

152. The method of claim 147, wherein the second transcript encodes a protein that functions in maintaining RNA stability.

153. The method of claim 147, wherein the protein is a nucleic acid binding protein.

154. The method of claim 153, wherein the nucleic acid binding protein is an RNA binding protein.

155. The method of claim 147, wherein the second transcript encodes a polymerase.

156. The method of claim 155, wherein the polymerase is an RNA polymerase.

157. The method of claim 155, wherein the polymerase is a DNA polymerase.

158. The method of claim 155, wherein the polymerase is a reverse transcriptase.

159. The method of claim 147, wherein either of both of the first and second transcripts are agent-specific transcripts, wherein the agent is an infectious agent.

160. The method of claim 147, wherein the first and second transcripts are agent-specific transcripts, wherein the agent is an infectious agent.

161. The method of claim 160, wherein the infectious agent is a virus.

162. The method of claim 161, wherein the virus is an influenza virus.

163. The method of claim 162, wherein the second transcript encodes either viral NP protein or viral PA protein.

164. The method of claim 163, wherein the first transcript encodes a protein selected from the group consisting of: M protein, HA protein, PB1 protein, PB2 protein, or NS protein.

165. A composition comprising: an RNAi-inducing entity, wherein the RNAi-inducing entity is targeted to an influenza virus transcript; and a delivery agent selected from the group consisting of: cationic polymers, modified cationic polymers, peptide molecular transporters, surfactants suitable for introduction into the lung, neutral or cationic lipids, liposomes, non-cationic polymers, modified non-cationic polymers, bupivacaine, and chloroquine.

166. The composition of claim 165, wherein the delivery agent comprises a delivery-enhancing moiety to enhance delivery to a cell of interest.

167. The composition of claim 165, wherein the delivery-enhancing moiety comprises an antibody, antibody fragment, or ligand that specifically binds to a molecule expressed by the cell of interest.

168. The composition of claim 167, wherein the cell of interest is a respiratory epithelial cell.

169. The composition of claim 165, wherein the delivery-enhancing moiety comprises a moiety selected to reduce degradation, clearance, or nonspecific binding of the delivery agent.

170. The composition of claim 165, wherein the RNAi-inducing entity comprises a viral vector.

171. The composition of claim 170, wherein the viral vector comprises a lentiviral vector.

172. The composition of claim 165, wherein the RNAi-inducing entity comprises a DNA vector.

173. The composition of claim 165, wherein the RNAi-inducing entity comprises a virus.

174. The composition of claim 173, wherein the RNAi-inducing entity comprises a lentivirus.

175. The composition of claim 165, wherein the RNAi-inducing entity comprises an siRNA.

176. The composition of claim 165, wherein the RNAi-inducing entity comprises an shRNA.

177. The composition of claim 165, wherein the RNAi-inducing entity comprises an RNAi-inducing vector whose presence within a cell results in production of an siRNA or shRNA targeted to an influenza virus transcript.

178. The composition of claim 165, wherein: the RNAi-inducing entity comprises an siRNA or shRNA or an RNAi-inducing vector whose presence within a cell results in production of an siRNA or shRNA, wherein the siRNA or shRNA comprises a portion that is perfectly complementary to a region of the target transcript, wherein the portion is at least 15 nucleotides in length.

179. The composition of claim 165, wherein: the RNAi-inducing entity comprises an siRNA or shRNA or an RNAi-inducing vector whose presence within a cell results in production of an siRNA or shRNA, wherein the siRNA or shRNA comprises a duplex portion selected from the group consisting of duplex portions of: NP-1496, NP-1496a, PA-2087, PB1-2257, PB1-129, PB2-2240, M-37, and M-598, or a variant of any of the foregoing, wherein the variant differs by at most one nucleotide from the corresponding siRNA or shRNA in either its sense portion, antisense portion, or both.

180. The composition of claim 179, wherein the siRNA or shRNA duplex portion comprises the duplex portion of NP-1496.

181. The composition of claim 179, wherein the siRNA or shRNA duplex portion comprises the duplex portion of NP-1496a.

182. The composition of claim 165, wherein: the RNAi-inducing entity comprises an siRNA or shRNA or an RNAi-inducing vector whose presence within a cell results in production of an siRNA or shRNA, wherein the siRNA or shRNA, wherein the sequence of the sense strand or portion of the siRNA or shRNA comprises a sequence selected from the group consisting of: the first 19 nucleotides of, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 83, SEQ ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 99, and SEQ ID NO: 188 reading in a 5′ to 3′ direction.

183. The composition of claim 182, wherein the sequence of the sense strand or portion of the siRNA or shRNA comprises the sequence of SEQ ID NO: 93.

184. The composition of claim 182, wherein the sequence of the sense strand or portion of the siRNA or shRNA comprises the sequence of SEQ ID NO: 188.

185. The composition of claim 165, wherein the delivery agent is selected from the group consisting of cationic polymers, modified cationic polymers, and surfactants suitable for introduction into the lung.

186. The composition of claim 185, wherein the cationic polymer is selected from the group consisting of polylysine, polyarginine, polyethyleneimine, polyvinylpyrrolidone, chitosan, and poly(β-amino ester) polymers.

187. The composition of claim 186, wherein the cationic polymer is polyethyleneimine.

188. The composition of claim 185, wherein the modified cationic polymer incorporates a modification selected to reduce the cationic nature of the polymer.

189. The composition of claim 188, wherein the modification comprises substitution with a group selected from the list consisting of: acetyl, imidazole, succinyl, and acyl.

190. The composition of claim 185, wherein between 25% and 75% of the residues of the modified cationic polymer are modified.

191. The composition of claim 190, wherein approximately 50% of the residues of the modified cationic polymer are modified.

192. The composition of claim 185, wherein the delivery agent comprises a surfactant suitable for introduction into the lung.

193. The composition of claim 192, wherein the surfactant is Infasurf®, Survanta®, or Exosurf®.

194. A method of treating or preventing influenza virus replication, pathogenicity, or infectivity comprising administering the composition of claim 165 to a subject at risk of or suffering from influenza virus infection.

195. The method of claim 194, wherein the composition is administered by a route selected from the group consisting of: intravenous injection, inhalation, intranasally, and as an aerosol.

196. The method of claim 194, wherein the composition is administered intravenously.

197. The method of claim 196, wherein the composition is administered using a conventional intravenous administration technique.

198. The method of claim 194, wherein the composition is administered by inhalation.

199. The method of claim 194, wherein the composition is administered intranasally.

200. The method of claim 194, wherein the composition is administered as an aerosol.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 60/414,457, filed Sep. 28, 2002, and U.S. Provisional Patent Application No. 60/446,377, filed Feb. 10, 2003. The contents of each of these applications is incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] The United States Government has provided grant support utilized in the development of the present invention. In particular, National Institutes of Health grant numbers 5-RO1-AI44477, 5-RO1-AI44478, 5-ROI-CA60686, and 1-RO1-AI50631 have supported development of this invention. The United States Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Influenza is one of the most widely spread infections worldwide. It can be deadly: an estimated 20 to 40 million people died during the 1918 influenza A virus pandemic. In the United States between 20 and 40 thousand people die from influenza A virus infection or its complications each year. During epidemics the number of influenza related hospitalizations may reach over 300,000 in a single winter season.

[0004] Several properties contribute to the epidemiological success of influenza virus. First, it is spread easily from person to person by aerosol (droplet infection). Second, small changes in influenza virus antigens are frequent (antigenic drift) so that the virus readily escapes protective immunity induced by a previous exposure to a different variant of the virus. Third, new strains of influenza virus can be easily generated by reassortment or mixing of genetic material between different strains (antigenic shift). In the case of influenza A virus, such mixing can occur between subtypes or strains that affect different species. The 1918 pandemic is thought to have been caused by a hybrid strain of virus derived from reassortment between a swine and a human influenza A virus.

[0005] Despite intensive efforts, there is still no effective therapy for influenza virus infection and existing vaccines are limited in value in part because of the properties of antigenic shift and drift described above. For these reasons, global surveillance of influenza A virus has been underway for many years, and the National Institutes of Health designates it as one of the top priority pathogens for biodefense. Although current vaccines based upon inactivated virus are able to prevent illness in approximately 70-80% of healthy individuals under age 65, this percentage is far lower in the elderly or immunocompromised. In addition, the expense and potential side effects associated with vaccine administration make this approach less than optimal. Although the four antiviral drugs currently approved in the United States for treatment and/or prophylaxis of influenza are helpful, their use is limited due to concerns about side effects, compliance, and possible emergence of resistant strains. Therefore, there remains a need for the development of effective therapies for the treatment and prevention of influenza infection.

SUMMARY OF THE INVENTION

[0006] The present invention provides novel therapeutics for the treatment of influenza due to influenza virus types A, B, and C based on the phenomenon of RNA interference (RNAi). In particular, the invention provides short interfering RNA (siRNA) and/or short hairpin RNA (shRNA) molecules targeted to one or more target transcripts involved in virus production, virus replication, virus infection, and/or transcription of viral RNA, etc. In addition, the invention provides vectors whose presence within a cell results in transcription of one or more RNAs that self-hybridize or hybridize to each other to form an shRNA or siRNA that inhibits expression of at least one target transcript involved in virus production, virus infection, virus replication, and/or transcription of viral mRNA, etc.

[0007] The invention further provides a variety of compositions containing the siRNAs, shRNAs, and/or vectors of the invention. In certain embodiments of the invention the siRNA comprises two RNA strands having complementary regions so that the strands hybridize to each other to form a duplex structure approximately 19 nucleotides in length, wherein each of the strands optionally comprises a single-stranded overhang. In certain embodiments of the invention the shRNA comprises a single RNA molecule having complementary regions that hybridize to each other to form a hairpin (stem/loop) structure with a duplex portion approximately 19 nucleotides in length and a single-stranded loop. Such RNA molecules are said to self-hybridize. The shRNA may optionally include one or more unpaired portions at the 5′ and/or 3′ portion of the RNA. The invention further provides compositions comprising the inventive siRNAs, shRNAs, and/or vectors, and methods of delivery of such compositions.

[0008] Thus in one aspect, the invention provides an siRNA or shRNA targeted to a target transcript, wherein the target transcript is an agent-specific transcript, which transcript is involved in the production of, replication of, pathogenicity of, and/or infection by an infectious agent, and/or involved in transcription of agent-specific RNA. For purposes of description an siRNA or shRNA that inhibits expression of a target transcript involved in the production of, replication of, pathogenicity of, and/or infection by an infectious agent, thereby inhibiting production of, replication of, pathogenicity of, and/or infection by the infectious agent will be said to inhibit the infectious agent. According to certain embodiments of the invention the infectious agent is a virus. According to certain preferred embodiments of the invention the infectious agent is a virus that infects cells of the respiratory passages and/or lungs, e.g., respiratory epithelial cells, such as an influenza virus. According to certain embodiments of the invention the target transcript encodes a protein selected from the group consisting of: a polymerase, a nucleocapsid protein, a neuraminidase, a hemagglutinin, a matrix protein, and a nonstructural protein. According to certain embodiments of the invention the target transcript encodes an influenza virus protein selected from the group consisting of hemagglutinin, neuraminidase, membrane protein 1, membrane protein 2, nonstructural protein 1, nonstructural protein 2, polymerase protein PB1, polymerase protein PB2, polymerase protein PA, polymerase protein NP.

[0009] In another aspect, the invention provides a vector comprising a nucleic acid operably linked to expression signals (e.g., a promoter or promoter/enhancer) active in a cell so that, when the construct is introduced into the cell, an siRNA or shRNA is produced inside the host cell that is targeted to an agent-specific transcript, which transcript is involved in production of, replication of, and/or infection by an infectious agent, and/or transcription of agent-specific RNA. In certain embodiments of the invention the infectious agent is a virus, e.g., an influenza virus. In certain preferred embodiments of the invention the siRNA or shRNA inhibits influenza virus. The siRNA or shRNA may be targeted to any of the transcripts mentioned above. In general, the vector may be a DNA plasmid or a viral vector such as a retrovirus (e.g., a lentivirus), adenovirus, adeno-associated virus, etc. whose presence within a cell results in transcription of one or more ribonucleic acids (RNAs) that self-hybridize or hybridize to each other to form a short hairpin RNA (shRNA) or short interfering RNA (siRNA) that inhibits expression of at least one influenza virus transcript in the cell. In certain embodiments of the invention the vector comprises a nucleic acid segment operably linked to a promoter, so that transcription from the promoter (i.e., transcription directed by the promoter) results in synthesis of an RNA comprising complementary regions that hybridize to form an shRNA targeted to the target transcript. In certain embodiments of the invention the lentiviral vector comprises a nucleic acid segment flanked by two promoters in opposite orientation, wherein the promoters are operably linked to the nucleic acid segment, so that transcription from the promoters results in synthesis of two complementary RNAs that hybridize with each other to form an siRNA targeted to the target transcript. The invention further provides compositions comprising the vector.

[0010] The invention also provides compositions comprising inventive siRNAs, shRNAs, and/or vectors described herein, wherein the composition further comprises any of a variety of substances (referred to herein as delivery agents) that facilitate delivery and/or uptake of the siRNA, shRNA, or vector. These substances include cationic polymers; peptide molecular transporters including arginine-rich peptides and histidine-rich peptides; cationic and neutral lipids; liposomes; certain non-cationic polymers; carbohydrates; and surfactant materials. The invention also encompasses the use of delivery agents that have been modified in any of a variety of ways, e.g., by addition of a delivery-enhancing moiety to the delivery agent.

[0011] In certain embodiments of the invention the delivery agent is modified in any of a number of ways to enhance stability, promote cellular uptake of the composition, promote release of siRNA, shRNA, and/or vectors within the cell, reduce cytotoxicity, or direct the composition to a particular cell type, tissue, or organ. For example, in certain embodiments of the invention the delivery agent is a modified cationic polymer (e.g., a cationic polymer substituted with one or more groups selected to reduce the cationic nature of the polymer and thereby reduce cytotoxicity). In certain embodiments of the invention the delivery agent comprises a delivery-enhancing moiety such as an antibody, antibody fragment, or ligand that specifically binds to a molecule that is present on the surface of a cell such as a respiratory epithelial cell.

[0012] The present invention further provides methods of treating or preventing infectious diseases, particularly infectious diseases of the respiratory system, e.g., influenza, by administering any of the inventive compositions to a subject within an appropriate time window prior to exposure to the infectious agent, while exposure is occurring, or following exposure, or at any point during which a subject exhibits symptoms of a disease caused by the infectious agent. The siRNAs or shRNAs may be chemically synthesized, produced using in vitro transcription, synthesized in vitro, produced intracellularly, etc. The compositions may be administered by a variety of routes including intravenous, inhalation, intranasally, as an aerosol, intraperitoneally, intramuscularly, intradermally, orally, etc.

[0013] The invention provides additional methods of treating or preventing a disease caused by an infectious agent, e.g., a disease caused by influenza virus, employing gene therapy. According to certain of these methods cells (either infected or noninfected) are engineered or manipulated to synthesize inventive siRNAs or shRNAs. According to certain embodiments of the invention the cells are engineered to contain a vector whose presence within the cell results in synthesis of one or more RNAs that hybridize with each other or self-hybridize within the cell to form one or more siRNAs or shRNAs targeted to an appropriate agent-specific target transcript. The cells may be engineered in vitro or while present within the subject to be treated, e.g., within the respiratory passages of the subject.

[0014] In another aspect, the invention provides methods for selecting and designing preferred siRNA or shRNA sequences to inhibit an infectious agent. The invention provides methods of selecting and designing siRNAs and shRNAs to inhibit infectious agents characterized in that multiple different strains or variants of the infectious agent exist, in particular wherein strain variation can occur by genetic reassortment or mixing. These methods find particular use in selecting and designing siRNA and shRNA sequences to combat infectious agents whose genomes consist of multiple different segments, wherein genetic reassortment can occur rapidly and unpredictably by substitution of an entire genomic segment from one subtype to another. These aspects of the invention are therefore particularly suited for infectious agents whose genome consists of multiple independent segments, meaning that the genome consists of physically distinct nucleic acid molecules that are not covalently joined to one another. The invention may also find particular utility for infectious agents that exchange genetic information by transfer of plasmids, e.g., plasmids encoding genes that confer resistance to therapeutic compounds.

[0015] The present invention also provides a system for identifying compositions comprising one or more RNAi-inducing entities such as siRNAs and/or shRNAs targeted to an influenza virus transcript, and/or comprising vector(s) whose presence within a cell results in production of one or more RNAs that hybridize with each other or self-hybridize to form an siRNA or shRNA that is targeted to an influenza virus transcript, wherein the compositions are useful for the inhibition of influenza virus.

[0016] The present invention further provides a system for the analysis and characterization of the mechanism of influenza replication and/or transcription of influenza virus RNAs, as well as for the characterization and analysis of relevant viral components involved in the viral life cycle.

[0017] In another aspect, the invention provides methods for designing siRNAs and/or shRNAs to inhibit an infectious agent in cases where multiple variants of the infectious agent exist. For example, the invention provides a method for designing an siRNA or shRNA molecule having a duplex portion, the method comprising steps of (i) identifying a portion of a target transcript, which portion is highly conserved among a plurality of variants of an infectious agent and comprises at least 15 consecutive nucleotides; and (ii) selecting an siRNA or shRNA, wherein the sense strand of the siRNA or the sense portion of the shRNA comprises the highly conserved sequence.

[0018] In another aspect, the invention provides siRNAs and siRNAs and methods for design thereof, wherein the siRNA or shRNA is targeted to a transcript whose inhibition results in inhibition of multiple (or all) other viral transcripts. In particular, the invention provides siRNA and shRNA compositions comprising siRNAs or shRNAs targeted to transcripts encoding viral polymerase (DNA or RNA polymerase) or nucleocapsid proteins.

[0019] This application refers to various patents, journal articles, and other publications, all of which are incorporated herein by reference. In addition, the following standard reference works are incorporated herein by reference: Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science , and Current Protocols in Cell Biology , John Wiley & Sons, N.Y., edition as of July 2002; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual , 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1A , adapted from Julkunen, I., et al., referenced elsewhere herein, presents a schematic of the influenza virus.

[0021] FIG. 1B , adapted from Fields' Virology, referenced elsewhere herein, shows the genome structure of the influenza virus and the transcripts derived from the influenza genome. Thin lines at the 5′ and 3′-termini of the mRNAs represent untranslated regions. Shaded or hatched areas represent coding regions in the 0 or +1 reading frames, respectively. Introns are depicted by V-shaped lines. Small rectangles at the 5′ ends of the mRNAs represent heterogenous cellular RNAs covalently linked to the viral nucleic acids. A _(n) symbolizes the polyA tail.

[0022] FIG. 2 , adapted from Julkunen, I., et al., referenced elsewhere herein, shows the influenza virus replication cycle.

[0023] FIG. 3 shows the structure of siRNAs observed in the Drosophila system.

[0024] FIG. 4 presents a schematic representation of the steps involved in RNA interference in Drosophila.

[0025] FIG. 5 shows a variety of exemplary siRNA and shRNA structures useful in accordance with the present invention.

[0026] FIG. 6 presents a representation of an alternative inhibitory pathway, in which the DICER enzyme cleaves a substrate having a base mismatch in the stem to generate an inhibitory product that binds to the 3′ UTR of a target transcript and inhibits translation.

[0027] FIG. 7 presents one example of a construct that may be used to direct transcription of both strands of an inventive siRNA.

[0028] FIG. 8 depicts one example of a construct that may be used to direct transcript of a single RNA molecule that hybridizes to form an shRNA in accordance with the present invention.

[0029] FIG. 9 shows a sequence comparison between six strains of influenza virus A that have a human host of origin. Dark shaded areas were used to design siRNAs that were tested as described in Example 2. The base sequence is the sequence of strain A/Puerto Rico/8/34. Lightly shaded letters indicate nucleotides that differ from the base sequence.

[0030] FIG. 10 shows a sequence comparison between two strains of influenza virus that have a human host of origin and five strains of influenza virus A that have an animal host of origin. Darkly shaded areas were used to design siRNAs that were tested as described in Example 2. The base sequence is the sequence of strain A/Puerto Rico/8/34. Lightly shaded letters indicate nucleotides that differ from the base sequence.

[0031] FIGS. 11A-11F show the results of experiments indicating that siRNA inhibits influenza virus production in MDCK cells. Six different siRNAs that target various viral transcripts were introduced into MDCK cells by electroporation, and cells were infected with virus 8 hours later. FIG. 11A is a time course showing viral titer in culture supernatants as measured by hemagglutinin assay at various times following infection with viral strain A/PR/8/34 (H1N1) (PR8), at a multiplicity of infection (MOI) of 0.01 in the presence or absence of the various siRNAs or a control siRNA. FIG. 11B is a time course showing viral titer in culture supernatants as measured by hemagglutinin assay at various times following infection with influenza virus strain A/WSN/33 (H1N1) (WSN) at an MOI of 0.01 in the presence or absence of the various siRNAs or a control siRNA. FIG. 11C shows a plaque assay showing viral titer in culture supernatants from virus infected cells that were either mock transfected or transfected with siRNA NP-1496. FIG. 11D shows inhibition of influenza virus production at different doses of siRNA. MDCK cells were transfected with the indicated amount of NP-1496 siRNA followed by infection with PR8 virus at an MOI of 0.01. Virus titer was measured 48 hours after infection. Representative data from one of two experiments are shown. FIG. 11E shows inhibition of influenza virus production by siRNA administered after virus infection. MDCK cells were infected with PR8 virus at an MOI of 0.01 for 2 hrs and then transfected with NP-1496 (2.5 nmol). Virus titer was measured at the indicated times after infection. Representative data from one of two experiments are shown.

[0032] FIG. 12 shows a sequence comparison between a portion of the 3′ region of NP sequences among twelve influenza A virus subtypes or isolates that have either a human or animal host of origin. The shaded area was used to design siRNAs that were tested as described in Examples 2 and 3. The base sequence is the sequence of strain A/Puerto Rico/8/34. Shaded letters indicate nucleotides that differ from the base sequence.

[0033] FIG. 13 shows positions of various siRNAs relative to influenza virus gene segments, correlated with effectiveness in inhibiting influenza virus.

[0034] FIG. 14A is a schematic of a developing chicken embryo indicating the area for injection of siRNA and siRNA/delivery agent compositions.

[0035] FIG. 14B shows the ability of various siRNAs to inhibit influenza virus production in developing chicken embryos.

[0036] FIG. 15 is a schematic showing the interaction of nucleoprotein with viral RNA molecules.

[0037] FIGS. 16A and 16B show schematic diagrams illustrating the differences between influenza virus vRNA, mRNA, and cRNA (template RNA) and the relationships between them. The conserved 12 nucleotides at the 3′ end and 13 nucleotides at the 5′ end of each influenza A virus vRNA segment are indicated in FIG. 16B . The mRNAs contain an m ⁷ GpppN ^m cap structure and, on average, 10 to 13 nucleotides derived from a subset of host cell RNAs. Polyadenylation of the mRNAs occurs at a site in the mRNA corresponding to a location 15 to 22 nucleotides before the 5′ end of the vRNA segment. Arrows indicate the positions of primers specific for each RNA species. (Adapted from ref. (1)).

[0038] FIG. 17 shows amounts of viral NP and NS RNA species at various times following infection with virus, in cells that were mock transfected or transfected with siRNA NP-1496 6-8 hours prior to infection.

[0039] FIG. 18A shows that inhibition of influenza virus production requires a wild type (wt) antisense strand in the duplex siRNA. MDCK cells were first transfected with siRNAs formed from wt and modified (m) strands and infected 8 hrs later with PR8 virus at MOI of 0.1. Virus titers in the culture supernatants were assayed 24 hrs after infection. Representative data from one of the two experiments are shown. FIG. 18B shows that M-specific siRNA inhibits the accumulation of specific mRNA. MDCK cells were transfected with M-37, infected with PR8 virus at MOI of 0.01, and harvested for RNA isolation 1, 2, and 3 hrs after infection. The levels of M-specific mRNA, cRNA, and vRNA were measured by reverse transcription using RNA-specific primers, followed by real time PCR. The level of each viral RNA species is normalized to the level of γ-actin mRNA (bottom panel) in the same sample. The relative levels of RNAs are shown as mean value ±S.D. Representative data from one of the two experiments are shown.

[0040] FIGS. 19 A-D show that NP-specific siRNA inhibits the accumulation of not only NP- but also M- and NS-specific mRNA, vRNA, and cRNA. MDCK (A-C) and Vero (D) cells were transfected with NP-1496, infected with PR8 virus at MOI of 0.1, and harvested for RNA isolation 1, 2, and 3 hrs after infection. The levels of mRNA, cRNA, and vRNA specific for NP, M, and NS were measured by reverse transcription using RNA-specific primers followed by real time PCR. The level of each viral RNA species is normalized to the level of γ-actin mRNA (not shown) in the same sample. The relative levels of RNAs are shown. Representative data from one of three experiments are shown.

[0041] FIGS. 19 E-G, right side in each figure, show that PA-specific siRNA inhibits the accumulation of not only PA- but also M- and NS-specific mRNA, vRNA, and cRNA. MDCK cells were transfected with PA-1496, infected with PR8 virus at MOI of 0.1, and harvested for RNA isolation 1, 2, and 3 hrs after infection. The levels of mRNA, cRNA, and vRNA specific for PA, M, and NS were measured by reverse transcription using RNA-specific primers followed by real time PCR. The level of each viral RNA species is normalized to the level of γ-actin mRNA (not shown) in the same sample. The relative levels of RNAs are shown.

[0042] FIG. 19H shows that NP-specific siRNA inhibits the accumulation of PB1-(top panel), PB2-(middle panel) and PA-(lower panel) specific mRNA. MDCK cells were transfected with NP-1496, infected with PR8 virus at MOI of 0.1, and harvested for RNA isolation 1, 2, and 3 hrs after infection. The levels of mRNA specific for PB1, PB2, and PA mRNA were measured by reverse transcription using RNA-specific primers followed by real time PCR. The level of each viral RNA species is normalized to the level of γ-actin mRNA (not shown) in the same sample. The relative levels of RNAs are shown.

[0043] FIG. 20A shows sequences of siRNA CD8-61 and its hairpin derivative CD8-61F.

[0044] FIG. 20B shows inhibition of CD8α expression by CD8-61 and CD8-61F. A CD8 ⁺ CD4 ⁺ T cell line was transfected with either CD8-61 or CD8-61F by electroporation. CD8α expression was assayed by flow cytometry 48 hrs later. Unlabeled line, mock transfection.

[0045] FIG. 20C shows a schematic diagram of the pSLOOP III vector, in which expression of CD8-61F hairpin RNA is driven by H1 RNA pol III promoter. Terminator, termination signal sequence.

[0046] FIG. 20D presents plots showing silencing of CD8α in HeLa cells using pSLOOP III. Untransfected cells did not express CD8α. Cells were transfected with the CD8α expression vector and either a promoterless pSLOOP III-CD8-61F construct, synthetic siRNA, or a pSLOOP III-CD8-61F containing a promoter.

[0047] FIG. 21A shows schematic diagrams of NP-1496 and GFP-949 siRNA and their hairpin derivatives/precursors.

[0048] FIG. 21B shows tandem arrays of NP-1496H and GFP-949H in two different orders.

[0049] FIG. 21C shows pSLOOP III expression vectors. Hairpin precursors of siRNA are cloned in the pSLOOP III vector alone (top), in tandem arrays (middle), or simultaneously with independent promoter and termination sequence (bottom).

[0050] FIG. 22A is a plot showing that siRNA inhibits influenza virus production in mice when administered together with the cationic polymer PEI prior to infection with influenza virus. Filled squares (no treatment); Open squares (GFP siRNA); Open circles (30 μg NP siRNA); Filled circles (60 μg NP siRNA). Each symbol represents an individual animal. p values between different groups are shown.

[0051] FIG. 22B is a plot showing that siRNA inhibits influenza virus production in mice when administered together with the cationic polymer PLL prior to infection with influenza virus. Filled squares (no treatment); Open squares (GFP siRNA); Filled circles (60 μg NP siRNA). Each symbol represents an individual animal. p values between different groups are shown.

[0052] FIG. 22C is a plot showing that siRNA inhibits influenza virus production in mice when administered together with the cationic polymer jetPEI prior to infection with influenza virus significantly more effectively than when administered in PBS. Open squares (no treatment); Open triangles (GFP siRNA in PBS); Filled triangles (NP siRNA in PBS); Open circles (GFP siRNA with jetPEI); Filled circles (NP siRNA with jetPEI). Each symbol represents an individual animal. p values between different groups are shown.

[0053] FIG. 23 is a plot showing that siRNAs targeted to influenza virus NP and PA transcripts exhibit an additive effect when administered together prior to infection with influenza virus. Filled squares (no treatment); Open circles (60 μg NP siRNA); Open triangles (60 μg PA siRNA); Filled circles (60 μg NP siRNA+60 μg PA siRNA). Each symbol represents an individual animal. p values between different groups are shown.

[0054] FIG. 24 is a plot showing that siRNA inhibits influenza virus production in mice when administered following infection with influenza virus. Filled squares (no treatment); Open squares (60 μg GFP siRNA); Open triangles (60 μg PA siRNA); Open circles (60 μg NP siRNA); Filled circles (60 μg NP+60 μg PA siRNA). Each symbol represents an individual animal. p values between different groups are shown.

[0055] FIG. 25A is a schematic diagram of a lentiviral vector expressing a shRNA. Transcription of shRNA is driven by the U6 promoter. EGFP expression is driven by the CMV promoter. SIN-LTR, Ψ, cPPT, and WRE are lentivirus components. The sequence of NP-1496 shRNA is shown.

[0056] FIG. 25B presents plots of flow cytometry results demonstrating that Vero cells infected with the lentivirus depicted in FIG. 25B express EGFP in a dose-dependent manner. Lentivirus was produced by co-transfecting DNA vector encoding NP-1496a shRNA and packaging vectors into 293T cells. Culture supernatants (0.25 ml or 1.0 ml) were used to infect Vero cells. The resulting Vero cell lines (Vero-NP-0.25 and Vero-NP-1.0) and control (uninfected) Vero cells were analyzed for GFP expression by flow cytometry. Mean fluorescence intensity of Vero-NP-0.25 (upper portion of figure) and Vero-NP-1.0 (lower portion of figure) cells are shown. The shaded curve represents mean fluorescence intensity of control (uninfected) Vero cells.

[0057] FIG. 25C is a plot showing inhibition of influenza virus production in Vero cells that express NP-1496 shRNA. Parental and NP-1496 shRNA expressing Vero cells were infected with PR8 virus at MOI of 0.04, 0.2 and 1. Virus titers in the supernatants were determined by hemagglutination (HA) assay 48 hrs after infection.

[0058] FIG. 26 is a plot showing that influenza virus production in mice is inhibited by administration of DNA vectors that express siRNA targeted to influenza virus transcripts. Sixty μg of DNA encoding RSV, NP-1496 (NP) or PB1-2257 (PB1) shRNA were mixed with 40 μl Infasurf and were administered into mice by instillation. For no treatment (NT) group, mice were instilled with 60 μl of 5% glucose. Thirteen hrs later, the mice were infected intranasally with PR8 virus, 12000 pfu per mouse. The virus titers in the lungs were measured 24 hrs after infection by MDCK/hemagglutinin assay. Each data point represents one mouse. p values between groups are indicated.

[0059] FIG. 27A shows results of an electrophoretic mobility shift assay for detecting complex formation between siRNA and poly-L-lysine (PLL). SiRNA-polymer complexes were formed by mixing 150 ng of NP-1496 siRNA with increasing amounts of polymer (0-1200 ng) for 30 min at room temperature. The reactive mixtures were then run on a 4% agarose gel and siRNAs were visualized with ethidium-bromide staining.

[0060] FIG. 27B shows results of an electrophoretic mobility shift assay for detecting complex formation between siRNA and poly-L-arginine (PLA). SiRNA-polymer complexes were formed by mixing 150 ng of NP-1496 siRNA with increasing amounts of polymer (0-1200 ng) for 30 min at room temperature. The reactive mixtures were then run on a 4% agarose gel and siRNAs were visualized with ethidium-bromide staining.

[0061] FIG. 28A is a plot showing cytotoxicity of siRNA/PLL complexes. Vero cells in 96-well plates were treated with siRNA (400 pmol)/polymer complexes for 6 hrs. The polymer-containing medium was then replaced with DMEM-10% FCS. The metabolic activity of the cells was measured 24 h later by using the MTT assay. Squares=PLL (MW˜8K); Circles=PLL (MW˜42K) Filled squares=25%; Open triangles=50%; Filled triangles=75%; X=95%. The data are shown as the average of triplicates.

[0062] FIG. 28B is a plot showing cytotoxicity of siRNA/PLA complexes. Vero cells in 96-well plates were treaed with siRNA (400 pmol)/polymer complexes for 6 hrs. The polymer-containing medium was then replaced with DMEM-10% FCS. The metabolic activity of the cells was measured 24 h later by using the MTT assay. The data are shown as the average of triplicates.

[0063] FIG. 29A is a plot showing that PLL stimulates cellular uptake of siRNA. Vero cells in 24-well plates were incubated with Lipofectamine+siRNA (400 pmol) or with siRNA (400 pmol)/polymer complexes for 6 hrs. The cells were then washed and infected with PR8 virus at a MOI of 0.04. Virus titers in the culture supernatants at different time points after infection were measured by HA assay. Polymer to siRNA ratios are indicated. Open circles=no treatment; Filled squares=Lipofectamine; Filled triangles=PLL (MW˜42K); Open triangles=PLL (MW˜8K).

[0064] FIG. 29B is a plot showing that poly-L-arginine stimulates cellular uptake of siRNA. Vero cells in 24-well plates were incubated with siRNA (400 pmol)/polymer complexes for 6 hrs. The cells were then washed and infected with PR8 virus at a MOI of 0.04. Virus titers in the culture supernatants at different time points after infection were measured by HA assay. Polymer to siRNA ratios are indicated. 0, 25, 50, 75, and 95% refer to percentage of ε-amino groups on PLL substituted with imidazole acetyl groups. Closed circles=no transfection; Open circles=Lipofectamine; Open and filled squares=0% and 25% (Note that the data points for 0% and 25% are identical); Filled triangles=50%; Open triangles=75%; X=95%.

ABBREVIATIONS

[0065] DNA: deoxyribonucleic acid

[0066] RNA: ribonucleic acid

[0067] vRNA: virion RNA in the influenza virus genome, negative strand

[0068] cRNA: complementary RNA, a direct transcript of vRNA, positive strand

[0069] mRNA: messenger RNA transcribed from vRNA or cellular genes, a template for protein synthesis

[0070] dsRNA: double-stranded RNA

[0071] siRNA: short interfering RNA

[0072] shRNA: short hairpin RNA

[0073] RNAi: RNA interference

Definitions

[0074] In general, the term antibody refers to an immunoglobulin, whether natural or wholly or partially synthetically produced. In certain embodiments of the invention the term also encompasses any protein comprising a immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. The antibody may be a fragment of an antibody such as an Fab′, F(ab′) ₂ , scFv (single-chain variable) or other fragment that retains an antigen binding site, or a recombinantly produced scFv fragment, including recombinantly produced fragments. See, e.g., Allen, T., Nature Reviews Cancer , Vol.2, 750-765, 2002, and references therein. In certain embodiments of the invention the term includes “humanized” antibodies in which for example, a variable domain of rodent origin is fused to a constant domain of human origin, thus retaining the specificity of the rodent antibody. It is noted that the domain of human origin need not originate directly from a human in the sense that it is first synthesized in a human being. Instead, “human” domains may be generated in rodents whose genome incorporates human immunoglobulin genes. See, e.g., Vaughan, et al., (1998), Nature Biotechnology , 16: 535-539. An antibody may be polyclonal or monoclonal, though for purposes of the present invention monoclonal antibodies are generally preferred.

[0075] As used herein, the terms approximately or about in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.

[0076] The term hybridize, as used herein, refers to the interaction between two complementary nucleic acid sequences. The phrase hybridizes under high stringency conditions describes an interaction that is sufficiently stable that it is maintained under art-recognized high stringency conditions. Guidance for performing hybridization reactions can be found, for example, in Current Protocols in Molecular Biology , John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1989, and more recent updated editions, all of which are incorporated by reference. See also Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual , 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. Aqueous and nonaqueous methods are described in that reference and either can be used. Typically, for nucleic acid sequences over approximately 50-100 nucleotides in length, various levels of stringency are defined, such as low stringency (e.g., 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for medium-low stringency conditions)); medium stringency (e.g., 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; high stringency hybridization (e.g., 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and very high stringency hybridization conditions (e.g., 0.5M sodium phosphate, 0.1% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.) Hybridization under high stringency conditions only occurs between sequences with a very high degree of complementarity. One of ordinary skill in the art will recognize that the parameters for different degrees of stringency will generally differ based upon various factors such as the length of the hybridizing sequences, whether they contain RNA or DNA, etc. For example, appropriate temperatures for high, medium, or low stringency hybridization will generally be lower for shorter sequences such as oligonucleotides than for longer sequences.

[0077] The term influenza virus is used here to refer to any strain of influenza virus that is capable of causing disease in an animal or human subject, or that is an interesting candidate for experimental analysis. Influenza viruses are described in Fields, B., et al., Fields' Virology , 4 ^th ed., Philadelphia: Lippincott Williams and Wilkins; ISBN: 0781718325, 2001. In particular, the term encompasses any strain of influenza A virus that is capable of causing disease in an animal or human subject, or that is an interesting candidate for experimental analysis. A large number of influenza A isolates have been partially or completely sequenced. Appendix A presents merely a partial list of complete sequences for influenza A genome segments that have been deposited in a public database (The Influenza Sequence Database (ISD), see Macken, C., Lu, H., Goodman, J., & Boykin, L., “The value of a database in surveillance and vaccine selection.” in Options for the Control of Influenza IV . A. D. M. E. Osterhaus, N. Cox & A. W. Hampson (Eds.) Amsterdam: Elsevier Science, 2001, 103-106). This database also contains complete sequences for influenza B and C genome segments. The database is available on the World Wide Web at the Web site having URL http://www.flu.lanl.gov/ along with a convenient search engine that allows the user to search by genome segment, by species infected by the virus, and by year of isolation. Influenza sequences are also available on Genbank. Sequences of influenza genes are therefore readily available to, or determinable by, those of ordinary skill in the art.

[0078] Isolated, as used herein, means 1) separated from at least some of the components with which it is usually associated in nature; 2) prepared or purified by a process that involves the hand of man; and/or 3) not occurring in nature.

[0079] Ligand, as used herein, means a molecule that specifically binds to a second molecule, typically a polypeptide or portion thereof, such as a carbohydrate moiety, through a mechanism other than an antigen-antibody interaction. The term encompasses, for example, polypeptides, peptides, and small molecules, either naturally occurring or synthesized, including molecules whose structure has been invented by man. Although the term is frequently used in the context of receptors and molecules with which they interact and that typically modulate their activity (e.g., agonists or antagonists), the term as used herein applies more generally.

[0080] Operably linked, as used herein, refers to a relationship between two nucleic acid sequences wherein the expression of one of the nucleic acid sequences is controlled by, regulated by, modulated by, etc., the other nucleic acid sequence. For example, the transcription of a nucleic acid sequence is directed by an operably linked promoter sequence; post-transcriptional processing of a nucleic acid is directed by an operably linked processing sequence; the translation of a nucleic acid sequence is directed by an operably linked translational regulatory sequence; the transport or localization of a nucleic acid or polypeptide is directed by an operably linked transport or localization sequence; and the post-translational processing of a polypeptide is directed by an operably linked processing sequence. Preferably a nucleic acid sequence that is operably linked to a second nucleic acid sequence is covalently linked, either directly or indirectly, to such a sequence, although any effective three-dimensional association is acceptable.

[0081] Purified, as used herein, means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure, where it is pure when it is removed from substantially all other compounds or entities, i.e., is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.

[0082] The term regulatory sequence is used herein to describe a region of nucleic acid sequence that directs, enhances, or inhibits the expression (particularly transcription, but in some cases other events such as splicing or other processing) of sequence(s) with which it is operatively linked. The term includes promoters, enhancers and other transcriptional control elements. In some embodiments of the invention, regulatory sequences may direct constitutive expression of a nucleotide sequence; in other embodiments, regulatory sequences may direct tissue-specific and/or inducible expression. For instance, non-limiting examples of tissue-specific promoters appropriate for use in mammalian cells include lymphoid-specific promoters (see, for example, Calame et al., Adv. Immunol . 43:235, 1988) such as promoters of T cell receptors (see, e.g., Winoto et al., EMBO J . 8:729, 1989) and immunoglobulins (see, for example, Banerji et al., Cell 33:729, 1983; Queen et al., Cell 33:741, 1983), and neuron-specific promoters (e.g., the neurofilament promoter; Byrne et al., Proc. Natl. Acad. Sci. USA 86:5473, 1989). Developmentally-regulated promoters are also encompassed, including, for example, the murine hox promoters (Kessel et al., Science 249:374, 1990) and the α-fetoprotein promoter (Campes et al., Genes Dev . 3:537, 1989). In some embodiments of the invention regulatory sequences may direct expression of a nucleotide sequence only in cells that have been infected with an infectious agent. For example, the regulatory sequence may comprise a promoter and/or enhancer such as a virus-specific promoter or enhancer that is recognized by a viral protein, e.g., a viral polymerase, transcription factor, etc. Alternately, the regulatory sequence may comprise a promoter and/or enhancer that is active in epithelial cells in the nasal passages, respiratory tract and/or the lungs.

[0083] As used herein, the term RNAi-inducing entity encompasses RNA molecules and vectors (other than naturally occurring molecules not modified by the hand of man) whose presence within a cell results in RNAi and leads to reduced expression of a transcript to which the RNAi-inducing entity is targeted. The term specifically includes siRNA, shRNA, and RNAi-inducing vectors.

[0084] As used herein, an RNAi-inducing vector is a vector whose presence within a cell results in transcription of one or more RNAs that self-hybridize or hybridize to each other to form an shRNA or siRNA. In various embodiments of the invention this term encompasses plasmids, e.g., DNA vectors (whose sequence may comprise sequence elements derived from a virus), or viruses, (other than naturally occurring viruses or plasmids that have not been modified by the hand of man), whose presence within a cell results in production of one or more RNAs that self-hybridize or hybridize to each other to form an shRNA or siRNA. In general, the vector comprises a nucleic acid operably linked to expression signal(s) so that one or more RNA molecules that hybridize or self-hybridize to form an siRNA or shRNA are transcribed when the vector is present within a cell. Thus the vector provides a template for intracellular synthesis of the RNA or RNAs or precursors thereof. For purposes of inducing RNAi, presence of a viral genome into a cell (e.g., following fusion of the viral envelope with the cell membrane) is considered sufficient to constitute presence of the virus within the cell. In addition, for purposes of inducing RNAi, a vector is considered to be present within a cell if it is introduced into the cell, enters the cell, or is inherited from a parental cell, regardless of whether it is subsequently modified or processed within the cell. An RNAi-inducing vector is considered to be targeted to a transcript if presence of the vector within a cell results in production of one or more RNAs that hybridize to each other or self-hybridize to form an siRNA or shRNA that is targeted. to the transcript, i.e., if presence of the vector within a cell results in production of one or more siRNAs or shRNAs targeted to the transcript.

[0085] A short, interfering RNA (siRNA) comprises an RNA duplex that is approximately 19 basepairs long and optionally further comprises one or two single-stranded overhangs. An siRNA may be formed from two RNA molecules that hybridize together, or may alternatively be generated from a single RNA molecule that includes a self-hybridizing portion. It is generally preferred that free 5′ ends of siRNA molecules have phosphate groups, and free 3′ ends have hydroxyl groups. The duplex portion of an siRNA may, but typically does not, contain one or more bulges consisting of one or more unpaired nucleotides. One strand of an siRNA includes a portion that hybridizes with a target transcript. In certain preferred embodiments of the invention, one strand of the siRNA is precisely complementary with a region of the target transcript, meaning that the siRNA hybridizes to the target transcript without a single mismatch. In other embodiments of the invention one or more mismatches between the siRNA and the targeted portion of the target transcript may exist. In most embodiments of the invention in which perfect complementarity is not achieved, it is generally preferred that any mismatches be located at or near the siRNA termini.

[0086] The term short hairpin RNA refers to an RNA molecule comprising at least two complementary portions hybridized or capable of hybridizing to form a double-stranded (duplex) structure sufficiently long to mediate RNAi (typically at least 19 base pairs in length), and at least one single-stranded portion, typically between approximately 1 and 10 nucleotides in length that forms a loop. The duplex portion may, but typically does not, contain one or more bulges consisting of one or more unpaired nucleotides. As described further below, shRNAs are thought to be processed into siRNAs by the conserved cellular RNAi machinery. Thus shRNAs are precursors of siRNAs and are, in general, similarly capable of inhibiting expression of a target transcript.

[0087] As used herein, the term specific binding refers to an interaction between a target polypeptide (or, more generally, a target molecule) and a binding molecule such as an antibody, ligand, agonist, or antagonist. The interaction is typically dependent upon the presence of a particular structural feature of the target polypeptide such as an antigenic determinant or epitope recognized by the binding molecule. For example, if an antibody is specific for epitope A, the presence of a polypeptide containing epitope A or the presence of free unlabeled A in a reaction containing both free labeled A and the antibody thereto, will reduce the amount of labeled A that binds to the antibody. It is to be understood that specificity need not be absolute but generally refers to the context in which the binding is performed. For example, it is well known in the art that numerous antibodies cross-react with other epitopes in addition to those present in the target molecule. Such cross-reactivity may be acceptable depending upon the application for which the antibody is to be used. One of ordinary skill in the art will be able to select antibodies having a sufficient degree of specificity to perform appropriately in any given application (e.g., for detection of a target molecule, for therapeutic purposes, etc). It is also to be understood that specificity may be evaluated in the context of additional factors such as the affinity of the binding molecule for the target polypeptide versus the affinity of the binding molecule for other targets, e.g., competitors. If a binding molecule exhibits a high affinity for a target molecule that it is desired to detect and low affinity for nontarget molecules, the antibody will likely be an acceptable reagent for immunodiagnostic purposes. Once the specificity of a binding molecule is established in one or more contexts, it may be employed in other, preferably similar, contexts without necessarily re-evaluating its specificity.

[0088] The term subject, as used herein, refers to an individual susceptible to infection with an infectious agent, e.g., an individual susceptible to infection with a virus such as the influenza virus. The term includes birds and animals, e.g., domesticated birds and animals (such as chickens, mammals, including swine, horse, dogs, cats, etc.), and wild animals, non-human primates, and humans.

[0089] An siRNA or shRNA or an siRNA or shRNA sequence is considered to be targeted to a target transcript for the purposes described herein if 1) the stability of the target transcript is reduced in the presence of the siRNA or shRNA as compared with its absence; and/or 2) the'siRNA or shRNA shows at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% precise sequence complementarity with the target transcript for a stretch of at least about 15, more preferably at least about 17, yet more preferably at least about 18 or 19 to about 21-23 nucleotides; and/or 3) one strand of the siRNA or one of the self-complementary portions of the shRNA hybridizes to the target transcript under stringent conditions for hybridization of small (<50 nucleotide) RNA molecules in vitro and/or under conditions typically found within the cytoplasm or nucleus of mammalian cells. An RNA-inducing vector whose presence within a cell results in production of an siRNA or shRNA that is targeted to a transcript is also considered to be targeted to the target transcript. Since the effect of targeting a transcript is to reduce or inhibit expression of the gene that directs synthesis of the transcript, an siRNA or shRNA targeted to a transcript is also considered to target the gene that directs synthesis of the transcript even though the gene itself (i.e., genomic DNA) is not thought to interact with the siRNA, shRNA, or components of the cellular silencing machinery. Thus as used herein, an siRNA, shRNA, or RNAi-inducing vector that targets a transcript is understood to target the gene that provides a template for synthesis of the transcript.

[0090] As used herein, treating includes reversing, alleviating, inhibiting the progress of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition.

[0091] In general, the term vector refers to a nucleic acid molecule capable of mediating entry of, e.g., transferring, transporting, etc., a second nucleic acid molecule into a cell. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (typically DNA molecules although RNA plasmids are also known), cosmids, and viral vectors. As is well known in the art, the term viral vector may refer either to a nucleic acid molecule (e.g., a plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer or integration of the nucleic acid molecule (examples include retroviral or lentiviral vectors) or to a virus or viral particle that mediates nucleic acid transfer (examples include retroviruses or lentiviruses). As will be evident to one of ordinary skill in the art, viral vectors may include various viral components in addition to nucleic acid(s).

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

[0092] I. Influenza Viral Life Cycle and Characteristics

[0093] Influenza viruses are enveloped, negative-stranded RNA viruses of the Orthomyxoviridae family. They are classified as influenza types A, B, and C, of which influenza A is the most pathogenic and is believed to be the only type able to undergo reassortment with animal strains. Influenza types A, B, and C can be distinguished by differences in their nucleoprotein and matrix proteins (see FIG. 1 ). As discussed further below, influenza A subtypes are defined by variation in their hemagglutinin (HA) and neuraminidase (NA) genes and usually distinguished by antibodies that bind to the corresponding proteins.

[0094] The influenza A viral genome consists of ten genes distributed in eight RNA segments. The genes encode 10 proteins: the envelope glycoproteins hemagglutinin (HA) and neuraminidase