Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/17—Immunomodulatory nucleic acids
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/321—2'-O-R Modification
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/322—2'-R Modification
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/50—Physical structure
  • C12N2310/53—Physical structure partially self-complementary or closed
  • C12N2310/531—Stem-loop; Hairpin

Definitions

• an immunomodulatory composition that selectively activates TLR7, comprising an RNA ligand and a pharmaceutically acceptable carrier, wherein the RNA ligand comprises: (a) a TLR7-selective motif comprising a nucleotide sequence selected from SEQ ID NOs: 44, 45, 46, or 47, (b) a first nucleotide sequence linked to the 5’ end of the TLR7-selective motif, and (c) a second nucleotide sequence linked to the 3’ end of the TLR7-selective motif.
• the first nucleotide sequence comprises one nucleoside, or at least two nucleosides linked by phosphorothioate linkages, wherein the first nucleotide sequence does not comprise a uridine nucleoside, and wherein the first nucleotide sequence is linked to the TLR7-selective motif by a phosphorothioate linkage.
• the first nucleotide sequence comprises eight nucleosides linked by phosphorothioate linkages.
• the first nucleotide sequence comprises the nucleotide sequence C*C*G*A*G*C*C*G (SEQ ID NO:
• the second nucleotide sequence does not comprise a uridine nucleoside, wherein the second nucleotide sequence comprises the nucleotide sequence G-G-G (SEQ ID NO: 69), wherein the nucleotide sequence G-G-G (SEQ ID NO: 69) is capable of base pairing with the TLR7-selective motif, and wherein the second nucleotide sequence is linked to the TLR7- selective motif by a phosphodiester linkage. In some embodiments, the second nucleotide sequence comprises about 15 nucleosides.
• the first nucleotide sequence comprises one nucleoside, or at least two nucleosides linked by phosphodiester linkages, wherein the first nucleotide sequence does not comprise a uridine nucleoside, and wherein the first nucleotide sequence is linked to the first TLR7- selective motif by a phosphodiester linkage.
• the RNA ligand comprises the nucleotide sequence of SEQ ID NO: 33, or a nucleotide sequence having at least about 85% identity to SEQ ID NO: 33 and comprising the first TLR7-selective motif of SEQ ID NO: 36 and/or the second TLR7-selective motif of SEQ ID NO: 36.
• the one or more additional TLR7-selective motifs comprise one, two, three, four, five, or more additional TLR7-selective motifs comprising Formula V. In some embodiments, the one or more additional TLR7-selective motifs comprise five additional TLR7-selective motifs comprising Formula V. In some embodiments, Ui of the first TLR7-selective motif has a 2 ⁇ - methyl modification (mU). In some embodiments, Ui of at least one of the one or more additional TLR7-selective motifs, or Ui of all of the one or more additional TLR7-selective motifs, has a 2’0-methyl modification (mU).
• the first TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 41. In some embodiments, at least one of the one or more additional TLR7-selective motifs, or all of the one or more additional TLR7-selective motifs comprise the nucleotide sequence of SEQ ID NO: 41.
• an immunomodulatory composition that selectively activates TLR7, comprising an RNA ligand and a pharmaceutically acceptable carrier, wherein the RNA ligand comprises: (a) a nucleotide sequence of SEQ ID NOs: 29 or 30; (b) a nucleotide sequence having at least about 85% identity to SEQ ID NO: 29 and comprising six TLR7- selective motifs of SEQ ID NO: 40; or (c) a nucleotide sequence having at least about 85% identity to SEQ ID NO: 30 and comprising six TLR7-selective motifs of SEQ ID NO: 41.
• an immunomodulatory composition that selectively activates TLR7, comprising an RNA ligand and a pharmaceutically acceptable carrier, wherein the RNA ligand comprises: (a) a TLR7-selective motif comprising Formula VI: Ci-U2-U3*U4- U5*C6 (Formula VI), wherein Ci and Ceare cytidine nucleosides, and U2, U3, U4, and Us are uridine nucleosides, wherein U2 has a 2’0-methyl modification (mU), a 2’-fluoro modification (fU), a 2 ’-amino modification, a 2’-deoxy modification, or a 2 ’-methoxy ethoxy modification, wherein Uihas a 2’0-methyl modification (mU), a 2’-fluoro modification (fU), a 2’-amino modification, a 2’-deoxy modification, or a 2’ -methoxy ethoxy modification, wherein Uihas a 2’
• U2 of the TLR7-selective motif has a 2’0-methyl modification (mU). In some embodiments, U4 of the TLR7-selective motif has a 2’0-methyl modification (mU). In some embodiments, U2 of the TLR7-selective motif has a 2’-fluoro modification (fU). In some embodiments, U4 of the TLR7- selective motif has a 2’-fluoro modification (fU). In some embodiments, U2 and U4 of the TLR7- selective motif have a 2’0-methyl modification (mU). In some embodiments, U2 and U4 of the TLR7-selective motif have a 2’-fluoro modification (fU).
• an immunomodulatory composition that selectively activates TLR7, comprising an RNA ligand and a pharmaceutically acceptable carrier, wherein the RNA ligand comprises: (a) a nucleotide sequence selected from SEQ ID NOs: 17, 18, 19, or 20; (b) a nucleotide sequence having at least about 85% identity to SEQ ID NO: 17 and comprising the TLR7-selective motif of SEQ ID NO: 48; (c) a nucleotide sequence having at least about 85% identity to SEQ ID NO: 18 and comprising the TLR7-selective motif of SEQ ID NO: 49; (d) a nucleotide sequence having at least about 85% identity to SEQ ID NO: 19 and comprising the TLR7-selective motif of SEQ ID NO: 50; or (e) a nucleotide sequence having at least about 85% identity to SEQ ID NO: 20 and comprising the TLR7-selective motif of
• the pharmaceutically acceptable carrier is a lipid nanoparticle (LNP).
• the LNP comprises a cationic lipid.
• the LNP comprises an ionizable lipid.
• the immunomodulatory composition increases secretion of IFN-a by one or more cells contacted with the immunomodulatory composition, as compared to corresponding one or more cells that are not contacted with the immunomodulatory composition.
• the increase in TLR7 activity in the one or more cells contacted with the immunomodulatory composition is an increase of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more, as compared to corresponding one or more cells that are not contacted with the immunomodulatory composition.
• the increase in secretion of IFN-a by the one or more cells contacted with the immunomodulatory composition is an increase of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more, as compared to corresponding one or more cells that are not contacted with the immunomodulatory composition.
• nucleic acid comprising the RNA ligand of any of the immunomodulatory compositions described herein.
• the nucleic acid is a DNA, RNA or a DNA/RNA molecule.
• RNA ligand of any of the immunomodulatory compositions described herein or any of the nucleic acids described herein.
• a pharmaceutical composition comprising any of the immunomodulatory compositions described herein.
• a vaccine composition comprising any of the immunomodulatory compositions described herein or any of the pharmaceutical compositions described herein.
• a method for selectively activating TLR7 in one or more cells comprising contacting one or more cells with any of the immunomodulatory compositions described herein, or any of the pharmaceutical compositions described herein.
• the one or more cells express TLR7.
• the one or more cells comprise peripheral blood mononuclear cells (PBMCs).
• the one or more cells comprise one or more plasmacytoid dendritic cells.
• the increase in TLR7 activity in the one or more cells is an increase of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more, as compared to corresponding one or more cells that are not contacted with the immunomodulatory composition, or the pharmaceutical composition.
• the increase in secretion by the one or more cells of IFN-a is an increase of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more, as compared to corresponding one or more cells that are not contacted with the immunomodulatory composition, or the pharmaceutical composition.
• contacting the one or more cells with the immunomodulatory composition, or the pharmaceutical composition results in an increase in TLR8 activity in the one or more cells of less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%, as compared to corresponding one or more cells that are not contacted with the immunomodulatory composition, or the pharmaceutical composition.
• contacting the one or more cells with the immunomodulatory composition, or the pharmaceutical composition results in an increase in secretion by the one or more cells of TNF-a of less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%, as compared to corresponding one or more cells that are not contacted with the immunomodulatory composition, or the pharmaceutical composition.
• administering to the individual an effective amount of the immunomodulatory composition, the pharmaceutical composition, or the vaccine composition results in an increase in TLR8 activity in the individual of less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%, as compared to a corresponding individual not administered the immunomodulatory composition, the pharmaceutical composition, or the vaccine composition.
• administering to the individual an effective amount of the immunomodulatory composition, the pharmaceutical composition, or the vaccine composition results in an increase in levels of TNF-a in the individual of less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%, as compared to a corresponding individual not administered the immunomodulatory composition, the pharmaceutical composition, or the vaccine composition.
• a method for stimulating an immune response in an individual comprising administering to the individual an effective amount of any of the immunomodulatory compositions described herein, any of the pharmaceutical compositions described herein, or any of the vaccine compositions described herein.
• administering to the individual an effective amount of the immunomodulatory composition, the pharmaceutical composition, or the vaccine composition results in an increase in TLR7 activity in the individual, as compared to a corresponding individual not administered the immunomodulatory composition, the pharmaceutical composition, or the vaccine composition.
• FIG. 7 shows the results of experiments that measured the level of TNF-a or IFN-a secretion by PBMCs in response to the RNA ligands described in Example 1.
• Human PBMCs were stimulated with poly-L-arginine (p-L-arginine)-complexed RNA ligands (indicated on the x-axis), either alone or in combination with guanosine (as indicated by the legend; “noG” indicates that the PBMCs were stimulated with the p-L-arginine-complexed RNA ligands alone, and “G” indicates that the PBMCs were co-stimulated with the p-L-arginine-complexed RNA ligands and guanosine).
• the second nucleotide sequence is linked to the 3’ end of the TLR7-selective motif by a phosphodiester linkage or a phosphorothioate linkage. In some embodiments, the second nucleotide sequence does not comprise a uridine nucleoside.
• the first, second, and/or third nucleotide sequences are linked to adjacent TLR7-selective motifs by phosphodiester linkages or phosphorothioate linkages. In some embodiments, the first, second, and/or third nucleotide sequences do not comprise a uridine nucleoside.
• the RNA ligand further comprises additional nucleotide sequences that flank the 5’ end of the TLR7-selective motif, the 3’ end of the TLR7-selective motif, or both.
• the additional nucleotide sequences do not comprise a uridine nucleoside.
• the first nucleotide sequence is linked to the 5’ end of the TLR7-selective motif by a phosphodiester linkage or a phosphorothioate linkage. In some embodiments, the first nucleotide sequence is linked to the 5’ end of the TLR7-selective motif by a phosphorothioate linkage.
• nucleosides 100, or more nucleosides, or between any of 100 and 125 nucleosides, 125 and 150 nucleosides, 150 and 175 nucleosides,
• the second nucleotide sequence comprises 2 nucleosides.
• RNA ligand comprising a TLR7-selective motif comprising a nucleotide sequence of SEQ ID NOs: 44, 45, 46, and 47.
• the RNA ligand comprises a TLR7-selective motif comprising a nucleotide sequence of SEQ ID NO: 44.
• the RNA ligand comprises a TLR7-selective motif comprising a nucleotide sequence of SEQ ID NO: 45.
• the RNA ligand comprises a TLR7-selective motif comprising a nucleotide sequence of SEQ ID NO: 46.
• the RNA ligand comprises a TLR7-selective motif comprising a nucleotide sequence of SEQ ID NO: 47. In some embodiments, the RNA ligand further comprises additional nucleotide sequences that flank the 5’ end of the TLR7-selective motif, the 3’ end of the TLR7-selective motif, or both. In some embodiments, the additional nucleotide sequences do not comprise a uridine nucleoside. In some embodiments, the RNA ligand further comprises a first nucleotide sequence that is linked to the 5’ end of the TLR7-selective motif.
• the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 58, or a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 58.
• the second nucleotide sequence comprises 2 nucleosides.
• the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 59, or a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 59.
• RNA ligand comprising a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 12 and comprising the TLR7-selective motif of SEQ ID NO: 44.
• the RNA ligand comprises the nucleotide sequence of SEQ ID NO: 12.
• Ci and C3 are cytidine nucleosides and U2 is a uridine nucleoside, and wherein represents a phosphodiester linkage.
• Ci comprises a modification at the 2’ position of the ribose.
• the modification is any modification at the 2’ position of the ribose known in the art or described herein.
• the modification is any modification at the 2’ position of the ribose that inhibits or blocks RNase activity known in the art or described herein.
• the modification is a 2’0-methyl modification, a 2’-fluoro modification, a 2’-amino modification, a 2’-deoxy modification, or a 2’ -methoxy ethoxy modification.
• the modification is a 2’0-methyl modification.
• the modification is a 2’-fluoro modification.
• the RNA ligand further comprises a first nucleotide sequence linked to the 5’ end of the TLR7-selective motif.
• the first nucleotide sequence does not comprise a uridine nucleoside.
• the first nucleotide sequence is linked to the TLR7-selective motif by a phosphodiester linkage.
• the first nucleotide sequence comprises at least one nucleoside.
• the first nucleotide sequence comprises at least two nucleosides.
• the first nucleotide sequence comprises any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
• the first nucleotide sequence comprises three nucleosides.
• the nucleosides within the first nucleotide sequence are linked by phosphodiester linkages or phosphorothioate linkages.
• the nucleosides within the first nucleotide sequence are linked by phosphodiester linkages.
• the first nucleotide sequence comprises the nucleotide sequence C-G-G (SEQ ID NO: 60), wherein represents a phosphodiester linkage.
• the RNA ligand further comprises a second nucleotide sequence linked to the 3’ end of the TLR7-selective motif.
• the second nucleotide sequence does not comprise a uridine nucleoside.
• the second nucleotide sequence comprises a nucleotide sequence that is capable of hybridizing to all or a part of the first nucleotide sequence, and/or all or a part of the TLR7-selective motif.
• the second nucleotide sequence comprises a nucleotide sequence that is capable of hybridizing to all or a part of the first nucleotide sequence, and/or all or a part of the TLR7-selective motif, wherein the hybridized sequences form a G:U wobble base pair.
• the second nucleotide sequence comprises the nucleotide sequence G-G-G (SEQ ID NO: 69).
• the nucleotide sequence G-G-G (SEQ ID NO: 69) is capable of base pairing with the TLR7-selective motif.
• the nucleotide sequence G-G-G hybridizes to the TLR7-selective motif and forms a G:U wobble base pair.
• the second nucleotide sequence is linked to the TLR7-selective motif by a phosphodiester linkage.
• the second nucleotide sequence comprises at least three nucleosides. In some embodiments, the second nucleotide sequence comprises any of
• the second nucleotide sequence comprises 15 nucleosides.
• nucleosides within the second nucleotide sequence are linked by phosphodiester linkages or phosphorothioate linkages. In some embodiments, the nucleosides within the second nucleotide sequence are linked by phosphodiester linkages.
• the second nucleotide sequence comprises a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 61 and comprising the nucleotide sequence of SEQ ID NO: 69, wherein the nucleotide sequence of SEQ ID NO: 69 is capable of base pairing with the TLR7-selective motif.
• the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 61.
• the RNA ligand is capable of adopting a double-stranded RNA hairpin structure.
• the RNA ligand comprises a G:U wobble base pair.
• the RNA ligand comprises the nucleotide sequence of SEQ ID NO: 23.
• the RNA ligand comprises a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 23 and comprising the TLR7-selective motif of SEQ ID NO: 42 and the nucleotide sequence of SEQ ID NO: 69, wherein the nucleotide sequence of SEQ ID NO: 69 is capable of base pairing with the TLR7-selective motif.
• the RNA ligand comprises the nucleotide sequence of SEQ ID NO: 24.
• the RNA ligand comprises a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 24 and comprising the TLR7-selective motif of SEQ ID NO: 43 and the nucleotide sequence of SEQ ID NO: 69, wherein the nucleotide sequence of SEQ ID NO: 69 is capable of base pairing with the TLR7-selective motif.
• the second nucleotide sequence comprises a nucleotide sequence that is capable of hybridizing to all or a part of the first nucleotide sequence, and/or all or a part of the TLR7-selective motif. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence that is capable of hybridizing to all or a part of the first nucleotide sequence, and/or all or a part of the TLR7- selective motif, wherein the hybridized sequences form a G:U wobble base pair. In some embodiments, the second nucleotide sequence comprises the nucleotide sequence G-G-G (SEQ ID NO: 69).
• the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 61.
• the RNA ligand is capable of adopting a double-stranded RNA hairpin structure.
• the RNA ligand comprises a G:U wobble base pair.
• U2 of the first TLR7-selective motif comprises a 2’-fluoro modification
• U2 of the second TLR7-selective motif comprises a 2’ -methoxy ethoxy modification.
• U2 of the first TLR7-selective motif comprises a 2’ -amino modification
• U2 of the second TLR7-selective motif comprises a 2’0-methyl modification.
• U2 of the first TLR7-selective motif comprises a 2’-amino modification
• U2 of the second TLR7-selective motif comprises a 2’-fluoro modification.
• U2 of the first TLR7- selective motif comprises a 2’-deoxy modification
• U2 of the second TLR7-selective motif comprises a 2 ’O-methyl modification
• U2 of the first TLR7-selective motif comprises a 2’-deoxy modification
• U2 of the second TLR7-selective motif comprises a 2’-fluoro modification
• U2 of the first TLR7-selective motif comprises a 2’-deoxy modification
• U2 of the second TLR7-selective motif comprises a 2’ -amino modification.
• the first TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 36.
• the second TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 36.
• the first TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 37.
• the second TLR7- selective motif comprises the nucleotide sequence of SEQ ID NO: 37.
• the first TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 36 and the second TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 37.
• nucleosides 100, or more nucleosides, or between any of 100 and 125 nucleosides, 125 and 150 nucleosides, 150 and
• the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 67.
• the RNA ligand comprises the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the RNA ligand comprises a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 34 and comprising a first TLR7-selective motif of SEQ ID NO: 37 and/or a second TLR7- selective motif of SEQ ID NO: 37.
• the RNA ligand comprises a first TLR7- selective motif comprising the nucleotide sequence of SEQ ID NO: 37 and a second TLR7- selective motif comprising the nucleotide sequence of SEQ ID NO: 36.
• the first nucleotide sequence comprises at least one nucleoside.
• the first nucleotide sequence comprises at least two nucleosides.
• the first nucleotide sequence comprises any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
• the second nucleotide sequence comprises five nucleosides. In some embodiments, the second nucleotide sequence does not comprise a uridine nucleoside. In some embodiments, the second nucleotide sequence is linked to the first and/or to the second TLR7-selective motif by a phosphodiester linkage.
• RNA ligand comprising a nucleotide sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 33 and comprising a first and a second TLR7-selective motif of SEQ ID NO: 36.
• Ui of the one or more TLR7-selective motifs comprising Formula V comprises a modification at the 2’ position of the ribose.
• the modification is any modification at the 2’ position of the ribose known in the art or described herein.
• the modification is any modification at the 2’ position of the ribose that inhibits or blocks RNase activity known in the art or described herein.
• the modification is a 2’0-methyl modification, a 2’-fluoro modification, a 2’- amino modification, a 2’-deoxy modification, or a 2 ’-methoxy ethoxy modification.
• the modification is a 2’0-methyl modification.
• Ui of the fourth TLR7-selective motif comprises any modification at the 2’ position of the ribose known in the art or described herein. In some embodiments, Ui of the fourth TLR7-selective motif comprises a 2’0-methyl modification, a 2’- fluoro modification, a 2’-amino modification, a 2’-deoxy modification, or a 2 ’-methoxy ethoxy modification. In some embodiments, Ui of the fifth TLR7-selective motif comprises any modification at the 2’ position of the ribose known in the art or described herein.
• Ui of the fifth TLR7-selective motif comprises a 2’0-methyl modification, a 2’- fluoro modification, a 2’-amino modification, a 2’-deoxy modification, or a 2 ’-methoxy ethoxy modification.
• Ui of the sixth TLR7-selective motif comprises any modification at the 2’ position of the ribose known in the art or described herein.
• Ui of the sixth TLR7-selective motif comprises a 2’0-methyl modification, a 2’- fluoro modification, a 2’-amino modification, a 2’-deoxy modification, or a 2 ’-methoxy ethoxy modification.
• the first TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 40.
• at least one of one or more additional TLR7-selective motifs e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional TLR7-selective motifs
• all of one or more additional TLR7-selective motifs e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional TLR7-selective motifs
• the first TLR7-selective motif comprises the nucleotide sequence of SEQ ID NO: 41.
• at least one of one or more additional TLR7-selective motifs e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional TLR7-selective motifs
• all of one or more additional TLR7-selective motifs e.g., any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional TLR7-selective motifs
• the RNA ligand further comprises one or more additional nucleotide sequences that flank the 5’ end of one or more of the TLR7-selective motifs, the 3’ end of one or more of the TLR7-selective motifs, or both.
• the additional nucleotide sequences do not comprise a uridine nucleoside.
• the first nucleotide sequence comprises two nucleosides.
• the induction or increase is of at least about any of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% or more.
• the one or more cells are human cells.
• the one or more cells are human peripheral blood mononuclear cells.
• the one or more cells are plasmacytoid dendritic cells.
• the induction or increase is of at least about any of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% or more.
• the one or more cells are human cells.
• the one or more cells are human peripheral blood mononuclear cells.
• the one or more cells are plasmacytoid dendritic cells.
• an immunomodulatory composition of the disclosure increases activity of TLR8 in one or more cells contacted and/or transfected with the immunomodulatory composition of the disclosure by less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1%, for example, as compared to corresponding one or more cells that are not contacted and/or transfected with the immunomodulatory composition (e.g., as compared to corresponding one or more cells treated with a negative control or with cell culture medium only).
• the one or more cells are human cells.
• the one or more cells are human peripheral blood mononuclear cells.
• the one or more cells are monocytes.
• the expression or secretion of B cell activation markers and/or cytokines by one or more B cells may assessed based on the mRNA levels of the markers or cytokines, e.g., using qPCR, RNA- sequencing, microarray-based methods, or any other suitable method for measuring mRNA known in the art.
• the levels of one or more ISGs may assessed based on the mRNA levels of the one or more ISGs, e.g., using qPCR, RNA-sequencing, microarray- based methods, or any other suitable method for measuring mRNA known in the art.
• the methods comprise contacting and/or transfecting the one or more cells with an immunomodulatory composition of the disclosure. In some embodiments, the methods further comprise contacting the one or more cells with guanosine or a guanosine derivative. In some embodiments, the guanosine derivative is 2’3’ cyclic GMP, 7-thia-8- oxoguanosine, 7-deazaguanosine, 7-allyl-8-oxoguanosine, 7-deaza-dG, 9-hexyl-guanine, or any combination thereof.
• an immunomodulatory composition of the disclosure induces or increases the expression or secretion of IFN-a by one or more cells contacted and/or transfected with the immunomodulatory composition of the disclosure, for example, as compared to corresponding one or more cells that are not contacted and/or transfected with the immunomodulatory composition (e.g., as compared to corresponding one or more cells treated with a negative control or with cell culture medium only).
• the induction or increase is of at least about any of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
• an immunomodulatory composition of the disclosure induces or increases the expression or secretion of IFN-d by one or more cells contacted and/or transfected with the immunomodulatory composition of the disclosure, for example, as compared to corresponding one or more cells that are not contacted and/or transfected with the immunomodulatory composition (e.g., as compared to corresponding one or more cells treated with a negative control or with cell culture medium only).
• the induction or increase is of at least about any of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% or more.
• the one or more cells are human cells.
• the one or more cells are human peripheral blood mononuclear cells.
• the one or more cells are plasmacytoid dendritic cells.
• an immunomodulatory composition of the disclosure induces or increases the expression or secretion of IFN-e by one or more cells contacted and/or transfected with the immunomodulatory composition of the disclosure, for example, as compared to corresponding one or more cells that are not contacted and/or transfected with the immunomodulatory composition (e.g., as compared to corresponding one or more cells treated with a negative control or with cell culture medium only).
• an immunomodulatory composition of the disclosure induces or increases the expression or secretion of IFN-co by one or more cells contacted and/or transfected with the immunomodulatory composition of the disclosure, for example, as compared to corresponding one or more cells that are not contacted and/or transfected with the immunomodulatory composition (e.g., as compared to corresponding one or more cells treated with a negative control or with cell culture medium only).
• an immunomodulatory composition of the disclosure increases activity of TLR8 in one or more cells contacted and/or transfected with the immunomodulatory composition of the disclosure by less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1%, for example, as compared to corresponding one or more cells that are not contacted and/or transfected with the immunomodulatory composition (e.g., as compared to corresponding one or more cells treated with a negative control or with cell culture medium only).
• the one or more cells are human cells.
• the one or more cells are human peripheral blood mononuclear cells.
• the one or more cells are monocytes.
• an immunomodulatory composition of the disclosure induces or increases the expression or secretion of NFk-B-dependent cytokines by one or more cells contacted and/or transfected with the immunomodulatory composition of the disclosure by less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1%, for example, as compared to corresponding one or more cells that are not contacted and/or transfected with the immunomodulatory composition (e.g., as compared to corresponding one or more cells treated with a negative control or with cell culture medium only).
• the one or more cells are human cells.
• the one or more cells are human peripheral blood mononuclear cells.
• the one or more cells are monocytes.
• the expression or secretion of type I IFNs, inflammatory cytokines, and/or NFk-B-dependent cytokines by one or more cells may be measured using any suitable method known in the art, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoassays, such as a Luminex assay (see, e.g., www.nKisvstfems coro/what- assay), a bead-based immunoassay, electrochemiluminescence-based methods such as Meso Scale Discovery (MSD), intracellular staining of cytokines analyzed by flow cytometry, or mass spectrometry.
• enzyme-linked immunosorbent assay ELISA
• immunoblotting immunoassays, such as a Luminex assay (see, e.g., www.nKisvstfems coro/what- assay)
• a bead-based immunoassay such as a Luminex assay
• the expression or secretion of type I IFNs, inflammatory cytokines, and/or NFk-B-dependent cytokines by one or more cells may assessed based on the mRNA levels of the type I IFNs, inflammatory cytokines, and/or NFk-B-dependent cytokines, e.g., using qPCR, RNA-sequencing, microarray-based methods, or any other suitable method for measuring mRNA known in the art.
• the expression or secretion of B cell activation markers and/or cytokines by one or more B cells may assessed based on the mRNA levels of the markers or cytokines, e.g., using qPCR, RNA- sequencing, microarray-based methods, or any other suitable method for measuring mRNA known in the art.
• an immunomodulatory composition of the disclosure modulates the levels of one or more interferon-stimulated genes (ISGs).
• the one or more ISGs include any ISG known in the art.
• the one or more ISGs include any of the ISGs described in Shamith et al, Nucleic Acids Research (2009) 37, suppl l; Schoggins and Rice, CurrOpin Virol (2011) l(6):519-525; Forster et al., Nucleic Acids Research (2013) (database issue): D1040-D1046; Liu et al, PNAS (2012) 109(11):4239-4244.
• the levels of one or more ISGs may assessed using any suitable method known in the art, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoassays, such as a Luminex assay (see, e.g., www.rndsystems.CGra/whai- hirmnex-assay), a bead-based immunoassay, electrochemiluminescence-based methods such as Meso Scale Discovery (MSD), intracellular staining of cytokines analyzed by flow cytometry, or mass spectrometry.
• enzyme-linked immunosorbent assay ELISA
• immunoblotting immunoassayssays, such as a Luminex assay (see, e.g., www.rndsystems.CGra/whai- hirmnex-assay), a bead-based immunoassay, electrochemiluminescence-based methods such as Meso Scale Discovery (MSD), intracellular staining of cytokines
• the levels of one or more ISGs may assessed based on the mRNA levels of the one or more ISGs, e.g., using qPCR, RNA-sequencing, microarray- based methods, or any other suitable method for measuring mRNA known in the art.
• Exemplary pharmaceutically acceptable excipients that may be used in the pharmaceutical compositions of the disclosure further include one or more interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).
• sHASEGP soluble neutral-active hyaluronidase glycoproteins
• rHuPH20 HYLENEX®, Baxter International, Inc.
• Certain exemplary sHASEGPs and methods of use, including rHuPH20 are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968 .
• a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
• Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
• the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
• the pharmaceutical compositions of the disclosure are adjuvant compositions.
• Adjuvant compositions refer to any substance or combination of substances that, upon administration, enhance an immune response, e.g., a cellular immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell.
• An adjuvant may be used for the prevention or treatment of a disease.
• Adjuvant compositions of the disclosure may include an RNA ligand of the disclosure, a pharmaceutically acceptable carrier, e.g., poly-L- arginine or DOTAP, and one or more of a pharmaceutically acceptable excipient, any additional agent, compound, molecule or ingredient described herein, and/or any suitable additional agent known in the art.
• an immunomodulatory composition of the disclosure may be used as an adjuvant for another treatment, such as a vaccine, e.g., an anti-viral vaccine, an anti-bacterial vaccine, or an anti cancer vaccine.
• a vaccine e.g., an anti-viral vaccine, an anti-bacterial vaccine, or an anti cancer vaccine.
• the pharmaceutical compositions of the disclosure may contain more than one active component as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
• an additional agent such as another immunomodulatory composition, a nucleic acid, a protein or polypeptide (e.g., an antibody or fragments thereof), a vaccine or vaccine composition, an adjuvant, and/or one or more drugs, e.g., a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant.
• a pharmaceutical composition of the disclosure comprises the immunomodulatory composition of the disclosure and one or more of an immunostimulatory agent, an anti-viral agent, an antibiotic, an anti-fungal agent, an anti-parasitic agent, an anti-bacterial agent, an anti-tumor agent, a chemokine, a growth factor, an anti-angiogenic factor, a chemotherapeutic agent, an antibody, a gene-silencing agent, or a cytokine (e.g., a type I IFN such as IFN-a and/or IFN-b).
• an immunostimulatory agent e.g., an anti-viral agent, an antibiotic, an anti-fungal agent, an anti-parasitic agent, an anti-bacterial agent, an anti-tumor agent, a chemokine, a growth factor, an anti-angiogenic factor, a chemotherapeutic agent, an antibody, a gene-silencing agent, or a cytokine (e.g., a type I
• isolated nucleic acids having a nucleotide sequence comprising any of the RNA ligands of the present disclosure are provided.
• one or more vectors comprising such nucleic acids are provided.
• a host cell comprising such nucleic acids or vectors is also provided.
• the host cell is eukaryotic or prokaryotic.
• Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.
• RNA ligand of the present disclosure methods of making an RNA ligand of the present disclosure are provided.
• the method includes culturing a host cell of the present disclosure comprising a nucleic acid or vector comprising the RNA ligand, under conditions suitable for expression of the RNA ligand.
• the RNA ligand is subsequently recovered from the host cell (or host cell culture medium).
• a nucleic acid comprising the RNA ligand is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures.
• Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript ⁇ e.g, pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
• plasmids and bacterial viruses e.g., pUC18, pUC19, Bluescript ⁇ e.g, pBS SK+
• mpl8 mpl9
• pBR322 mpl9
• ColEl ColEl
• pCRl pCRl
• RP4 phage DNAs
• shuttle vectors such as pSA3 and pAT28.
• monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al. Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
• Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells and myeloma cell lines.
• kits for selectively activating TLR7 in an individual using the immunomodulatory compositions and/or pharmaceutical compositions of the disclosure comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition and/or pharmaceutical compositions of the disclosure.
• the immunomodulatory compositions and/or pharmaceutical compositions of the disclosure are administered as single agents, or in combination with one or more additional therapeutic agents, e.g., as described herein.
• the methods for selectively activating TLR7 in an individual provided herein may find use in the prevention or treatment of certain diseases, disorders, or conditions such as infections, immune disorders, cancers; and/or in the modulation of the immune system in an individual.
• preventing,” “prevention,” “prevent,” and the like include providing prophylaxis with respect to occurrence or recurrence of a particular disease, disorder, or condition in an individual.
• An individual may be predisposed to, susceptible to a particular disease, disorder, or condition, or at risk of developing such a disease, disorder, or condition, but has not yet been diagnosed with the disease, disorder, or condition.
• An individual “at risk” of developing a disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein.
• treat refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition.
• An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.
• a “therapeutically effective amount” is at least the minimum amount of an agent, such as an immunomodulatory composition or pharmaceutical composition of the disclosure, required to effect a measurable improvement of a particular disease, disorder, or condition.
• a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the agent to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
• kits for preventing or treating a viral infection in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents for preventing or treating the viral infection.
• bacterial infections examples include, without limitation, Streptococcus agalactiae, Escherichia coli, Listeria monocytogenes, Streptococcus pneumonia, Haemophilus influenza, Neisseria meningitides, Klebsiella spp., Staphylococcus aureus, Streptococcus pneumonia, Mycobacterium tuberculosis, Cryptococcus neoformans, or Clostridium tetanus.
• kits for preventing or treating a parasitic infection in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents for preventing or treating the parasitic infection.
• parasitic infections that may be prevented or treated using the immunomodulatory compositions and/or pharmaceutical compositions of the disclosure include, without limitation, worm infections, such as intestinal worm infections.
• parasitic infections include, without limitation, Acanthamoeba, African Sleeping Sickness (African trypanosomiasis), alveolar echinococcosis (Echinococcosis, Hydatid Disease), amebiasis (Entamoeba histolytica), American trypanosomiasis (Chagas Disease), ancylostomiasis (Hookworm), angiostrongyliasis (Angiostrongylus), anisakiasis (Anisakis, Pseudoterranova), ascariasis (Ascaris, intestinal roundworms), babesiosis (Babesia), balantidiasis (Balantidium), balamuthia, Baylisascariasis (Baylisascaris, Raccoon Roundworm), bilharzia (Schistosomiasisis
• Pneumocystis jirovecii pneumonia Pseudoterranova (Anisakiasis, Anisakis), baylisascariasis (Baylisascaris), Sappinia, Sarcocystosis (Sarcocystosis),
• Toll-like receptors such as TLR7, as well as Type I IFNs have several known roles in the immunopathology of immune disorders, such as immunodeficiencies, autoimmune disorders or allergies. See, e.g., Uematsu and Akira, Expert Opin Biol Ther (2006) 6(3):203-14; Crow el ah, Annual Review of Pathology: Mechanisms of Disease - Type I Interferons in Autoimmune Disease (2019) 14:369-393; and Wang etal, Front Immunol (2017) 8:1431. Accordingly, immunomodulatory compositions and/or pharmaceutical compositions of the disclosure that selectively activate TLR7 may find use in preventing or treating immune disorders, such as immunodeficiencies, autoimmune disorders or allergies.
• kits for preventing or treating an immune disorder in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents for preventing or treating the immune disorder.
• immune disorders that may be prevented or treated using the immunomodulatory compositions and/or pharmaceutical compositions of the disclosure include, without limitation, autoimmune diseases, immunodeficiencies, and allergies.
• Non-limiting examples of allergies that may be prevented or treated using the immunomodulatory compositions and/or pharmaceutical compositions of the disclosure include seasonal allergies, mastocytosis, perennial allergies, anaphylaxis, food allergies, allergic rhinitis, or atopic dermatitis.
• Toll-like receptors such as TLR7, as well as Type I IFNs have several known roles in regulating the anti-tumor immune response. See, e.g., Urban-Wojciuk et al, Front. Immunol (2019) 10:2388; Musella etal, (2017) 6(5):el314424; and Zitvogel etal, Nature Reviews Immunology (2015) 15:405-414. Accordingly, immunomodulatory compositions and/or pharmaceutical compositions of the disclosure that selectively activate TLR7 may find use in preventing or treating cancers.
• sarcomas of primary cutaneous origin e.g., dermatofibrosarcoma protuberans
• lymphomas of primary cutaneous origin e.g., mycosis fungoides
• thoracic and respiratory cancers such as bronchial adenomas/carcinoids, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma, and thymic carcinoma
• AIDS-related cancers Kaposi sarcoma
• desmoplastic small round cell tumor and liposarcoma desmoplastic small round cell tumor and liposarcoma.
• Toll-like receptors such as TLR7, as well as Type I IFNs have several known roles in regulating the immune system, including, but not limited to regulation of humoral immunity, adaptive immunity, and antigen-specific T-cell responses. See, e.g., Le Bon et al, Immunity (2001) 14(4):461-470; Fitzgerald and Kagan, Cell (2020) 180(6):1044-1066; and Fi et al, Front Immunol (2019) 10:2191. Accordingly, immunomodulatory compositions and/or pharmaceutical compositions of the disclosure that selectively activate TFR7 may find use in increasing, inducing, or enhancing humoral immunity, adaptive immunity, B cell responses, and/or antigen- specific T-cell responses in an individual.
• kits for increasing, inducing, or enhancing antigen-specific T-cell responses comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• kits for increasing, inducing, or enhancing B cell activation and/or proliferation in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• kits for increasing, inducing, or enhancing dendritic cell e.g., cDC, plasmacytoid dendritic cell
• the methods comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• methods for increasing, inducing, or enhancing IL- 12 production in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• kits for increasing, inducing, or enhancing T cell priming in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• kits for increasing, inducing, or enhancing B cell class switch recombination in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• kits for increasing, inducing, or enhancing B cell antibody secretion in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• kits for increasing, inducing, or enhancing B cell cytokine secretion in an individual comprise administering to the individual a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure; or a therapeutically effective amount of an immunomodulatory composition or pharmaceutical composition of the disclosure in combination with one or more additional agents.
• the methods further comprise measuring the level of TLR7 activity in a sample obtained from the individual before and/or after the individual has received one or more doses of the immunomodulatory composition and/or pharmaceutical composition of the disclosure.
• the methods further comprise measuring the level of one or more interferon- stimulated genes (ISGs) in a sample obtained from the individual before and/or after the individual has received one or more doses of the immunomodulatory composition and/or pharmaceutical composition of the disclosure.
• ISGs interferon- stimulated genes
• the one or more ISGs include any ISG known in the art, e.g., one or more of the ISGs described in Shamith et al., Nucleic Acids Research (2009) 37, suppl_l; Schoggins and Rice, CurrOpin Virol (2011) l(6):519-525; Forster et al, Nucleic Acids Research (2013) (database issue): D1040-D1046; Liu et al., PNAS (2012) 109(11):4239-4244.
• ISGs examples include, without limitation, IFIT1, CXCL10, CXCL11, ISG15, CCL8, 2’5’OAS, APOBEC3G, APOBEC3A, PKR, ISG56, Mx2, MDA5, IFI44, IRF7, OASL1, ISG20 IFIT2, IFIT3, IFITM3, OAS2, OAS3, IFI16, IRF1, MX1, or IDO.
• the sample is a blood sample, a plasma sample, or a serum sample.
• the sample is a tissue biopsy, e.g., from spleen, one or more lymph nodes, or from a tumor.
• the levels of type I IFNs may be measured using any suitable method known in the art, such as enzyme-linked immunosorbent assay, immunoblotting, immunoassays, such as a Luminex assay (see, assay), a bead-based immunoassay, electrochemiluminescence-based methods such as Meso Scale Discovery (MSD), intracellular staining of cytokines analyzed by flow cytometry, or mass spectrometry.
• enzyme-linked immunosorbent assay such as a Luminex assay (see, assay)
• a bead-based immunoassay such as a bead-based immunoassay
• electrochemiluminescence-based methods such as Meso Scale Discovery (MSD), intracellular staining of cytokines analyzed
• the levels of type I IFNs may assessed based on the mRNA levels of the type I IFNs, e.g., using qPCR, RNA-sequencing, microarray-based methods, or any other suitable method for measuring mRNA known in the art.
• the levels of type I IFNs may assessed by fluorescence in situ hybridization (FISH), qPCR or any other suitable method known in the art for measuring RNA levels in tissue samples.
• FISH fluorescence in situ hybridization
• the levels of one or more ISGs may assessed using any suitable method known in the art, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoassays, such as a Luminex assay (see, e.g., ww w rn dsv stem s . com/ what- lu i n ex- assay) , a bead-based immunoassay, electrochemiluminescence-based methods such as Meso Scale Discovery (MSD), intracellular staining of cytokines analyzed by flow cytometry, or mass spectrometry.
• ELISA enzyme-linked immunosorbent assay
• immunoassays such as a Luminex assay (see, e.g., ww w rn dsv stem s . com/ what- lu i n ex- assay)
• a bead-based immunoassay such as a bead-based immunoassay
• the levels of one or more ISGs may assessed based on the mRNA levels of the one or more ISGs, e.g., using qPCR, RNA-sequencing, microarray-based methods, or any other suitable method for measuring mRNA known in the art.
• a clinical symptom of any of the diseases, disorders, or conditions of the present disclosure can be monitored.
• the level of TLR7 activity may be measured in a sample obtained from the individual before and/or after the individual has received one or more doses of the immunomodulatory composition and/or pharmaceutical composition of the disclosure.
• the level of one or more type I IFNs may be measured in a sample obtained from the individual before and/or after the individual has received one or more doses of the immunomodulatory composition and/or pharmaceutical composition of the disclosure.
• the level of IFN-a may be measured in a sample obtained from the individual before and/or after the individual has received one or more doses of the immunomodulatory composition and/or pharmaceutical composition of the disclosure.
• the level of one or more interferon-stimulated genes may be measured in a sample obtained from the individual before and/or after the individual has received one or more doses of the immunomodulatory composition and/or pharmaceutical composition of the disclosure.
• ISGs examples include, without limitation, IFIT1, CXCL10, CXCL11, ISG15, CCL8, 2’5’OAS, APOBEC3G, APOBEC3A, PKR, ISG56, Mx2, MDA5, IFI44, IRF7, OASL1, ISG20 IFIT2, IFIT3, IFITM3, OAS2, OAS3, IFI16, IRF1, MX1, or IDO.
• the sample is a blood sample, a plasma sample, or a serum sample.
• the sample is a tissue biopsy, e.g., from spleen, one or more lymph nodes, or from a tumor.
• the levels of type I IFNs may be measured using any suitable method known in the art, such as enzyme-linked immunosorbent assay, immunoblotting, immunoassays, such as a Luminex assay (see, e.g., www rndsystems.
• the levels of one or more ISGs may assessed based on the mRNA levels of the one or more ISGs, e.g., using qPCR, RNA-sequencing, microarray-based methods, or any other suitable method for measuring mRNA known in the art.
• Administration of an immunomodulatory composition or pharmaceutical composition of the disclosure can be continuous or intermittent, depending, for example, on the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
• the administration of an immunomodulatory composition or pharmaceutical composition of the disclosure may be essentially continuous over a preselected period of time or may be in a series of spaced doses.
• dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
• Immunomodulatory compositions and/or pharmaceutical compositions of the present disclosure containing an RNA ligand of the present disclosure may be administered to an individual in need of thereof, in accordance with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, intracranial, intraspinal, subcutaneous, intra-articular, intrasynovial, intravenous, intraarterial, intrathecal, oral, topical, or inhalation routes.
• an immunomodulatory composition and/or pharmaceutical composition of the present disclosure is administered by intravenous infusion.
• an immunomodulatory composition and/or pharmaceutical composition of the present disclosure is administered by local injection, e.g., into a tissue or into an organ.
• Administration of an immunomodulatory composition or pharmaceutical composition of the present disclosure may be performed using any suitable method known in the art, such as using catheters, syringes, or like devices to deliver the immunomodulatory composition or pharmaceutical composition into a target organ or tissue.
• Immunomodulatory compositions or pharmaceutical compositions of the present disclosure may be administered in any suitable form known in the art.
• an immunomodulatory composition or pharmaceutical composition of the present disclosure may be formulated for administration as a liposome, a polymeric microparticle, or an emulsion.
• an immunomodulatory composition or a pharmaceutical composition of the present disclosure is formulated for administration as liposomes.
• Various amphiphilic lipids can form bilayers in an aqueous environment to encapsulate a RNA-ligand- containing aqueous core as a liposome. These lipids can have an anionic, cationic or zwitterionic hydrophilic head groups.
• Lipids that may be used include any lipid described herein, e.g., in the “Carriers” section, or know in the art.
• liposomes of the disclosure include one or more phospholipids, such as, but not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols.
• phospholipids such as, but not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols.
• liposomes of the disclosure include one or more cationic lipids, such as, but not limited to, DOTAP, l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 1,2-dioleyloxy- N,Ndimethyl-3-aminopropane (DODMA), l,2-dilinoleyloxy-N,N-dimethy 1-3 -aminopropane (DLinDMA), DOTMA, or l,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA).
• DSDMA 1,2-dioleyloxy- N,Ndimethyl-3-aminopropane
• DODMA 1,2-dioleyloxy- N,Ndimethyl-3-aminopropane
• DLinDMA 1,2-dioleyloxy-N,Ndimethyl-3-aminopropane
• liposomes of the disclosure include one or more Zwitterionic lipids, such as, but not limited to, acyl zwitterionic lipids, ether zwitterionic lipids, DPPC, DOPC or dodecylphosphocholine.
• liposomes of the disclosure include saturated and/or unsaturated lipds.
• liposomes of the disclosure include a single lipid or a mixture of lipids.
• the mixture may comprise (i) a mixture of anionic lipids, (ii) a mixture of cationic lipids, (iii) a mixture of zwitterionic lipids, (iv) a mixture of anionic lipids and cationic lipids, (v) a mixture of anionic lipids and zwitterionic lipids, (vi) a mixture of zwitterionic lipids and cationic lipids, or (vii) a mixture of anionic lipids, cationic lipids and zwitterionic lipids.
• liposomes of the disclosure include non- amphiphilic lipids, such as cholesterol.
• lipids within liposomes of the disclosure may be modified by covalent attachment of a polyethylene glycol (i.e., PEGylated). See, e.g., Heyes et al. (2005) J Controlled Release 107:276-87.
• liposomes of the disclosure are multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), or mixtures thereof.
• MLVs have multiple bilayers in each vesicle, forming several separate aqueous compartments.
• SUVs and LUVs have a single bilayer encapsulating an aqueous core.
• SUVs typically have a diameter ⁇ 50nm, and LUVs have a diameter >50nm.
• Any suitable method for the preparation of liposomes may be used, for example, as described in Weissing V (ed.). Liposomes: Methods and Protocols, Vol. 1, Springer, 12, 29-50 (2010); and Lunctional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes) (eds. Arshady & Guyot). Citus Books, 2002.
• an immunomodulatory composition or a pharmaceutical composition of the present disclosure is formulated for administration as polymeric microparticles.
• Various polymers can form microparticles to encapsulate or adsorb RNA.
• Polymers that may be used include any polymer or polymeric molecule or agent described herein, e.g., in the “Carriers” section, or know in the art.
• polymers examples include, without limitation, poly(alpha-hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones, polyester-amides, polyamino acids (e.g., poly-L- arginine, poly-L-lysine, poly-L-ornithine), and combinations thereof. Techniques for preparing suitable microparticles are well known in the art.
• a microparticle may include a cationic surfactant and/or lipid.
• An alternative way of making polymeric microparticles is by molding and curing e.g., as disclosed in W02009/132206.
• an immunomodulatory composition or a pharmaceutical composition of the present disclosure is formulated for administration as an emulsion.
• Emulsions of the disclosure may comprise one or more oils, such as, without limitation, oils from an animal, vegetable or synthetic source.
• the emulsion may comprise a combination of oils.
• the aqueous component of the emulsion may be water, e.g., water for injection, and may include further components e.g., solutes. For instance, it may include salts to form a buffer e.g., citrate or phosphate salts, such as sodium salts.
• Typical buffers include a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer.
• the emulsion comprises a cationic lipid, such as any cationic lipid described herein or known in the art.
• the emulsion comprises a non-ionic surfactant and/or a zwitterionic surfactant.
• Administration of an immunomodulatory composition and/or a pharmaceutical composition of the disclosure may be performed in combination or in conjunction with another compound, composition, or agent. Such administration includes simultaneous administration and/or administration at different times. Administration in conjunction or in combination also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.
• kits comprising an immunomodulatory composition, a nucleic acid, a vector, a pharmaceutical composition, or a vaccine composition of the present disclosure.
• Kits of the present disclosure may include one or more containers comprising an immunomodulatory composition, a nucleic acid, a vector, a pharmaceutical composition, or a vaccine composition of the present disclosure.
• the kits further include instructions for use in accordance with the methods of this disclosure.
• these instructions comprise a description of administration or use of the immunomodulatory composition, nucleic acid, vector, pharmaceutical composition, or vaccine composition of the present disclosure to prevent, reduce risk, or treat an individual having a disease, disorder, or condition described herein according to any methods of this disclosure.
• the instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
• the kit may further include an additional agent (e.g., in the same or in one or more additional containers), such as another immunomodulatory composition, a nucleic acid, a protein or polypeptide (e.g., an antibody or fragments thereof), a vaccine or vaccine composition, an adjuvant, and/or one or more drugs, e.g., a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant.
• an additional agent e.g., in the same or in one or more additional containers
• another immunomodulatory composition e.g., in the same or in one or more additional containers
• a nucleic acid e.g., a protein or polypeptide (e.g., an antibody or fragments thereof), a vaccine or vaccine composition, an adjuvant, and/or one or more drugs, e.g., a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormon
• the kit may further include (e.g., in the same or in one or more additional containers) one or more of an immunostimulatory agent, an anti-viral agent, an antibiotic, an anti-fungal agent, an anti-parasitic agent, an anti-bacterial agent, an anti-tumor agent, a chemokine, a growth factor, an anti-angiogenic factor, a chemotherapeutic agent, an antibody, a gene-silencing agent, or a cytokine (e.g., a type I IFN such as IFN-a and/or IFN-b).
• an immunostimulatory agent e.g., an anti-viral agent, an antibiotic, an anti-fungal agent, an anti-parasitic agent, an anti-bacterial agent, an anti-tumor agent, a chemokine, a growth factor, an anti-angiogenic factor, a chemotherapeutic agent, an antibody, a gene-silencing agent, or a cytokine (e.g.
• the kit may further include instructions for using the immunomodulatory composition, nucleic acid, vector, pharmaceutical composition, or vaccine composition of the present disclosure in combination with an additional agent, such as another immunomodulatory composition, a nucleic acid, a protein or polypeptide (e.g., an antibody or fragments thereof), a vaccine or vaccine composition, an adjuvant, one or more drugs, e.g., a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, cardioprotectant, an immunostimulatory agent, an anti-viral agent, an antibiotic, an anti-fungal agent, an anti-parasitic agent, an anti-bacterial agent, an anti-tumor agent, a chemokine, a growth factor, an anti- angiogenic factor, a chemotherapeutic agent, an antibody, a gene-silencing agent, or a cytokine (e.g., a type I IFN such as IFN-a and/or IFN-
• kits in the kits may be unit doses, bulk packages (e.g. , multi-dose packages) or sub-unit doses.
• kits of the present disclosure are typically written instructions on a label or package insert (e.g. , a paper sheet included in the kit), but machine-readable instructions (e.g. , instructions carried on a magnetic or optical storage disk) are also included in the disclosure.
• the label or package insert indicates that the immunomodulatory composition, nucleic acid, vector, pharmaceutical composition, or vaccine composition, and any additional agents, are used for treating or preventing, e.g., a disease, disorder, or condition of the present disclosure. Instructions may be provided for practicing any of the methods described herein.
• the kits of this disclosure are in suitable packaging.
• Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
• This Example describes the design of TLR7-selective ligands with RNA modifications that block RNase cleavage at certain nucleotides to avoid the generation of uridine monomers that have the potential to activate TLR8.
• oligoribonucleotide 7013 has the nucleotide sequence C*C*G*A*G*C*C*G*C*U*U*U*C*C*C*C (SEQ ID NO: 1) and was derived from Forsbach et al., J Immunology (2008) 180:3729-3738.
• ORN7013 contains four uridines flanked by non-stimulatory sequences and is fully phosphorothioate-modified.
• ORN7023 The second selected RNA sequence was derived from R2153, as described by Hartmann et al in W02010/105819, and is referred to herein as ORN7023.
• ORN7023 has the nucleotide sequence C-G-G-C-U-C-G-G-C-A-G-A-A-G-C-C-G-G-G-C-C (SEQ ID NO: 2) and forms a short canonical RNA hairpin by pairing of the underlined sequences, including a G:U wobble base pair (FIG. 4).
• ORN7005 which has the nucleotide sequence U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-U-C-C-U-C-C-U-C (SEQ ID NO: 4).
• ORN7005 and ORN7009 were derived from pUlC and pU2C, respectively (see, e.g., Zhang et al., 2018, Cell Reports 25, 3371-3381).
• ORN7001 has the nucleotide sequence C-A-G-A-C-C-U-U-C-C-A-G-A-C-C-U-U-C (SEQ ID NO: 5).
• ORN7001 also contains a C/U-rich C-U-U-C motif (SEQ ID NO: 32) with two potential motifs for TLR7 Binding Site #2: C-U-U (SEQ ID NO: 54) and U-U-C (SEQ ID NO: 55).
• Table 1 Selected RNA sequences for generation of TLR7-selective ligands.
• the uridine in the center of the RNA trimer (U1-U 2 -U3) bound by TLR7 at Binding Site #2 is deeply embedded in the binding pocket, including the 2 ⁇ H (2 ⁇ H2). Suggesting that modification of this 2 ⁇ H could affect TLR7 activity, and is therefore likely not available for modification.
• the 2 ⁇ H moieties of the flanking nucleotides (2’OHi and 2 ⁇ H3) appear to be located to more open parts of the binding pocket (FIG. 5). Therefore, modification of the nucleotides directly 5’ of all uridines that could become uridine monomers was pursued.
• RNA modifications that specifically block RNase-mediated cleavage at certain nucleotides were introduced into the selected RNA sequences described above.
• the aim of the modifications was to enable the generation of RNA trimer degradation products that bind to TLR7 Binding Site #2 (i.e., having the sequence N-U-N (SEQ ID NO: 6), N*U*N (SEQ ID NO: 63), or N-U*N (SEQ ID NO: 64) wherein N represents any ribonucleotide), and that cannot be degraded to monomeric U, which is a ligand for TLR8 Binding Site #1.
• the C*U*U*U*U*C motif (SEQ ID NO: 7) can be degraded to the TLR7 Binding Site #2 motifs C*U*U (SEQ ID NO: 8), U*U*U (SEQ ID NO: 9), or U*U*C (SEQ ID NO: 10), and can be further degraded to monomeric U, which is a TLR8 Binding Site #1 ligand.
• ORN7014-7017 corresponding to SEQ ID NOs: 12-15.
• Table 2 Modifications of ORN7013 to generate a TLR7-selective ligand.
• ORN7013 derived from Forsbach et al., J Immunology (2008) 180:3729-3738, was also modified to reduce phosphorothioate modifications. Specifically, all nucleotides, except for nucleotides in the C*U*U*U*U*C motif (SEQ ID NO: 7), were linked by a phosphodiester backbone instead of phosphorothioate-modified backbone. This resulted in ORN7022, which has the nucleotide sequence C-C-G-A-G-C-C-G-C*U*U*U*U*U*C-C-C (SEQ ID NO: 16). Modified ORNs equivalent to ORN7014-7017 were designed based on ORN7022, resulting in ORN7018- ORN7021, corresponding to SEQ ID NOs: 17-20 (Table 3).
• nucleotide sequence of ORN7023 (SEQ ID NO: 2), derived from W02010/105819 (Hartmann et al), contains only one TLR7 Binding Site #2 motif (underlined), which is composed of nucleotides 4-6 of ORN7023: C-U-C (SEQ ID NO: 21):
• ORN7023 An additional version of ORN7023 was generated, containing a 2’ unmodified sequence and the backbone converted from phosphodiester to phosphorothioate linkages (ORN7024; SEQ ID NO: 25).
• ORN7005 derived from pUlC (see, Zhang et al., 2018, Cell Reports 25, 3371-3381), has the nucleotide sequence:
• ORN7005 In the nucleotide sequence of ORN7005 (SEQ ID NO: 3), all C residues were modified with either 2’0-methyl modifications or 2’-fluoro modifications to avoid cleavage and preserve unmodified U residues. This resulted in the 2’0-methyl-modified ORN7006 (SEQ ID NO: 26), and the 2’-fluoro-modified ORN7007 (SEQ ID NO: 27). In addition, to test whether the limited RNase resistance conferred by phosphorothioate modification could increase activity, a fully phosphorothioate-modified version of ORN7005 was generated, resulting in ORN7008 (SEQ ID NO: 28).
• the nucleotide sequence of ORN7009 contains multiple U-U-C repeats (SEQ ID NO: 39).
• the first U in each U-U-C repeat (SEQ ID NO: 39) in ORN7009 was modified with either 2’0-methyl or 2’-fluoro modifications. This resulted in the 2’0-methyl- modified ORN7010 (SEQ ID NO: 29), with the modified mU-U-C repeat (SEQ ID NO: 40); and the 2’-fluoro-modified ORN7011 (SEQ ID NO: 30), with the modified fU-U-C repeat (SEQ ID NO: 41).
• ORN7012 SEQ ID NO: 31.
• ORN7001 The first U in the C-U-U-C motifs (SEQ ID NO: 32) in ORN7001 was modified with 2- fluoro or 2’-0-methyl modifications, resulting in ORN7002 (SEQ ID NO: 33), with the modified motif C-mU-U-C (SEQ ID NO: 36); and ORN7003 (SEQ ID NO: 34) with the modified motif C-fU-U-C (SEQ ID NO: 37).
• ORN7001 a modified ORN was generated from ORN7001 in which the entire C-U-U-C motif (SEQ ID NO: 32) was modified with phosphorothioates, resulting in ORN7004 (SEQ ID NO: 35), with the modified motif C*U*U*C (SEQ ID NO: 38).
• the modified ORNs derived from ORN7001 are summarized in Table 7.
• Example 2 Generation and functional characterization of TLR7 -selective RN A ligands.
• This Example describes experiments that evaluated the TLR7-selectivity and activity of RNA ligands described in Example 1. The results of these experiments showed that certain modified RNA ligands derived from ORN7013 (derived from Forsbach et al, J Immunology (2008) 180:3729-3738) and ORN7023 (derived from W02010/105819) had enhanced TLR7- selectivity and activity.
• RNA oligomers were synthesized and HPLC-purified by Integrated DNA Technologies (IDT).
• PBMCs Peripheral blood mononuclear cells
• PBS phosphate- buffered saline
• PBMCs were isolated via density gradient centrifugation with Lymphoprep density gradient media (STEMCELL Technologies) and SepMate-50 tubes (STEMCELL Technologies). Samples were centrifuged at 1000 g for 15 minutes. After centrifugation, the upper layers were collected by pouring into a new tube, diluted with PBS, and centrifuged at 450 x g for 5 minutes.
• ORN7023 derived from W02010/105819
• ORN7024 the phosphorothioate- modified RNA ligand derived from ORN7023 had reduced activity.
• ORN7001 (tie novo designed ligand) induced TLR7 and TLR8 activity, indicated by IFN-a and TNF-a secretion, respectively.
• ORN7002 which is a 2’O-methyl modified version of ORN7001
• ORN7003 which is a 2’-fluoro-modified version of ORN7001
• ORN7004 which is a phosphorothioate-linked version of ORN7001, had mildly increased TLR7 activity and markedly increased TLR8 activity.

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