Claims
- 1. An immunostimulatory composition, comprising:
an isolated RNA oligomer 5-40 nucleotides long having a base sequence comprising at least one guanine (G) and at least one uracil (U), and optionally a cationic lipid.
- 2. The composition of claim 1, wherein the isolated RNA oligomer is a G,U-rich RNA.
- 3. The composition of claim 1, wherein the base sequence comprises 5′-RURGY-3′, wherein R represents purine, U represents uracil, G represents guanine, and Y represents pyrimidine.
- 4. The composition of claim 1, wherein the base sequence comprises 5′-GUAGU-3′, wherein A represents adenine.
- 5. The composition of claim 1, wherein the base sequence comprises 5′-GUAGUGU-3′.
- 6. The composition of claim 1, wherein the base sequence comprises 5′-GUUGB-3′, wherein B represents U, G, or C, wherein C represents cytosine.
- 7. The composition of claim 1, wherein the base sequence comprises 5′-GUGUG-3′.
- 8. The composition of claim 1, wherein the base sequence comprises 5′-GUGUUUAC-3′.
- 9. The composition of claim 1, wherein the base sequence comprises 5′-GUAGGCAC-3′.
- 10. The composition of claim 1, wherein the base sequence comprises 5′-CUAGGCAC-3′.
- 11. The composition of claim 1, wherein the base sequence comprises 5′-CUCGGCAC-3′.
- 12. The composition of claim 1, wherein the oligomer is 5-12 nucleotides long.
- 13. The composition of claim 1, wherein the base sequence is free of CpG dinucleotide.
- 14. The composition of claim 1, wherein the base sequence is at least 50 percent self-complementary.
- 15. The composition of claim 1, wherein the oligomer is a plurality of oligomers.
- 16. The composition of claim 15, wherein the plurality of oligomers comprises an oligomer having a first base sequence and an oligomer having a second base sequence, wherein the first base sequence and the second base sequence are at least 50 percent complementary.
- 17. The composition of claim 15, wherein the plurality of oligomers comprises an oligomer having a base sequence comprising 5′-GUGUUUAC-3′ and an oligomer having a base sequence comprising 5′-GUAGGCAC-3′.
- 18. The composition of claim 1, wherein the oligomer comprises a non-natural backbone linkage.
- 19. The composition of claim 18, wherein the non-natural backbone linkage is a phosphorothiate linkage.
- 20. The composition of claim 1, wherein the oligomer comprises a modified base selected from the group consisting of 7-deazaguanosine, 8-azaguanosine, 5-methyluracil, and pseudouracil.
- 21. The composition of claim 1, wherein the oligomer comprises a modified sugar.
- 22. The composition of claim 1, wherein the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP).
- 23. The composition of claim 1, further comprising an antigen.
- 24. The composition of claim 23, wherein the antigen is an allergen.
- 25. The composition of claim 23, wherein the antigen is a cancer antigen.
- 26. The composition of claim 23, wherein the antigen is a microbial antigen.
- 27. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
- 28. A method of activating an immune cell, comprising:
contacting an immune cell with the composition of any one of claims 1-27 in an effective amount to induce activation of the immune cell.
- 29. The method of claim 28, wherein the activation of the immune cell comprises secretion of a cytokine or chemokine by the immune cell.
- 30. The method of claim 29, wherein the cytokine is selected from the group consisting of interleukin 6 (IL-6), interleukin 12 (IL-12), an interferon (IFN), and tumor necrosis factor (TNF).
- 31. The method of claim 29, wherein the chemokine is interferon-gamma-inducible protein 10 (IP-10).
- 32. The method of claim 28, wherein the activation of the immune cell comprises activation of a MyD88-dependent signal transduction pathway.
- 33. The method of claim 32, wherein the MyD88-dependent signal transduction pathway is associated with a Toll-like receptor (TLR).
- 34. The method of claim 33, wherein the TLR is TLR8.
- 35. The method of claim 33, wherein the TLR is TLR7.
- 36. A method of inducing an immune response in a subject, comprising:
administering to a subject a composition of any one of claims 1-27 in an effective amount to induce an immune response in the subject.
- 37. The method of claim 36, wherein the subject has or is at risk of having a cancer.
- 38. The method of claim 36, wherein the subject has or is at risk of having an infection with an agent selected from the group consisting of viruses, bacteria, fungi, and parasites.
- 39. The method of claim 36, wherein the subject has or is at risk of having a viral infection.
- 40. A method of inducing an immune response in a subject, comprising:
administering to a subject an antigen; and administering to the subject a composition of any one of claims 1-22 or 27 in an effective amount to induce an immune response to the antigen.
- 41. The method of claim 40, wherein the antigen is an allergen.
- 42. The method of claim 40, wherein the antigen is a cancer antigen.
- 43. The method of claim 40, wherein the antigen is a microbial antigen.
- 44. A method of inducing an immune response in a subject, comprising:
isolating dendritic cells of a subject; contacting the dendritic cells ex vivo with the composition of any one of claims 1-22 or 27; contacting the dendritic cells ex vivo with an antigen; and administering the contacted dendritic cells to the subject.
- 45. The method of claim 44, wherein the antigen is an allergen.
- 46. The method of claim 44, wherein the antigen is a cancer antigen.
- 47. The method of claim 44, wherein the antigen is a microbial antigen.
- 48. A composition, comprising:
an effective amount of a ligand for Toll-like receptor 8 (TLR8) to induce TLR8 signaling, and an effective amount of a ligand for a second TLR selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9 and TLR10 to induce signaling by the second TLR.
- 49. The composition according to claim 48, wherein the second TLR is TLR3.
- 50. The composition according to claim 48, wherein the second TLR is TLR7.
- 51. The composition according to claim 48, wherein the second TLR is TLR9.
- 52. The composition according to claim 48, wherein the ligand for TLR8 and the ligand for the second TLR are linked.
- 53. The composition according to claim 48, further comprising a pharmaceutically acceptable carrier.
- 54. A composition, comprising:
an effective amount of a ligand for TLR7 to induce TLR7 signaling, and an effective amount of a ligand for a second TLR selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR8, TLR9, and TLR10 to induce signaling by the second TLR.
- 55. The composition according to claim 54, wherein the second TLR is TLR3.
- 56. The composition according to claim 54, wherein the second TLR is TLR8.
- 57. The composition according to claim 54, wherein the second TLR is TLR9.
- 58. The composition according to claim 54, wherein the ligand for TLR7 and the ligand for the second TLR are linked.
- 59. The composition according to claim 54, further comprising a pharmaceutically acceptable carrier.
- 60. A composition, comprising:
a DNA:RNA conjugate, wherein DNA of the conjugate comprises a CpG motif effective for stimulating TLR9 signaling and RNA of the conjugate comprises RNA effective for stimulating signaling by TLR3, TLR7, TLR8, or any combination thereof.
- 61. The composition according to claim 60, wherein the conjugate comprises a chimeric DNA:RNA backbone.
- 62. The composition according to claim 61, wherein the chimeric backbone comprises a cleavage site between the DNA and the RNA.
- 63. The composition according to claim 60, wherein the conjugate comprises a double-stranded DNA:RNA heteroduplex.
- 64. The composition according to claim 60, further comprising a pharmaceutically acceptable carrier.
- 65. A method for stimulating TLR8 signaling, comprising:
contacting TLR8 with an isolated RNA in an effective amount to stimulate TLR8 signaling.
- 66. The method according to claim 65, wherein the RNA is double-stranded RNA.
- 67. The method according to claim 65, wherein the RNA is ribosomal RNA.
- 68. The method according to claim 65, wherein the RNA is transfer RNA.
- 69. The method according to claim 65, wherein the RNA is messenger RNA.
- 70. The method according to claim 65, wherein the RNA is viral RNA.
- 71. The method according to claim 65, wherein the RNA is G,U-rich RNA.
- 72. The method according to claim 65, wherein the RNA consists essentially of G and U.
- 73. A method for stimulating TLR8 signaling, comprising:
contacting TLR8 with a mixture of nucleosides consisting essentially of G and U in a ratio between 1G:50U and 10G:1U, in an amount effective to stimulate TLR8 signaling.
- 74. The method according to claim 73, wherein the nucleosides are ribonucleosides.
- 75. The method according to claim 73, wherein the nucleosides comprise a mixture of ribonucleosides and deoxyribonucleosides.
- 76. The method according to claim 73, wherein the G is a guanosine derivative selected from the group consisting of: 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, 7-allyl-8-oxoguanosine, guanosine ribonucleoside vanadyl complex, inosine, and nebularine.
- 77. A method for stimulating TLR8 signaling, comprising:
contacting TLR8 with a mixture of ribonucleoside vanadyl complexes.
- 78. The method according to claim 77, wherein the mixture comprises guanosine ribonucleoside vanadyl complexes.
- 79. A method for stimulating TLR8 signaling, comprising:
contacting TLR8 with an isolated G,U-rich oligonucleotide comprising a sequence selected from the group consisting of: UUGUGG, UGGUUG, GUGUGU, and GGGUUU, in an amount effective to stimulate TLR8 signaling.
- 80. The method according to claim 79, wherein the oligonucleotide is an oligoribonucleotide.
- 81. The method according to claim 79, wherein the oligonucleotide is 7-50 bases long.
- 82. The method according to claim 79, wherein the oligonucleotide is 12-24 bases long.
- 83. The method according to claim 79, wherein the oligonucleotide has a sequence 5′-GUUGUGGUUGUGGUUGUG-3′ (SEQ ID NO:1).
- 84. A method for stimulating TLR8 signaling, comprising:
contacting TLR8 with an at least partially double-stranded nucleic acid molecule comprising at least one G-U base pair, in an amount effective to stimulate TLR8 signaling.
- 85. A method for supplementing a TLR8-mediated immune response, comprising:
contacting TLR8 with an effective amount of a TLR8 ligand to induce a TLR8-mediated immune response; and contacting a TLR other than TLR8 with an effective amount of a ligand for the TLR other than TLR8 to induce an immune response mediated by the TLR other than TLR8.
- 86. A method for supplementing a TLR8-mediated immune response in a subject, comprising:
administering to a subject in need of an immune response an effective amount of a TLR8 ligand to induce a TLR8-mediated immune response; and administering to the subject an effective amount of a ligand for a TLR other than TLR8 to induce an immune response mediated by the TLR other than TLR8.
- 87. The method according to claim 86, wherein the TLR other than TLR8 is TLR9.
- 88. The method according to claim 87, wherein the ligand for TLR9 is a CpG nucleic acid.
- 89. The method according to claim 88, wherein the CpG nucleic acid has a stabilized backbone.
- 90. The method according to claim 87, wherein the ligand for TLR8 and the ligand for TLR9 are a conjugate.
- 91. The method according to claim 90, wherein the conjugate comprises a double-stranded DNA:RNA heteroduplex.
- 92. The method according to claim 90, wherein the conjugate comprises a chimeric DNA:RNA backbone.
- 93. The method according to claim 92, wherein the chimeric backbone comprises a cleavage site between the DNA and the RNA.
- 94. A method for stimulating TLR7 signaling, comprising:
contacting TLR7 with an isolated guanosine ribonucleoside in an effective amount to stimulate TLR7 signaling.
- 95. The method according to claim 94, wherein the guanosine ribonucleoside is a guanosine ribonucleoside derivative selected from the group consisting of: 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine, 7-allyl-8-oxoguanosine, guanosine ribonucleoside vanadyl complex, inosine, and nebularine.
- 96. The method according to claim 95, wherein the guanosine ribonucleoside derivative is 8-oxoguanosine.
- 97. The method according to claim 94, wherein the guanosine nucleoside is a ribonucleoside.
- 98. The method according to claim 97, wherein the guanosine nucleoside comprises a mixture of ribonucleosides and deoxyribonucleosides.
- 99. A method for stimulating TLR7 signaling, comprising:
contacting TLR7 with an isolated nucleic acid comprising a terminal oxidized or halogenized guanosine in an effective amount to stimulate TLR7 signaling.
- 100. The method according to claim 99, wherein the oxidized or halogenized guanosine is 8-oxoguanosine
- 101. A method for stimulating TLR7 signaling, comprising:
contacting TLR7 with an isolated RNA in an effective amount to stimulate TLR7 signaling.
- 102. The method according to claim 101, wherein the RNA is double-stranded RNA.
- 103. The method according to claim 101, wherein the RNA is ribosomal RNA.
- 104. The method according to claim 101, wherein the RNA is transfer RNA.
- 105. The method according to claim 101, wherein the RNA is messenger RNA.
- 106. The method according to claim 101, wherein the RNA is viral RNA.
- 107. The method according to claim 101, wherein the RNA is G-rich RNA.
- 108. The method according to claim 107, further wherein the RNA is part of a DNA:RNA heteroduplex.
- 109. The method according to claim 107, wherein the RNA consists essentially of guanosine ribonucleoside.
- 110. A method for stimulating TLR7 signaling, comprising:
contacting TLR7 with a mixture of ribonucleoside vanadyl complexes.
- 111. The method according to claim 110, wherein the mixture comprises guanosine ribonucleoside vanadyl complexes.
- 112. A method for supplementing a TLR7-mediated immune response, comprising:
contacting TLR7 with an effective amount of a TLR7 ligand to induce a TLR7-mediated immune response; and contacting a TLR other than TLR7 with an effective amount of a ligand for the TLR other than TLR7 to induce an immune response mediated by the TLR other than TLR7.
- 113. A method for supplementing a TLR7-mediated immune response in a subject, comprising:
administering to a subject in need of an immune response an effective amount of a TLR7 ligand to induce a TLR7-mediated immune response; and administering to the subject an effective amount of a ligand for a TLR other than TLR7 to induce an immune response mediated by the TLR other than TLR7.
- 114. The method according to claim 113, wherein the TLR other than TLR7 is TLR9.
- 115. The method according to claim 114, wherein the ligand for TLR9 is a CpG nucleic acid.
- 116. The method according to claim 115, wherein the CpG nucleic acid has a stabilized backbone.
- 117. The method according to claim 114, wherein the ligand for TLR7 and the ligand for TLR9 are a conjugate.
- 118. The method according to claim 117, wherein the conjugate comprises a double-stranded DNA:RNA heteroduplex.
- 119. The method according to claim 117, wherein the conjugate comprises a chimeric DNA:RNA backbone.
- 120. The method according to claim 119, wherein the chimeric backbone comprises a cleavage site between the DNA and the RNA.
- 121. A method for screening candidate immunostimulatory compounds, comprising:
measuring a TLR8-mediated reference signal in response to an RNA reference; measuring a TLR8-mediated test signal in response to a candidate immunostimulatory compound; and comparing the TLR8-mediated test signal to the TLR8-mediated reference signal.
- 122. A method for screening candidate immunostimulatory compounds, comprising:
measuring a TLR8-mediated reference signal in response to an imidazoquinoline reference; measuring a TLR8-mediated test signal in response to a candidate immunostimulatory compound; and comparing the TLR8-mediated test signal to the TLR8-mediated reference signal.
- 123. The method according to claim 122, wherein the imidazoquinoline is resiquimod (R-848).
- 124. The method according to claim 122, wherein the imidazoquinoline is imiquimod (R-837).
- 125. A method for screening candidate immunostimulatory compounds, comprising:
measuring a TLR7-mediated reference signal in response to a 7-allyl-8-oxoguanosine reference; measuring a TLR7-mediated test signal in response to a candidate immunostimulatory compound; and comparing the TLR7-mediated test signal to the TLR7-mediated reference signal.
- 126. A method for screening candidate immunostimulatory compounds, comprising:
measuring a TLR7-mediated reference signal in response to an imidazoquinoline reference; measuring a TLR7-mediated test signal in response to a candidate immunostimulatory compound; and comparing the TLR7-mediated test signal to the TLR7-mediated reference signal.
- 127. The method according to claim 126, wherein the imidazoquinoline is resiquimod (R-848).
- 128. The method according to claim 126, wherein the imidazoquinoline is imiquimod (R-837).
RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. 60/421,966, filed Oct. 29, 2002, and U.S. 60/370,515, filed Apr. 4, 2002, the contents of which are incorporated herein by reference in their entirety.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60370515 |
Apr 2002 |
US |
|
60421966 |
Oct 2002 |
US |