Modified oligonucleotides

Abstract

The present invention relates to oligonucleotides that inhibit Toll-like receptor 7 (TLR7) and / or Toll-like receptor 8 (TLR8) or enhance TLR8 and uses thereof.

Description

Technical Field

[0001] The present invention relates to oligonucleotides that inhibit Toll-like receptor 7 (TLR7) and / or Toll-like receptor 8 (TLR8) or enhance TLR8, and uses thereof.

[0002] Related Applications

[0003] This application claims the benefit of the priority of Australian Provisional Application No. 2022902992, filed on October 12, 2022, and Australian Provisional Application No. 2023901499, filed on May 16, 2023, the entire disclosures of which are incorporated herein by reference. Background Art

[0004] RNA-targeting therapeutic agents based on synthetic oligonucleotides have received extensive attention, obtained several regulatory approvals in the United States and the European Union, and have received billions of dollars in licensing deals in recent years. To ensure their basic functions related to gene-targeting activity, oligonucleotide-based therapeutic agents need to increase their affinity for their targets and stability against nuclease activity.

[0005] Understanding the complex relationship between synthetic oligonucleotides and nucleic acid sensors of the innate immune system, such as Toll-like receptors (TLRs), is important for the design of oligonucleotide therapeutic agents. These oligonucleotides are designed to evade the activation of innate immune sensors and avoid strong off-target pro-inflammatory immune responses in patients. From this perspective, chemical modification may have the dual benefits of increasing the targeting efficacy of oligonucleotides while reducing their immunostimulatory effects.

[0006] Nonetheless, it has been clear for some time that selected phosphorothioate (PS)-modified DNA oligonucleotides (ODNs) have broad immunosuppressive effects. This is best exemplified by the PS-ODN A151 containing "TTAGGG", which involves inhibition of TLR9, TLR7, absent in melanoma 2 (AIM2), and cyclic GMP-AMP synthase (cGAS). These effects are sequence-dependent, with some PS-DNA ODNs showing limited immunosuppressive activity on individual immune sensors. These observations suggest the complexity of chemically modified oligonucleotide immunosuppression, where the sequence determines the activity of the chemically modified oligonucleotide on nucleic acid sensors. Since only a few ODNs have been studied across different receptors to date, there is currently a lack of detailed understanding of the immunosuppressive sequence determinants of ODNs. In addition, understanding of the immunosuppressive effects of oligonucleotides with combined base and / or backbone modifications, as seen in most approved and developing oligonucleotide therapeutics, is almost non-existent. While potentially helpful in generating anti-inflammatory ODNs, characterizing the immunosuppressive effects of therapeutic RNAs is becoming increasingly important to help avoid increased susceptibility to infection in the large patient populations starting ODN therapy.

[0007] There is a need for new or improved inhibitors of Toll-like receptor 7 (TLR7) and / or Toll-like receptor 8 (TLR8) activity, or new or improved molecules that enhance TLR8 activity.

[0008] The citation of any prior art in this specification does not represent an admission or implication that such prior art constitutes a part of the common general knowledge in any jurisdiction, nor that such prior art can be reasonably understood, regarded as relevant, and / or combined with other prior art by a person skilled in the art. SUMMARY OF THE INVENTION

[0009] In a first aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0010] [mX / modified mX]* y X A * z X B

[0011] Wherein:

[0012] * y and * z Each independently represents an internucleotide linkage, where * y and * z At least one of them is not a phosphorodiamidate;

[0013] X A and X BEach independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0014] wherein mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide containing a morpholine ring; and

[0015] where when [mX / modified mX] is mX, at least one of X A and X B is not mX;

[0016] where when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate nucleotide internucleotide bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleotide bond;

[0017] wherein the sequence is optionally functionalized.

[0018] Preferably, * y and * z are not phosphorodiamides.

[0019] Preferably, X A is independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX; and X B is independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X.

[0020] In one embodiment of the first aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0021] [mX / modified mX]* y X A * z X B

[0022] wherein:

[0023] * y and* z Each independently represents an internucleotide bond;

[0024] X A and X B Each is independently selected from the group consisting of mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX and modified fX;

[0025] wherein mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide comprising a 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, and fX is a nucleotide comprising a 2'-fluorine and / or 3'-fluorine modification; and

[0026] Wherein when [mX / modified mX] is mX, X A and X B At least one of is not mX;

[0027] Wherein when [mX / modified mX] is 3'-OMe N7-methylated guanosine, * y is not a 5'-5'-triphosphate internucleotide bond, and* z Not a 3'-5'-phosphodiester internucleotide bond;

[0028] wherein the sequence is optionally functionalized.

[0029] Preferably,* y and* z More preferably, at least one of is not phosphoric acid diamide. y and* z Neither is phosphoramide.

[0030] Any oligonucleotide of the first aspect inhibits TLR7 activity, preferably human TLR7 activity. In a preferred embodiment, the oligonucleotide of the first aspect does not enhance TLR8 activity, preferably human TLR8 activity. In a particularly preferred embodiment, the oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity. In an alternative preferred embodiment, the oligonucleotide of the first aspect enhances TLR8 activity, preferably human TLR8 activity.

[0031] Each internucleotide bond may be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond, and 5'-2'-bond. Preferably, each internucleotide bond may be selected from: 3'-5'-bond and 5'-5'-bond. Preferably, each internucleotide bond is a 3'-5'-bond.

[0032] In a preferred embodiment of the first aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0033] 5'-[mX / modified mX]* y X A * z X B -3'

[0034] Wherein:

[0035] * y and * z each independently represents an internucleotide bond, wherein * y and * z at least one of them is not a phosphorodiamidate;

[0036] X A and X B each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0037] Wherein mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide containing a morpholine ring; and

[0038] wherein when [mX / modified mX] is mX, X A and X B at least one of them is not mX;

[0039] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate internucleotide bond, and * z is not a 3'-5'-phosphodiester internucleotide bond;

[0040] wherein said sequence is optionally functionalized.

[0041] In a particularly preferred embodiment, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0042] 5'-[mX / modified mX]* y X A * z X B -3'

[0043] wherein:

[0044] * y and * z each independently represents an internucleotide bond;

[0045] X A and X B each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, and modified fX;

[0046] where mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing a 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, and fX is a nucleotide containing a 2'-fluoro and / or 3'-fluoro modification; and

[0047] where when [mX / modified mX] is mX, at least one of A X B and X

[0048] is not mX;

[0049] wherein said sequence is optionally functionalized.

[0050] Preferably, each internucleotide bond is a 3'-5' bond.

[0051] Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is phosphorothioate.

[0052] In one embodiment, the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers. In another embodiment, the oligonucleotide of the first aspect comprises a single phosphorothioate stereoisomer, preferably wherein * y is in the S configuration.

[0053] Preferably, mX is a nucleotide comprising a 2'-OMe modification.

[0054] Preferably, moX is a nucleotide comprising a 2'-MOE modification.

[0055] Preferably, fX is a nucleotide comprising a 2'-fluoro modification.

[0056] Modified dX, modified rX, and modified morpholino comprise at least one modification or substitution at the base and / or sugar position. Modified mX, modified moX, modified LX, and modified fX comprise at least one additional modification or substitution at an additional position of the base and / or sugar. Preferably, the modification or substitution is selected from the group consisting of: pseudouridine, 3'-deoxy, hydroxy, deamino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0057] Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mU3, mC1, and m7 G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mU3 is N3-Me-U (3-methyluridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), and m7 G is 3'-OMe-N7-methylated guanosine. Preferably, the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, and mC1. Most preferably, the modified mX is mC1.

[0058] Exemplary modified dXs include, but are not limited to: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, PSU, N3-Me-dC, 5-I-dC, dI, 8-Br-dG, 7-deaza-dG, 8-Br-dA, 8-oxo-dA, O6-Me-dG, 8-NH2-dG, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, PSU is pseudouridine, N3-Me-dC is 3-methyl deoxycytidine, 5-I-dC is 5-iodo-deoxycytidine, dI is deoxyinosine, 8-Br-dG is 8-bromo-deoxyguanosine, 7-deaza-dG is 7-deaza-deoxyguanosine, 8-Br-dA is 8-bromo-deoxyadenosine, 8-oxo-dA is 8-oxo-deoxyadenosine, O6-Me-dG is O6-methyl deoxyguanosine, and 8-NH2-dG is 8-amino-deoxyguanosine.

[0059] Exemplary modified rXs include, but are not limited to, PSU, 2'-NH2-rX, and ara-rX, where 2'-NH2-rX is an RNA base modified with 2'-amino, and ara-rX is an RNA base modified with arabinose. Exemplary 2'-NH2-rXs include, but are not limited to, 2'-NH2-U and 2'-NH2-C, where 2'-NH2-U is 2'-NH2-uridine, and 2'-NH2-C is 2'-NH2-cytidine. Exemplary ara-rX is ara-C (cytarabine).

[0060] In one embodiment, [mX / modified mX] is selected from the group consisting of: mG, mI, mG1, and mU. Preferably, [mX / modified mX] is mG or mI. In one embodiment, [mX / modified mX] is modified mX. Preferably, the modified mX is mI or mG1, preferably mI. Preferably, the modified mX is not 2'-OMe-N1-Me-G (2'-O-methyl-N1-methyl guanosine). In another embodiment, [mX / modified mX] is mX. Preferably, mX is mG or mU, preferably mG.

[0061] In a preferred embodiment, [mX / modified mX] is [mG / modified mG]. Preferably, the modified mG is not 2'-OMe-N1-Me-G (2'-O-methyl-N1-methylguanosine). The modified mG includes but is not limited to: mG1 and mI, where mG1 is 2'-OMe-2,6-diaminopurine, and mI is 2'-OMe-I (2'-O-methylinosine). Preferably, [mG / modified mG] is [mG / mI]. In one embodiment, [mG / modified mG] is mG. In another embodiment, [mG / modified mG] is mI.

[0062] In a preferred embodiment, X A and X B each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, and morpholino-X. In a particularly preferred embodiment, X A and X B each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, and modified dX.

[0063] In one embodiment, X A is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified rX. Preferably, X A is selected from the group consisting of: mX, dX, rX, modified mX, modified dX, and modified rX. Preferably, X A is selected from the group consisting of: mU, mU1, mU2, mU3, PSU, mG, mA, mC, dT, dG, dA, dC, rU, 2'-NH2-rU, 8-Br-dA, and 8-oxo-dA. In one embodiment, X A is selected from the group consisting of: mX, dX, rX, and modified mX. Preferably, X A is selected from the group consisting of: mU, mU1, mU2, PSU, mG, mA, mC, dT, dG, dA, dC, and rU. More preferably, X A is mU.

[0064] In one embodiment, X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, and morpholino-X. Preferably, X BSelected from the group consisting of: dA, dC, dG, dT, mC, mC1, mG, rC, moC, LC, LA, LT, LG, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, N3-Me-dC, 5-I-dC, 2'-NH2-C, ara-C, morpholino-C, N3-Me-mU, dI, 8-Br-dG, 7-deaza-dG, O6-Me-dG and 8-NH2-dG. In one embodiment, X B Selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX and modified dX. Preferably, X B Selected from the group consisting of: dA, dC, dG, dT, mC, mC1, mG, rC, moC, LC, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC and pdC. In a preferred embodiment, X B Selected from the group consisting of: mX, dX, LX, modified mX, modified dX and modified rX. Preferably, X B Selected from the group consisting of: LC, dC, 5-Me-dC, 5-Br-dC, mC, mC1, ara-C. In a particularly preferred embodiment, X B Selected from the group consisting of: LX, modified mX, modified dX and modified rX. Preferably, X B Selected from the group consisting of: LC, 5-Me-dC, 5-Br-dC and mC1. In an even more preferred embodiment, X B is LX, preferably LC.

[0065] In one embodiment, X A and X B at least one of which is LX. In one embodiment, X A and X B are independently LX. In another embodiment, X A and X B one of which is LX. Preferably, X B is LX. Preferably, X B is LX, and X A is mX.

[0066] In one embodiment, X A and X B at least one of which is dX. In one embodiment, X A and X B are independently dX. In another embodiment, X A and X BOne of them is dX. Preferably, X B is dX. Preferably, X B is dX, and X A is mX. More preferably, X B is dX, and X A is mU.

[0067] In one embodiment, X A and X B at least one of which is rX. In one embodiment, X A and X B are independently rX. In another embodiment, X A and X B one of which is rX. Preferably, X A is rX. Preferably, X B is mX or rX, and X A is rX. More preferably, X B is mX, and X A is rU; X A is rA, and X B is rA; or X A is rU, and X B is rC. Preferably, when X A and X B at least one of which is rX, each internucleotide bond is a 3'-5'-thioester bond.

[0068] In a particularly preferred embodiment, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0069] 5'-[mG / mI]*mU*X B -3'

[0070] Wherein:

[0071] * each independently represents a 3'-5'-thioester bond;

[0072] X B is selected from the group consisting of: dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX and modified morpholino-X;

[0073] wherein dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide containing a morpholine ring; and

[0074] Wherein said sequence is optionally functionalized.

[0075] Preferably, X B is selected from the group consisting of: mX, dX, LX, modified mX, modified rX, and modified dX.

[0076] In one embodiment, the sequence can be functionalized. Preferably, the functionalized sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclic group, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclic group, substituted arylalkyl, and hydrophobic lipid. Preferably, the hydrophobic lipid is selected from cholesterol and tocopherol. Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, and tocopherol.

[0077] In one embodiment, the compound is directly conjugated to the sequence. In another embodiment, the compound is conjugated to the sequence through a linker. The linker can be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0078] Preferably, the compound is conjugated to the terminal nucleotide of the sequence, preferably the terminal 3'-nucleotide. Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'-position.

[0079] The functionalized sequence can comprise functionalized nucleotides selected from the group consisting of: dX-TEG, dX-Chol, and dX-Toco, where dX-TEG is a DNA base having triethylene glycol covalently linked to the 3'-position through a monophosphate group, dX-Chol is a DNA base having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position through a monophosphate group, and dX-Toco is a DNA base having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position through a monophosphate group. Preferably, the functionalized sequence can comprise functionalized nucleotides selected from the group consisting of: dC-TEG, dC-Chol, dC-Toco, where dC-TEG is deoxycytidine having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position through a monophosphate group, dC-Chol is deoxycytidine having triethylene glycol covalently linked to the 3'-position through a monophosphate group, and dC-Toco is deoxycytidine having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position through a monophosphate group.

[0080] Preferably, the sequence is selected from the group consisting of:

[0081]

[0082]

[0083]

[0084] In one embodiment, [mX / modified mX] is [mG / mI]; X A is mU; and X B is selected from the group consisting of mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified rX, and modified dX, where the sequence is optionally functionalized. Preferably, X B is selected from the group consisting of LX, mX, dX, modified mX, modified rX, and modified dX. More preferably, X B is selected from the group consisting of LC, mC, ara-C, dC, mC1, and modified dC. Preferably, modified dC is selected from the group consisting of 5-Me-dC, 5-Br-dC, and 5-I-dC. Preferably, the sequence is selected from the group consisting of mG*mU*LC, mI*mU*LC, mG*mU*mC1, mG*mU*5-Me-dC, mG*mU*5-Br-dC, mG*mU*dC, mG*mU*dC-TEG, mI*mU*mC, mG*mU*dC-Chol, mG*mU*dC-Toco, mG*mU*ara-C, and mG*mU*5-I-dC.

[0085] In another preferred embodiment, [mX / modified mX] is [mG / mI]; X A is mU; and X B is selected from the group consisting of mX, dX, rX, LX, modified mX, modified rX, and modified dX. Preferably, X B is selected from the group consisting of LC, mC1, dC, mC, and modified dC. Preferably, modified dC is selected from the group consisting of 5-Me-dC, 5-Br-dC, and 5-I-dC. Preferably, the sequence is selected from the group consisting of mG*mU*LC, mI*mU*LC, mG*mU*mC1, mG*mU*5-Me-dC, mG*mU*5-Br-dC, and mG*mU*5-I-dC.

[0086] In another preferred embodiment, [modified mX] is [mG / mI]; and X A and X BIt is rX. Preferably, the sequence is selected from the group consisting of: mG*rA*rA, mG*rU*rC, mG*rG*rA, mG*rU*rA, mG*rU*rU, mG*rA*rG, mG*rG*rC, mG*rA*rU, mG*rG*rG. More preferably, the sequence is selected from: mG*rA*rA, mG*rU*rC and mG*rG*rA.

[0087] In another particularly preferred embodiment, the oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity. Preferably, the oligonucleotide that further inhibits TLR8 activity comprises or consists of the sequence mI*mU*mC or mI*mA*dG.

[0088] In one embodiment, the oligonucleotide consists of a sequence.

[0089] In another embodiment, the oligonucleotide comprises a sequence. Preferably, the oligonucleotide comprising the sequence has a length of no more than 20 bases, preferably a length of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 bases. Preferably, the sequence is located at the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In a preferred embodiment, the oligonucleotide comprises the sequence 5'-mG*mU*X B -3', where X B is dX, preferably X B is dC. Even more preferably, the oligonucleotide comprises the sequence 5'-mG*mU*dC*dC*dC*dC-3'.

[0090] In another embodiment of the first aspect, a method for modifying the TLR7 activity of an oligonucleotide is provided, the method comprising modifying the oligonucleotide by adding a sequence to the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In one embodiment, the method reduces the TLR7 enhancing activity of the oligonucleotide. In another embodiment, the method increases the TLR7 inhibitory activity of the oligonucleotide.

[0091] In another embodiment of the first aspect, a fusion oligonucleotide is provided, which comprises two or more oligonucleotides according to the first aspect connected by a cleavable linker. Preferably, the fusion oligonucleotide comprises:

[0092] A-[Y-A] n

[0093] where

[0094] each A independently represents an oligonucleotide according to the first aspect, and each A can be the same or different;

[0095] Y represents a cleavable linker, and each Y can be the same or different; and

[0096] n is equal to or greater than 1.

[0097] Preferably, Y is enzymatically cleavable. Preferably, Y is selected from the group consisting of: TEG linker, carbon spacer (such as C3, C6, C9, C 12 ), glycerol, and PolydT. More preferably, Y is a TEG linker.

[0098] Preferably, each A is independently bound to Y via an internucleotide bond (*). Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is a phosphorothioate.

[0099] Each internucleotide bond can be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond, and 5'-2'-bond. Preferably, each internucleotide bond can be selected from: 3'-5'-bond and 5'-5'-bond. Preferably, each internucleotide bond is a 3'-5'-bond. More preferably, each internucleotide bond is a 3'-5'-phosphorothioate bond.

[0100] In one embodiment, the fusion oligonucleotide comprises or consists of the sequence 5'-mG*mU*dC-3'-*TEG*-3'-dC*mU*mG-5'.

[0101] In another embodiment of the first aspect, there is provided a modified oligonucleotide comprising an agent linked via a linker to the oligonucleotide or fusion oligonucleotide according to the first aspect. The agent can be a therapeutic agent and / or a diagnostic agent. Preferably, the agent is a therapeutic agent, and more preferably, a therapeutic RNA selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamer, antisense oligonucleotide, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In a preferred embodiment, the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. The linker can be cleavable or non-cleavable. Preferably, the linker is a cleavable linker.

[0102] In another embodiment of the first aspect, there is provided a composition comprising the oligonucleotide or fusion oligonucleotide according to the first aspect and a pharmaceutically acceptable excipient.

[0103] In one embodiment, the composition is an immunogenic composition comprising a therapeutic RNA and the oligonucleotide or fusion oligonucleotide according to the first aspect. Preferably, the therapeutic RNA is selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamer, antisense oligonucleotide, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the immunogenic composition comprises a modified oligonucleotide according to the first aspect. Preferably, the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0104] In one embodiment, the composition may comprise at least one additional active agent, and the at least one additional active agent includes, but is not limited to, an anti-inflammatory agent.

[0105] In another embodiment of the first aspect, there is provided a method for inhibiting TLR7 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect, thereby inhibiting TLR7 activity in the subject.

[0106] In another embodiment of the first aspect, there is provided a method for inhibiting TLR7 activity in a cell, the method comprising contacting the cell with the oligonucleotide, fusion oligonucleotide or composition according to the first aspect, thereby inhibiting TLR7 activity in the cell.

[0107] In another embodiment of the first aspect, there is provided the use of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect in the preparation of a medicament for inhibiting TLR7 activity in a subject.

[0108] In another embodiment of the first aspect, there is provided the use of a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect for inhibiting TLR7 activity in a subject.

[0109] In another embodiment of the first aspect, there is provided a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect for inhibiting TLR7 activity in a subject.

[0110] In another embodiment of the first aspect, a method of inhibiting TLR7 activation in a subject caused by a therapeutic RNA is provided, the therapeutic RNA being selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide, or composition according to the first aspect, thereby inhibiting TLR7 activation in the subject. In one embodiment, the method includes administering an immunogenic composition comprising the oligonucleotide or fusion oligonucleotide according to the first aspect, and a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the immunogenic composition comprises a modified oligonucleotide according to the first aspect, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0111] In one embodiment, the oligonucleotide, fusion oligonucleotide, or composition substantially does not reduce the translation of the therapeutic RNA.

[0112] In one embodiment, the therapeutic RNA comprises pseudouridine.

[0113] In another embodiment, the therapeutic RNA does not comprise pseudouridine.

[0114] In another embodiment of the first aspect, there is provided the use of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect in the preparation of a medicament for inhibiting TLR7 activation caused by therapeutic RNA in a subject, wherein the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof. In one embodiment, the subject has received, is receiving or will receive therapeutic RNA. In another embodiment, there is provided the use of a first composition and a second composition in the preparation of a medicament for inhibiting TLR7 activation caused by therapeutic RNA in a subject, wherein the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof, wherein the first composition comprises the oligonucleotide, fusion oligonucleotide or composition according to the first aspect, and the second composition comprises therapeutic RNA. In another embodiment, there is provided the use of an immunogenic composition comprising the modified oligonucleotide according to the first aspect in the preparation of a medicament for inhibiting TLR7 activation caused by therapeutic RNA in a subject, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof.

[0115] In another embodiment of the first aspect, there is provided the use of a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect for inhibiting TLR7 activation caused by therapeutic RNA in a subject, wherein the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof. In one embodiment, the subject has received, is receiving or will receive therapeutic RNA. In another embodiment, there is provided the use of a therapeutically effective amount of an immunogenic composition comprising the modified oligonucleotide according to the first aspect for inhibiting TLR7 activation caused by therapeutic RNA in a subject, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof.

[0116] In another embodiment of the first aspect, there is provided a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect for inhibiting TLR7 activation in a subject caused by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the subject has received, is receiving, or will receive a therapeutic RNA. In another embodiment, there is provided a therapeutically effective amount of an immunogenic composition comprising a modified oligonucleotide according to the first aspect for inhibiting TLR7 activation in a subject caused by a therapeutic RNA, wherein the therapeutic agent is a therapeutic agent selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0117] In another embodiment of the first aspect, there is provided a method of treating or preventing a disease, disorder or condition in a subject responsive to TLR7 inhibition, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0118] In another embodiment of the first aspect, there is provided the use of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect in the preparation of a medicament for treating or preventing a disease, disorder or condition in a subject responsive to TLR7 inhibition.

[0119] In another embodiment of the first aspect, there is provided the use of a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect for treating or preventing a disease, disorder or condition in a subject responsive to TLR7 inhibition.

[0120] In another embodiment of the first aspect, there is provided a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the first aspect for preventing or treating a disease, disorder or condition in a subject responsive to TLR7 inhibition.

[0121] Preferably, the disease, disorder or condition responsive to TLR7 inhibition is selected from the group consisting of: inflammation-related diseases, allergic diseases, infections, cancers, and autoimmune diseases.

[0122] In a second aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0123] X C * y X D *z X E

[0124] wherein:

[0125] * y and * z each independently represents an internucleotide bond;

[0126] X C is selected from the group consisting of: mX, modified mX, dG, and morpholino-X;

[0127] X D and X E each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0128] wherein mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide containing a morpholine ring; and

[0129] wherein:

[0130] when X C is mX, at least one of X D and X E is not mX;

[0131] when X C is dG, at least one of X D and X E is not dX;

[0132] when X C is mG and when:

[0133] X D is dG, X E is not dA or dC;

[0134] X D is dT or mU, X E is not dC or dT;

[0135] X D is mC, X E is not dT, dG, or dC; and

[0136] X DWhen X is mG or dC, E it is not dX; or

[0137] When X C is dG, X E is not mG;

[0138] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y it is not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z it is not a 3'-5'-phosphodiester nucleotide internucleoside bond;

[0139] wherein the sequence is optionally functionalized.

[0140] Preferably, X C is selected from the group consisting of: mX, modified mX, dG; X D is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX; and X E is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X.

[0141] In one embodiment of the second aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0142] X C * y X D * z X E

[0143] wherein:

[0144] * y and * z each independently represents an internucleoside bond;

[0145] X C is selected from the group consisting of: mX, modified mX, and dG;

[0146] X D and X E each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, and modified fX;

[0147] wherein mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide comprising a 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, and fX is a nucleotide comprising a 2'-fluoro and / or 3'-fluoro modification; and

[0148] wherein:

[0149] when X C is mX, at least one of X D and X E is not mX;

[0150] when X C is dG, at least one of X D and X E is not dX;

[0151] when X C is mG and when:

[0152] X D is dG, X E is not dA or dC;

[0153] X D is dT or mU, X E is not dC or dT;

[0154] X D is mC, X E is not dT, dG or dC; and

[0155] X D is mG or dC, X E is not dX; or

[0156] when X C is dG, X E is not mG;

[0157] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate internucleotide bond, and * z is not a 3'-5'-phosphodiester internucleotide bond;

[0158] wherein the sequence is optionally functionalized.

[0159] Any oligonucleotide of the second aspect inhibits TLR8 activity, preferably human TLR8 activity. In a preferred embodiment, the oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activity. In an alternative preferred embodiment, the oligonucleotide of the second aspect substantially does not inhibit TLR7 activity, preferably human TLR7 activity.

[0160] Each internucleotide bond may be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond, and 5'-2'-bond. Preferably, each internucleotide bond may be selected from: 3'-5'-bond and 5'-5'-bond. Preferably, each internucleotide bond is a 3'-5'-bond.

[0161] In a particularly preferred embodiment of the second aspect, an oligonucleotide is provided that comprises or consists of a sequence consisting of:

[0162] 5'-X C * y X D * z X E -3'

[0163] Wherein:

[0164] * y And * z Each independently represents an internucleotide bond;

[0165] X C Is selected from the group consisting of: mX, modified mX, dG, and morpholino-X;

[0166] X D And X E Each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0167] Wherein mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide comprising a 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, fX is a nucleotide comprising a 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide comprising a morpholine ring; and

[0168] Wherein:

[0169] When XC When X is mX, X D and X E at least one of which is not mX;

[0170] When X C is dG, X D and X E at least one of which is not dX;

[0171] When X C is mG and when:

[0172] X D is dG, X E is not dA or dC;

[0173] X D is dT or mU, X E is not dC or dT;

[0174] X D is mC, X E is not dT, dG or dC; and

[0175] X D is mG or dC, X E is not dX; or

[0176] When X C is dG, X E is not mG;

[0177] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleoside bond;

[0178] wherein the sequence is optionally functionalized.

[0179] In a particularly preferred embodiment of the second aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0180] 5'-X C * y X D * z X E -3'

[0181] wherein:

[0182] * y and * z each independently represents a nucleotide internucleoside bond;

[0183] XC selected from the group consisting of: mX, modified mX, and dG;

[0184] X D and X E each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, and modified fX;

[0185] wherein mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification; and

[0186] wherein:

[0187] when X C is mX, at least one of X D and X E is not mX;

[0188] when X C is dG, at least one of X D and X E is not dX;

[0189] when X C is mG and when:

[0190] X D is dG, X E is not dA or dC;

[0191] X D is dT or mU, X E is not dC or dT;

[0192] X D is mC, X E is not dT, dG or dC; and

[0193] X D is mG or dC, X E is not dX; or

[0194] when X C is dG, X E is not mG;

[0195] wherein the sequence is optionally functionalized.

[0196] Preferably, each internucleotide bond is a 3'-5' bond.

[0197] Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, phosphodiester, phosphoramidate, and phosphorodiamidate. Each internucleotide bond can be the same or different. In one preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is phosphorothioate.

[0198] In a particularly preferred embodiment, each internucleotide bond is a 3'-5'-phosphorothioate bond.

[0199] In one embodiment, the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers. In another embodiment, the oligonucleotide comprises a single stereoisomer.

[0200] Preferably, mX is a nucleotide comprising a 2'-OMe modification.

[0201] Preferably, moX is a nucleotide comprising a 2'-MOE modification.

[0202] Preferably, fX is a nucleotide comprising a 2'-fluoro modification.

[0203] Modified dX, modified rX, and modified morpholino-X comprise at least one modification or substitution at the base and / or sugar position. Modified mX, modified moX, modified LX, and modified fX comprise at least one additional modification or substitution at an additional position of the base and / or sugar. Preferably, the modification or substitution is selected from the group consisting of: pseudouridine, 3'-deoxy, hydroxy, deamino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0204] Exemplary modified mX includes, but is not limited to: mG1, mI, mU1, mU2, mU3, mC1, and m7 G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mU3 is N3-Me-U (3-methyluridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), and m7 G is N7-methylated guanosine. Preferably, the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, and mC1.

[0205] Exemplary modified dXs include, but are not limited to: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, PSU, dI, 8-Br-dG, N1-Me-dG, 7-deaza-dG, 8-Br-dA, 8-oxo-dA, O6-Me-dG, and 8-NH2-dG, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, PSU is pseudouridine, dI is deoxyinosine, 8-Br-dG is 8-bromodeoxyguanosine, N1-Me-dG is 1-methyl deoxyguanosine, 7-deaza-dG is 7-deaza-deoxyguanosine, 8-Br-dA is 8-bromodeoxyadenosine, 8-oxo-dA is 8-oxodeoxyadenosine, O6-Me-dG is O6-methyl deoxyguanosine, and 8-NH2-dG is 8-amino deoxyguanosine.

[0206] Exemplary modified rXs include, but are not limited to: PSU, 2'-NH2-rX, and ara-rX, where 2'-NH2-rX is an RNA base modified with 2'-amino, and ara-rX is an RNA base modified with arabinose. Exemplary 2'-NH2-rXs include, but are not limited to: 2'-NH2-U and 2'-NH2-C, where 2'-NH2-U is 2'-NH2-uridine, and 2'-NH2-C is 2'-NH2-cytidine. Exemplary ara-rX is ara-C (cytarabine).

[0207] In one embodiment, X C is selected from the group consisting of: mG, mU, mC, mI, mG1, and dG.

[0208] In one embodiment, X C is selected from the group consisting of: mX and modified mX. Preferably, mX is selected from the group consisting of: mG, mC, and mU, more preferably mG; and the modified mX is mI. In a preferred embodiment, X C is selected from the group consisting of: mG and mI. In a particularly preferred embodiment, X C is mI.

[0209] In one embodiment, X D is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified rX. Preferably, X DSelected from the group consisting of: mA, mU, mC, dA, dT, dG, mU1, mU2, mU3, PSU, 8-Br-dA, 8-oxo-dA, rA, rG, rU, and 2'-NH2-rU. In one embodiment, X D Selected from the group consisting of: mX, dX, LX, modified mX, and modified dX. Preferably, X D Selected from the group consisting of: mX, dX, modified mX, and modified dX. Preferably, X D Selected from the group consisting of: mA, mU, mC, dA, dT, dG, mU1, mU2, and PSU. More preferably, X D Selected from the group consisting of: mA, mU, dA, dT, and dG.

[0210] In one embodiment, X E Selected from the group consisting of: mX, dX, rX, morpholino-X, moX, LX, fX, rX, modified mX, modified dX, and modified rX. Preferably, X E Selected from the group consisting of: dA, dC, dG, dT, rG, mC, mC1, mG, mU3, moC, LA, LC, LG, LT, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, dI, 8-Br-dG, N1-Me-dG, 7-deaza-dG, O6-Me-dG, 8-NH2-dG, morpholino-G, rA, rG, rU, rC, N3-Me-dC, 5-I-dC, 2'-NH2-C, ara-C, and morpholino-C. In one embodiment, X E Selected from the group consisting of: mX, dX, moX, LX, fX, rX, modified mX, and modified dX. Preferably, X E Selected from the group consisting of: dA, dC, dG, dT, rG, mC, mC1, mG, moC, LA, LC, LG, LT, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, and pdC. More preferably, X E Selected from the group consisting of: dA, dC, dG, dT, rG, mC, mC1, mG, moC, LA, LC, LG, LT, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, and pdC. In one embodiment, X E Selected from the group consisting of: mX, dX, rX, and LX. Preferably, X E Selected from the group consisting of: dA, dC, dG, dT, rG, mC, LA, LC, LG, and LT. Even more preferably, X ESelected from the group consisting of: mX and dX. Preferably, X E Selected from the group consisting of: dC, dG, dT, and mC.

[0211] In one embodiment, X D and X E At least one of which is LX. In one embodiment, X D and X E Are independently LX. In another embodiment, X D and X E One of which is LX. Preferably, X E Is LX. Preferably, X E Is LX, and X D Is mX.

[0212] In one embodiment, X D and X E At least one of which is dX. In one embodiment, X D and X E Are independently dX. In another embodiment, X D and X E One of which is dX. Preferably, X E Is dX. Preferably, X E Is dX, and X D Is mX.

[0213] In one embodiment, X D and X E At least one of which is rX. In one embodiment, X D and X E One of which is rX. In another embodiment, X D and X E Are each independently rX. Preferably, X D Is selected from rA and rG, and X E Is selected from rA, rG, and rC. Preferably, X D Is rA, and X E Is rA; X D Is rG, and X E Is rA; X D Is rA, and X E Is rG; X D Is rA, and X E Is rC. Preferably, when X A and X B At least one of which is rX, each internucleotide bond is a 3'-5'-thiophosphate bond.

[0214] In one embodiment, the sequence can be functionalized. Preferably, the functionalized sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclic group, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclic group, substituted arylalkyl, and hydrophobic lipid. Preferably, the hydrophobic lipid is selected from cholesterol and tocopherol. Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, and tocopherol.

[0215] In one embodiment, the compound is directly conjugated to the sequence. In another embodiment, the compound is conjugated to the sequence through a linker. The linker can be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0216] Preferably, the compound is conjugated to the terminal nucleotide of the sequence, preferably the terminal 3'-nucleotide. Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'-position.

[0217] The functionalized sequence can comprise functionalized nucleotides selected from the group consisting of: dX-TEG, dX-Chol, and dX-Toco, where dX-TEG is a DNA base having triethylene glycol covalently linked to the 3'-position through a monophosphate group, dX-Chol is a DNA base having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position through a monophosphate group, and dX-Toco is a DNA base having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position through a monophosphate group.

[0218] Preferably, the sequence is selected from the group consisting of:

[0219]

[0220]

[0221]

[0222] Preferably, the sequences are selected from the group consisting of: mI*mA*dG, mI*mU*mC, mG*dA*dG, mG*mA*dT, mG*mA*dG, mG*mA*dC, mG*mA*LG, mG*mA*rG, mG*mA*LT, mG*mA*LC, mU*dT*dC, mU*dA*dC, mG*mA*LA, mU*dA*dG, mC*dA*dG, mU*dT*dT, mU*dA*dT, mU*dA*dA, mC*dT*dA, mU*dG*dT, mC*dT*dC, mC*dA*dT, mU*dG*dG, mC*dT*dT, mC*dT*dG, mU*dT*dA, mU*dT*dG, mG*mA*O6-Me-dG, mG*rA*rA, mG*rG*rA, mG*rA*rG, and mG*rA*rU.

[0223] More preferably, the sequences are selected from the group consisting of: mI*mA*dG, mI*mU*mC, mG*dA*dG, mG*mA*dT, mG*mA*dG, mG*mA*dC, mG*rA*rA, and mG*rG*rA. Even more preferably, the sequences are selected from the group consisting of: mI*mA*dG and mI*mU*mC.

[0224] In another particularly preferred embodiment, the oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activity. Preferably, the oligonucleotide that further inhibits TLR7 activity comprises or consists of the sequences mI*mU*mC or mI*mA*dG.

[0225] In one embodiment, the oligonucleotide consists of a sequence.

[0226] In another embodiment, the oligonucleotide comprises a sequence. Preferably, the oligonucleotide comprising the sequence has a length of no more than 20 bases, preferably a length of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 bases. Preferably, the sequence is located at the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end.

[0227] In another embodiment of the second aspect, a method of modifying the TLR8 activity of an oligonucleotide is provided, the method comprising modifying the oligonucleotide by adding a sequence to the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In one embodiment, the method reduces the TLR8 enhancing activity of the oligonucleotide. In another embodiment, the method increases the TLR8 inhibitory activity of the oligonucleotide.

[0228] In another embodiment of the second aspect, a fusion oligonucleotide is provided, which comprises two or more oligonucleotides according to the second aspect connected by a cleavable linker. Preferably, the fusion oligonucleotide comprises:

[0229] A-[Y-A] n

[0230] wherein

[0231] each A independently represents an oligonucleotide according to the second aspect, and each A may be the same or different;

[0232] Y represents a cleavable linker, and each Y may be the same or different; and

[0233] n is equal to or greater than 1.

[0234] Preferably, Y is cleavable by an enzyme. Preferably, Y is selected from the group consisting of: TEG linker, carbon spacer (such as C3, C6, C9, C 12 ), glycerol, and PolydT. More preferably, Y is a TEG linker.

[0235] Preferably, each A is independently bound to Y by an internucleotide bond (*). Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond may be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is a phosphorothioate.

[0236] In another embodiment of the second aspect, a modified oligonucleotide is provided, which comprises an agent linked to an oligonucleotide or fusion oligonucleotide according to the second aspect through a linker. The agent can be a therapeutic agent and / or a diagnostic agent. Preferably, the agent is a therapeutic agent, and more preferably, a therapeutic RNA selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamer, antisense oligonucleotide, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In a preferred embodiment, the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. The linker can be cleavable or non-cleavable. Preferably, the linker is a cleavable linker.

[0237] In another embodiment of the second aspect, a composition is provided, which comprises an oligonucleotide or fusion oligonucleotide according to the second aspect, and a pharmaceutically acceptable excipient.

[0238] In one embodiment, the composition is an immunogenic composition comprising a therapeutic RNA and an oligonucleotide or fusion oligonucleotide according to the second aspect. Preferably, the therapeutic RNA is selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamer, antisense oligonucleotide, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the immunogenic composition comprises a modified oligonucleotide according to the second aspect. Preferably, the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0239] In one embodiment, the composition may comprise at least one additional active agent, and the at least one additional active agent includes but is not limited to an anti-inflammatory agent.

[0240] In another embodiment of the second aspect, a method of inhibiting TLR8 activity in a cell is provided, the method comprising contacting the cell with an oligonucleotide, fusion oligonucleotide, or composition according to the second aspect, thereby inhibiting TLR8 activity in the cell.

[0241] In another embodiment of the second aspect, a method of inhibiting TLR8 activity in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide, or composition according to the second aspect, thereby inhibiting TLR8 activity in the subject.

[0242] In another embodiment of the second aspect, the use of an oligonucleotide, fusion oligonucleotide, or composition according to the second aspect in the preparation of a medicament for inhibiting TLR8 activity in a subject is provided.

[0243] In another embodiment of the second aspect, the use of a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide, or composition according to the second aspect for inhibiting TLR8 activity in a subject is provided.

[0244] In another embodiment of the second aspect, a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide, or composition according to the second aspect for inhibiting TLR8 activity in a subject is provided.

[0245] In another embodiment of the second aspect, there is provided a method of inhibiting TLR8 activation in a subject caused by a therapeutic RNA selected from the group consisting of RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide, or composition according to the second aspect, thereby inhibiting TLR8 activation in the subject. In one embodiment, the method includes administering an immunogenic composition comprising the oligonucleotide or fusion oligonucleotide according to the second aspect and a therapeutic RNA selected from the group consisting of RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the immunogenic composition comprises a modified oligonucleotide according to the second aspect, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0246] In one embodiment, the oligonucleotide, fusion oligonucleotide, or composition substantially does not reduce the translation of the therapeutic RNA.

[0247] In one embodiment, the therapeutic RNA comprises pseudouridine.

[0248] In another embodiment, the therapeutic RNA does not comprise pseudouridine.

[0249] In another embodiment of the second aspect, there is provided the use of an oligonucleotide, fusion oligonucleotide or composition according to the second aspect in the preparation of a medicament for inhibiting TLR8 activation caused by a therapeutic RNA in a subject, wherein the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the subject has received, is receiving, or will receive a therapeutic RNA. In another embodiment, there is provided the use of a first composition and a second composition in the preparation of a medicament for inhibiting TLR8 activation caused by a therapeutic RNA in a subject, wherein the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof, wherein the first composition comprises an oligonucleotide, fusion oligonucleotide or composition according to the second aspect, and the second composition comprises a therapeutic RNA. In another embodiment, there is provided the use of an immunogenic composition comprising a modified oligonucleotide according to the second aspect in the preparation of a medicament for inhibiting TLR8 activation caused by a therapeutic RNA in a subject, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0250] On the other hand, there is provided the use of a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the second aspect for inhibiting TLR8 activation caused by a therapeutic RNA in a subject, wherein the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the subject has received, is receiving, or will receive a therapeutic RNA. In another embodiment, there is provided the use of a therapeutically effective amount of an immunogenic composition comprising a modified oligonucleotide according to the second aspect for inhibiting TLR8 activation caused by a therapeutic RNA in a subject, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0251] On the other hand, there is provided a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the second aspect for inhibiting TLR8 activation in a subject caused by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, guide RNA, self-amplifying RNA, circular RNA and combinations thereof. In one embodiment, the subject has received, is receiving or will receive a therapeutic RNA. In another embodiment, there is provided a therapeutically effective amount of an immunogenic composition comprising a modified oligonucleotide according to the second aspect for inhibiting TLR8 activation in a subject caused by a therapeutic RNA, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, guide RNA, self-amplifying RNA, circular RNA and combinations thereof.

[0252] In another embodiment of the second aspect, there is provided a method of treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the second aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0253] In another embodiment of the second aspect, there is provided the use of the oligonucleotide, fusion oligonucleotide or composition according to the second aspect in the preparation of a medicament for treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition.

[0254] In another embodiment of the second aspect, there is provided the use of a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the second aspect for treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition.

[0255] In another embodiment of the second aspect, there is provided a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the second aspect for preventing or treating a disease, disorder or condition in a subject responsive to TLR8 inhibition.

[0256] Preferably, the disease, disorder or condition responsive to TLR8 inhibition is selected from the group consisting of: inflammation-related diseases, allergic diseases, infections, cancers and autoimmune diseases.

[0257] On the other hand, there is provided a fusion oligonucleotide comprising at least one first oligonucleotide according to the first aspect linked to at least one second oligonucleotide according to the second aspect by a cleavable linker. Preferably, the fusion oligonucleotide comprises:

[0258] A-[Y-A] n

[0259] wherein

[0260] each A independently represents an oligonucleotide according to the first aspect or the second aspect, and each A may be the same or different, provided that at least one A is an oligonucleotide according to the first aspect and at least one additional A is an oligonucleotide according to the second aspect;

[0261] Y represents a cleavable linker, and each Y may be the same or different; and

[0262] n is equal to or greater than 1.

[0263] Preferably, Y is enzymatically cleavable. Preferably, Y is selected from the group consisting of: TEG linker, carbon spacer (such as C3, C6, C9, C 12 ), glycerol, and PolydT. More preferably, Y is a TEG linker.

[0264] Preferably, each A is independently bound to Y by an internucleotide bond (*). Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond may be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is phosphorothioate.

[0265] In another embodiment, a modified oligonucleotide is provided that comprises a synthetic oligonucleotide linked to at least one first oligonucleotide according to the first aspect and at least one second oligonucleotide according to the second aspect through one or more linkers. In another embodiment, a modified oligonucleotide is provided that comprises a synthetic oligonucleotide linked to a fusion oligonucleotide through a linker, wherein the fusion oligonucleotide comprises at least one first oligonucleotide according to the first aspect linked to at least one second oligonucleotide according to the second aspect through a linker. Preferably, the synthetic oligonucleotide is a therapeutic oligonucleotide and / or a diagnostic oligonucleotide, and more preferably a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. The linker may be cleavable or non-cleavable. Preferably, the linker is a cleavable linker.

[0266] The fusion oligonucleotide and the modified oligonucleotide can be used in the methods and uses of the first aspect and the second aspect described herein, and the fusion oligonucleotide and the modified oligonucleotide comprise at least one first oligonucleotide according to the first aspect linked to at least one second oligonucleotide according to the second aspect.

[0267] In a third aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0268] [mX / modified mX]* y X F * z X G

[0269] wherein:

[0270] * y and * z each independently represents an internucleotide bond;

[0271] X F and X G each independently is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified LX; where X F and X G at least one of which is dX, LX, rX, modified dX, or modified LX;

[0272] where mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0273] when mX is mC and X F is dG, X G is not mG or dG; or

[0274] when mX is mG and when:

[0275] X F is mU, mC, or dG, X G is not dT, dA, dG;

[0276] X F is dT, dA, or mA, X G is not dX;

[0277] X F is dG, X G is not dA, dG, dT;

[0278] where when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate internucleotide bond, and * z is not a 3'-5'-phosphodiester internucleotide bond;

[0279] wherein the sequence is optionally functionalized.

[0280] In one embodiment of the third aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0281] mX* y X F * z X G

[0282] wherein:

[0283] * y and * z each independently represents an internucleotide bond;

[0284] X F and X G each independently is selected from the group consisting of mX, dX, LX, modified mX, modified dX, and modified LX; wherein X F and X G at least one of which is dX, LX, modified dX, or modified LX;

[0285] wherein mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0286] when mX is mC and X F is dG, X G is not mG or dG; or

[0287] when mX is mG and when:

[0288] X F is mU, mC, or dG, X G is not dT, dA, dG;

[0289] X F is dT, dA, or mA, X G is not dX;

[0290] X F is dG, X G is not dA, dG, dT;

[0291] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate internucleotide bond, and * z is not a 3'-5'-phosphodiester internucleotide bond;

[0292] wherein the sequence is optionally functionalized.

[0293] Any oligonucleotide of the third aspect enhances TLR8 activity, preferably human TLR8 activity. In a preferred embodiment, the oligonucleotide of the third aspect substantially does not inhibit TLR7 activity, preferably human TLR7 activity. In an alternative preferred embodiment, the oligonucleotide of the third aspect inhibits TLR7 activity, preferably human TLR7 activity.

[0294] Each internucleotide bond may be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond and 5'-2'-bond. Preferably, each internucleotide bond may be selected from: 3'-5'-bond and 5'-5'-bond. Preferably, each internucleotide bond is a 3'-5'-bond.

[0295] In a particularly preferred embodiment of the third aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0296] 5'-[mX / modified mX]* y X F * z X G -3'

[0297] wherein:

[0298] * y and * z each independently represents an internucleotide bond;

[0299] X F and X G each independently is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX and modified LX; where X F and X G at least one of which is dX, rX, LX, modified dX or modified LX;

[0300] where mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0301] when mX is mC and X F is dG, X G is not mG or dG; or

[0302] when mX is mG and when:

[0303] X F is mU, mC or dG, X G is not dT, dA, dG;

[0304] X F When it is dT, dA or mA, X G is not dX;

[0305] X F When it is dG, X G is not dA, dG, dT;

[0306] wherein said sequence is optionally functionalized.

[0307] In a particularly preferred embodiment of the third aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0308] 5'-mX* y X F * z X G -3'

[0309] wherein:

[0310] * y and * z each independently represents an internucleotide bond;

[0311] X F and X G each independently is selected from the group consisting of mX, dX, LX, modified mX, modified dX and modified LX; wherein X F and X G at least one of which is dX, LX, modified dX or modified LX;

[0312] wherein mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0313] When mX is mC, X F is dG, X G is not mG or dG; or

[0314] When mX is mG and when:

[0315] X F is mU, mC or dG, X G is not dT, dA, dG;

[0316] X F is dT, dA or mA, X G is not dX;

[0317] X F is dG, X G is not dA, dG, dT;

[0318] wherein said sequence is optionally functionalized.

[0319] Preferably, each internucleotide bond is a 3'-5' bond.

[0320] Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is a phosphorothioate.

[0321] In a particularly preferred embodiment, each internucleotide bond is a 3'-5'-phosphorothioate bond.

[0322] In one embodiment, the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers. In another embodiment, the oligonucleotide of the third aspect comprises a single phosphorothioate stereoisomer, preferably wherein * z is in the R configuration.

[0323] Preferably, mX is a nucleotide comprising a 2'-OMe modification.

[0324] Modified dX and modified rX comprise at least one modification or substitution at the base and / or sugar position. Modified mX, modified moX, modified LX, and modified fX comprise at least one additional modification or substitution at an additional position of the base and / or sugar. Preferably, the modification or substitution is selected from the group consisting of: pseudouridine, 3'-deoxy, hydroxy, deamino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0325] Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mC1, m7 G, and N1-Me-G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), m7G is 3'-OMe-N7-methylated guanosine, and N1-Me-G (1-methylguanosine). Preferably, the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, and mC1.

[0326] Exemplary modified dX includes, but is not limited to: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, and PSU, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, and PSU is pseudouridine.

[0327] In one embodiment, X F and X G each independently is selected from the group consisting of: mX, dX, and LX; where X F and X G at least one of which is dX or LX.

[0328] In one embodiment, mX is selected from the group consisting of: mG, mC, and mU. In a preferred embodiment, mX is mG. In another preferred embodiment, mX is mC. In yet another preferred embodiment, mX is mU.

[0329] In one embodiment, X F is selected from the group consisting of: mX and dX. Preferably, X F is selected from the group consisting of: dC, dG, dA, dT, mG, mC, and mU. Preferably, X F is selected from the group consisting of: dC, dG, and mG. In a preferred embodiment, X F is dX, preferably dC.

[0330] In one embodiment, X G is selected from the group consisting of: dX and LX. Preferably, X G is selected from the group consisting of: dC, dT, dA, dG, LG, LC, LT, and LA. More preferably, X G is selected from the group consisting of: dX and LG, preferably, dX.

[0331] In one embodiment, X F and X G at least one of which is dX. In one embodiment, X F and X G one of which is dX. In a preferred embodiment, X F and XG Independently, it is dX.

[0332] In one embodiment, X F is selected from the group consisting of: dX and mX; and X G is dX. In another embodiment, X F is mX; and X G is selected from the group consisting of: dX and LX. In yet another embodiment, when X F is dC or mG, X F is dX or LX.

[0333] In one embodiment, the sequence can be functionalized. Preferably, the functionalized sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclic group, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclic group, substituted arylalkyl, and hydrophobic lipid. Preferably, the hydrophobic lipid is selected from cholesterol and tocopherol. Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, and tocopherol.

[0334] In one embodiment, the compound is directly conjugated to the sequence. In another embodiment, the compound is conjugated to the sequence via a linker. The linker can be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0335] Preferably, the compound is conjugated to the terminal nucleotide of the sequence, preferably the terminal 3'-nucleotide. Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'-position.

[0336] The functionalized sequence can comprise functionalized nucleotides selected from the group consisting of: dX-TEG, dX-Chol, and dX-Toco, where dX-TEG is a DNA base having triethylene glycol covalently linked to the 3'-position via a monophosphate group, dX-Chol is a DNA base having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position via a monophosphate group, and dX-Toco is a DNA base having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position via a monophosphate group.

[0337] Preferably, the sequence is selected from the group consisting of:

[0338]

[0339]

[0340] Preferably, the sequences are selected from the group consisting of: mG*dC*dC, mC*dC*dT, mG*dC*dA, mG*dC*dG, mC*dC*dC, mU*dC*dC, mC*dG*dC, mG*dC*dT, mG*mG*dA, mU*dC*dG, mU*mG*LG, mU*dC*dA, and mU*dC*dT.

[0341] More preferably, the sequences are selected from the group consisting of: mG*dC*dC, mC*dC*dT, mU*mG*LG, mC*dC*dC, mU*dC*dC, mG*dC*dA, mG*dC*dG, and mG*dC*dT. Even more preferably, the oligonucleotide is mG*dC*dC.

[0342] In one embodiment, the oligonucleotide consists of a sequence.

[0343] In another embodiment, the oligonucleotide contains a sequence. Preferably, the oligonucleotide containing the sequence has a length not exceeding 20 bases, preferably a length of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 bases. Preferably, the sequence is located at the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In a preferred embodiment, the oligonucleotide contains the sequence 5'-mG*mU*dC-3'. Even more preferably, the oligonucleotide contains the sequence 5'-mG*mU*dC*dC*dC*dC-3'.

[0344] In another embodiment of the third aspect, a method of modifying the TLR8 activity of an oligonucleotide is provided, the method comprising modifying the oligonucleotide by adding a sequence to the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In one embodiment, the method increases the TLR8 enhancing activity of the oligonucleotide. In another embodiment, the method reduces the TLR8 inhibitory activity of the oligonucleotide.

[0345] In another embodiment of the third aspect, a fusion oligonucleotide is provided, which comprises two or more oligonucleotides according to the third aspect linked by a cleavable linker. Preferably, the fusion oligonucleotide comprises:

[0346] A-[Y-A] n

[0347] where

[0348] each A independently represents an oligonucleotide according to the third aspect, and each A can be the same or different;

[0349] Y represents a cleavable linker, and each Y can be the same or different; and

[0350] n is equal to or greater than 1.

[0351] Preferably, Y is enzymatically cleavable. Preferably, Y is selected from the group consisting of: TEG linker, carbon spacer (such as C3, C6, C9, C 12 ), glycerol, and PolydT. More preferably, Y is a TEG linker.

[0352] Preferably, each A is independently bound to Y by an internucleotide bond (*). Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is a phosphorothioate.

[0353] In another embodiment of the third aspect, a modified oligonucleotide is provided, which comprises an agent linked to the oligonucleotide or fusion oligonucleotide according to the third aspect through a linker. The agent can be a therapeutic agent and / or a diagnostic agent. Preferably, the agent is a therapeutic agent, and more preferably, a therapeutic RNA selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamer, antisense oligonucleotide, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In a preferred embodiment, the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. The linker can be cleavable or non-cleavable. Preferably, the linker is a cleavable linker.

[0354] In another embodiment of the third aspect, a composition is provided, which comprises the oligonucleotide or fusion oligonucleotide according to the third aspect, and a pharmaceutically acceptable excipient.

[0355] In one embodiment, the composition is an immunogenic composition comprising a therapeutic RNA and the oligonucleotide or fusion oligonucleotide according to the third aspect. Preferably, the therapeutic RNA is selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamer, antisense oligonucleotide, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the immunogenic composition comprises the modified oligonucleotide according to the third aspect. Preferably, the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0356] In one embodiment, the composition can comprise at least one additional active agent selected from the following: gene targeting agents and TLR8 agonists.

[0357] In one embodiment of the third aspect, a method of enhancing TLR8 activity in a cell is provided, the method comprising contacting the cell with an oligonucleotide, fusion oligonucleotide or composition according to the third aspect, thereby enhancing TLR8 activity in a subject.

[0358] In another embodiment of the third aspect, a method of enhancing TLR8 activity in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the third aspect, thereby enhancing TLR8 activity in the subject.

[0359] In another embodiment of the third aspect, use of an oligonucleotide, fusion oligonucleotide or composition according to the third aspect in the preparation of a medicament for enhancing TLR8 activity in a subject is provided.

[0360] In another embodiment of the third aspect, use of a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the third aspect for enhancing TLR8 activity in a subject is provided.

[0361] In another embodiment of the third aspect, a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the third aspect for enhancing TLR8 activity in a subject is provided.

[0362] In another embodiment of the third aspect, a method of enhancing TLR8 activation in a subject caused by a therapeutic RNA is provided, the therapeutic RNA being selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the third aspect, thereby enhancing TLR8 activation in the subject. In one embodiment, the method includes administering an immunogenic composition comprising an oligonucleotide or fusion oligonucleotide according to the third aspect and a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof. In one embodiment, the immunogenic composition comprises a modified oligonucleotide according to the third aspect, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof.

[0363] In another embodiment of the third aspect, there is provided the use of an oligonucleotide, fusion oligonucleotide or composition according to the third aspect in the preparation of a medicament for enhancing TLR8 activation in a subject caused by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof. In one embodiment, the subject has received, is receiving or will receive a therapeutic RNA. In another embodiment, there is provided the use of a first composition and a second composition in the preparation of a medicament for enhancing TLR8 activation in a subject caused by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof, wherein the first composition comprises an oligonucleotide, fusion oligonucleotide or composition according to the third aspect, and the second composition comprises a therapeutic RNA. In another embodiment, there is provided the use of an immunogenic composition comprising a modified oligonucleotide according to the third aspect in the preparation of a medicament for enhancing TLR8 activation in a subject caused by a therapeutic RNA, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof.

[0364] On the other hand, there is provided the use of a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the third aspect for enhancing TLR8 activation in a subject caused by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof. In one embodiment, the subject has received, is receiving or will receive a therapeutic RNA. In another embodiment, there is provided the use of a therapeutically effective amount of an immunogenic composition comprising a modified oligonucleotide according to the third aspect for enhancing TLR8 activation in a subject caused by a therapeutic RNA, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA and combinations thereof.

[0365] On the other hand, there is provided a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the third aspect for enhancing TLR8 activation in a subject caused by a therapeutic RNA, wherein the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In one embodiment, the subject has received, is receiving, or will receive a therapeutic RNA. In another embodiment, there is provided a therapeutically effective amount of an immunogenic composition comprising the modified oligonucleotide according to the third aspect for enhancing TLR8 activation in a subject caused by a therapeutic RNA, wherein the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof.

[0366] In one embodiment, the oligonucleotide, fusion oligonucleotide or composition substantially does not increase the translation of the therapeutic RNA.

[0367] In one embodiment, the therapeutic RNA comprises pseudouridine.

[0368] In another embodiment, the therapeutic RNA does not comprise pseudouridine. In one embodiment of the third aspect, there is provided a method of treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signaling, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the third aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0369] In another embodiment of the third aspect, there is provided the use of the oligonucleotide, fusion oligonucleotide or composition according to the third aspect in the preparation of a medicament for treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signaling.

[0370] In another embodiment of the third aspect, there is provided the use of a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the third aspect for treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signaling.

[0371] In another embodiment of the third aspect, there is provided a therapeutically effective amount of the oligonucleotide, fusion oligonucleotide or composition according to the third aspect for preventing or treating a disease, disorder or condition in a subject responsive to increased TLR8 signaling.

[0372] Preferably, the oligonucleotide, fusion oligonucleotide or composition according to the third aspect activates or increases TLR8 signaling.

[0373] Preferably, the diseases, disorders or conditions responsive to increased TLR8 signaling are selected from the group consisting of: cancer, chronic viral (e.g., HBV) and bacterial infections.

[0374] In another embodiment of the third aspect, the method or use further comprises administering a TLR8 agonist. Preferably, the TLR8 agonist is administered at a sub-therapeutic dose.

[0375] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. Description of the Drawings

[0376] Figure 1 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 100 nM of the indicated oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and Dunnett's multiple comparison to the C2Mut1-dC condition are shown. All internucleotide linkages were phosphorothioates (only the first 4 internucleotide linkages are indicated by *).

[0377] Figure 2 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the indicated oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and Dunnett's multiple comparison to the 5-short-Mut1-Hyb condition are shown.

[0378] Figure 3: HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the indicated trimers for approximately 60 minutes and then stimulated overnight with R848 (1 μg / ml). The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as Dunnett's multiple comparison to the 2'OMe GUC condition are shown.

[0379] Figure 4 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the indicated trimers for approximately 60 minutes and then stimulated overnight with or without R848 (1 μg / ml). The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as Dunnett's multiple comparison to the R848 condition are shown.

[0380] Figure 5 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated dose of trimers for approximately 60 minutes and then stimulated overnight with R848 (1 μg / ml). mG*mU*mC was used at 500 nM in (A). The data shown are the mean of 1 independent experiment (A) or 3 independent experiments (B, C) in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as Dunnett's multiple comparison to the GUC condition are shown.

[0381] Figure 6 : Chemical structures of the nucleotides, trimers, and linked trimers used in the study. Chemical structures of the modified nucleotides compared to the parental GUC (mG*mU*mC) or parental GAG (mG*mA*mA) are shown.

[0382] Figure 7: (A and B) HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated trimers at 5 μM for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA, as well as Dunnett's multiple comparisons to the GUC (mG*mU*mC) condition, are shown.

[0383] Figure 8 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated dose of oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848.

[0384] Figure 9 : (A) HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. (B) HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated dose of oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA, as well as Dunnett's multiple comparisons to the R848 condition, are shown.

[0385] Figure 10: A) HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. B and C) HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated doses of the oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. A, B, and C) All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as Dunnett's multiple comparisons to the R848 (A, B) or NT (C) conditions are shown.

[0386] Figure 11 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated doses of the oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 3 biological replicates per screen. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. U should be understood as T of the DNA trimer.

[0387] Figure 12 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated doses of the oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 3 biological replicates per screen. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848.

[0388] Figure 13 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with the indicated doses of the oligonucleotide for approximately 60 minutes and then stimulated with R848 (indicated dose) overnight. The data shown are the mean of 3 biological replicates per screen. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848.

[0389] Figure 14: RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 μM oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as Dunnett's multiple comparison with the GGC or GAG condition are shown.

[0390] Figure 15 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with the indicated dose of oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM is shown.

[0391] Figure 16 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 500 nM GGC or non-GGC (NT) for approximately 60 minutes and then transfected with 500 nM B406AS1 ssRNA with DOTAP overnight. The data shown are the mean of 2 independent experiments in biological triplicate. The ELAM-luciferase values were reported relative to the NT condition. SEM and two-tailed unpaired t-test are shown.

[0392] Figure 17 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 μM DNA or 2'-OMe trimer for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 3 biological replicates per screen. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848.

[0393] Figure 18 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with the indicated dose of oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848.

[0394] Figure 19 : A) THP-1 was pretreated with the indicated doses of oligonucleotides for approximately 60 minutes, then stimulated with R848 (1 μg / mL) for 8 hours, and the supernatants were analyzed by IP-10 ELISA. B) HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated doses of oligonucleotides for approximately 60 minutes, then stimulated with Motolimod (600 nM) overnight. The data shown are the means of 3 biological replicates per screen. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values in B were reported relative to the Motolimod-only condition. All trimer conditions in A and B were co-stimulated with R848 or Motolimod, respectively.

[0395] Figure 20 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene (A, C) were pretreated with 5 μM of the indicated oligonucleotides for approximately 60 minutes, then stimulated with Motolimod (Mo) (600 nM) overnight. The data shown are the means of 2 independent experiments with biological triplicates. The NF-κB-luciferase values were reported relative to the Motolimod condition. All trimer conditions were co-stimulated with Motolimod. SEM and one-way ANOVA as well as Dunnett's multiple comparisons to the Motolimod-only condition are shown. THP-1 cells (B, D) were pretreated with 5 μM for approximately 60 minutes, then stimulated with 1 μg / ml of R848 overnight. Supernatants were collected and analyzed for IP-10 production by ELISA. The data shown are the means of 2 independent experiments with biological triplicates. SEM and one-way ANOVA as well as Dunnett's multiple comparisons to the R848-only (B) or NT (D) conditions are shown.

[0396] Figure 21 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated doses of oligonucleotides for approximately 60 minutes, then stimulated with Motolimod (400 for MOE and 600 nM for DNA) overnight. The data shown are the means of 3 biological replicates per screen. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the Motolimod condition. All trimer conditions were co-stimulated with Motolimod.

[0397] Figure 22: A) HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the oligonucleotide for approximately 60 minutes and then stimulated with motolimod (600 nM) overnight. The data shown are the mean of 3 biological replicates per screen. B) HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 500 nM of the selected oligonucleotide for approximately 60 minutes and then stimulated with motolimod (600 nM) overnight. The data shown are the mean of 2 independent experiments with biological triplicates. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod.

[0398] Figure 23 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 50 nM of the indicated oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments with biological triplicates. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons to the R848 condition are shown. All internucleotide linkages were phosphorothioates.

[0399] Figure 24 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with various doses of the indicated oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments with biological triplicates (except for GUC-v1, where data are from n = 1). Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM is shown.

[0400] Figure 25 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 2 μM of the indicated oligonucleotide for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments with biological triplicates. The NF-κB-luciferase values were reported relative to the R848 condition (background correction was not applied here). All trimer conditions were co-stimulated with R848. SEM is shown.

[0401] Figure 26: HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the trimer (A) modified with third-base LNA at a concentration of 5 μM or 400 nM or the trimer (B) modified with either full 2'-OMe or third-base LNA at 400 nM for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. All internucleotide linkages were phosphorothioates.

[0402] Figure 27 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated amounts of oligonucleotides (A, 200 nM, and B, 100 nM) for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. Except for the GUC-v1 sequence in.b from biological triplicate, the data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons of the R848 condition were shown. Figure 27 .b in the GUC-v1 sequence, the data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons of the R848 condition were shown.

[0403] Figure 28 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated amounts of oligonucleotides (A, 5 μM, and B, 1 μM) for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown in (A) and (B) are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons of the R848 condition were shown.

[0404] Figure 29 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with various doses of the indicated oligonucleotides or Enpatoran for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons of the R848 condition were shown.

[0405] Figure 30 : HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated amount of oligonucleotide (5 μM) for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 1 independent experiment in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM is shown.

[0406] Figure 31 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with a 100 nM dose of oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons to the R848 condition are shown. All internucleotide linkages are phosphorothioates (only the first 4 / 5 internucleotide linkages are indicated by *).

[0407] Figure 32 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with a 5 μM dose of oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons to the R848 condition are shown. All internucleotide linkages are phosphorothioates (only the first few internucleotide linkages are indicated by *).

[0408] Figure 33 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 μM of oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons to the R848 condition (non-significant comparisons are not shown) are shown.

[0409] Figure 34: RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 500 nM oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as multiple comparisons to the R848 condition are shown.

[0410] Figure 35 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 μM oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as multiple comparisons to the R848 condition are shown.

[0411] Figure 36 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 mM oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 1 experiment in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM is shown.

[0412] Figure 37 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 μM oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are from one experiment performed on two independent plates. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848.

[0413] Figure 38 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 1 μM oligonucleotide for approximately 60 minutes and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are from two independent experiments performed in biological triplicate. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848.

[0414] Figure 39 : TLR7 kika / wt BMDMs were treated overnight with 5 μM GGC-v1 or 200 nM emapatrilat, and RNA was analyzed by RNAseq (using 3 mice / condition). Typically, for each sample, there were approximately 2.8 million counts across 18,000 genes. One untreated sample from the Kika group was excluded from further analysis due to low counts. Statistical comparisons were performed using the contrasts.fit function from the limma package (v3.48.3), and empirical Bayes moderated t-tests were conducted. p-values were obtained using eBayes, and untreated TLR7 kika / wt BMDM cells were used as a reference.

[0415] Figure 40 : TLR7 kika / wt and WT BMDMs were treated overnight with 5 μM GGC-v1 or 200 nM emapatrilat, and RNA was analyzed by RTqPCR. Gene expression was normalized to 18S and then reported relative to the WT-NT condition. Each point represents RNA data from one mouse per condition.

[0416] Figure 41 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 μM oligonucleotide or 50 nM emapatrilat for approximately 60 minutes, then stimulated overnight with 0.5 μg / ml gardiquimod (A) and 0.5 μg / ml CL075 (B). The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then ELAM-luciferase values were reported relative to the gardiquimod (A) and CL075 (B) conditions. All trimer and emapatrilat conditions were co-stimulated with gardiquimod or CL075. SEM and one-way ANOVA and multiple comparisons with agonist conditions are shown.

[0417] Figure 42: A) 200 μg of GGCv1 trimer conjugated to JetPei (or PBS - grey and black dots) was injected i.v. into WT mice for 1 hour, followed by intraperitoneal treatment with or without 25 μg of R848. Serum was collected 2 hours after R848 treatment, and TNF levels were analyzed using beads by flow cytometry. RTqPCR analysis of the indicated genes normalized to 18S was performed on splenic mRNA. One - way ANOVA comparisons are shown. B) Six WT mice per group were treated with 20 μg of trimeric oligonucleotides dissolved in PBS, where 30% Pluronic F - 127 was applied on the ear, or 60 μg of trimeric oligonucleotides was applied on the back, followed by daily application of Aldara cream for 4 days. The Vaseline group (without Aldara) was used as a control. The ear thickness was measured daily with calipers, and ear redness and back hyperkeratosis were scored. On day 5, mice were sacrificed, and RNA was collected from the dorsal skin of 4 mice per group. The expression of the indicated genes relative to 18S in dorsal skin RNA was analyzed by RTqPCR and further reported relative to the Aldara + vehicle or Vaseline conditions. One - way ANOVA comparisons are shown.

[0418] Figure 43 : HEK - TLR8 cells expressing the NF - κB - luciferase reporter gene were pretreated with 5 μM of oligonucleotides for approximately 60 minutes and then stimulated with motolimod (600 nM) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF - κB - luciferase values were reported relative to the motolimod condition. All trimer conditions were co - stimulated with motolimod. SEM and one - way ANOVA as well as multiple comparisons with the GUC condition are shown.

[0419] Figure 44: A) HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 500 nM of the oligonucleotide for approximately 60 minutes and then stimulated with motolimod (600 nM) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod. SEM and one-way ANOVA as well as multiple comparisons with the GAG-v4 condition are shown. B) HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated concentrations of the GUC-v16 oligonucleotide for approximately 60 minutes and then stimulated with motolimod (600 nM) overnight. The data shown are the mean of 3 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod. SEM is shown.

[0420] Figure 45 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated doses of the oligonucleotide (A = 5 μM, B = 1 μM, C = 1 μM, and D = various doses) for approximately 60 minutes and then stimulated with motolimod (600 nM) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod. SEM and one-way ANOVA as well as multiple comparisons with the motolimod-only (A, B) or parental 2'OMe GAG (C) conditions are shown.

[0421] Figure 46 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 1 μM of the oligonucleotide (A) or 5 μM of the oligonucleotide (A and B) for approximately 60 minutes and then stimulated with motolimod (600 nM) overnight. The data shown are from two independent screens performed in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod.

[0422] Figure 47: HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 1 μM of the oligonucleotide or 5 μM of the oligonucleotide for approximately 60 minutes and then stimulated overnight with motolimod (600 nM). The data shown are from two independent screens performed in biological triplicates. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod. GAG-v1 (mGmAdG and mGdCdC) were used as positive controls for TLR8 inhibition and enhancement, respectively. The 5'-ends of all trimers were 2'-OMe.

[0423] Figure 48 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 1 μM of the oligonucleotide for approximately 60 minutes and then stimulated overnight with motolimod (600 nM). The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod. SEM and one-way ANOVA and multiple comparisons with the motolimod condition are shown.

[0424] Figure 49 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the oligonucleotide for approximately 60 minutes and then stimulated overnight with uridine (20 mM). The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the uridine condition. All trimer conditions were co-stimulated with uridine. SEM and one-way ANOVA and multiple comparisons with the uridine condition are shown.

[0425] Figure 50 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with the indicated dose of the oligonucleotide for approximately 60 minutes and then stimulated overnight with motolimod (600 nM). The data shown are the mean of 3 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the motolimod condition. All trimer conditions were co-stimulated with motolimod. SEM is shown.

[0426] Figure 51: HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the oligonucleotide for approximately 60 minutes and then stimulated with uridine (20 mM) overnight. The data shown are the mean of 2 independent experiments in biological triplicate. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the uridine condition. All trimer conditions were co-stimulated with uridine. SEM and one-way ANOVA as well as multiple comparisons with the uridine condition are shown.

[0427] Figure 52 : HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of mGdCdC or 1 μM dT20 for approximately 60 minutes and transfected overnight with 5 μg of total mouse RNA using DOTAP. The data shown are the mean of 2 independent experiments in biological triplicate. The NF-κB-luciferase values were reported relative to the NT control. SEM and unpaired t-test are shown.

[0428] Figure 53 : PMA-differentiated and IFNγ-sensitized THP-1 cells were transfected overnight with 750 nM of the indicated oligonucleotide with DOTAP, and the supernatants were analyzed by ELISA. The data shown are the mean of 2 independent experiments in biological triplicate. SEM and one-way ANOVA as well as multiple comparisons with the ssRNA40 condition are shown. All bases of the sequences are RNA with a PS backbone, and the bold bases are 2'-OMe.

[0429] Figure 54 : A) Undifferentiated THP-1 cells were pretreated with 5 μM of serum-free CleanCap AG trimer (GAG-linked) for approximately 60 minutes and then stimulated with R848 (1 μg / ml) for 8 hours and the supernatants were analyzed by ELISA. The data shown are the mean of 3 independent experiments in biological triplicate. B) HEK-TLR8 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of serum-free CleanCap AG trimer for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. The data shown are the mean of 2 (B) and 3 (A) independent experiments in biological triplicate. SEM and one-way ANOVA as well as multiple comparisons with the R848 condition are shown.

[0430] Figure 55: A) HEK-TLR7 cells expressing the NF-κB-luciferase reporter gene were pretreated with 5 μM of the indicated oligonucleotides without serum for approximately 60 minutes, and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF-κB-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as multiple comparisons to the R848 condition are shown. B) RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pretreated with 5 μM of the oligonucleotide without serum for approximately 60 minutes, and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as multiple comparisons to the R848 condition are shown.

[0431] Figure 56 : PMA-differentiated and IFNγ-sensitized THP-1 cells pretreated or not pretreated with 5 μM of GUC-v16 were transfected overnight with 1 μg of CleanCap AG EGFP mRNA (Trilink) with DOTAP, and the supernatants were analyzed by ELISA. The data shown are the mean of 2 independent experiments in biological triplicates. SEM and one-way ANOVA as well as multiple comparisons to the EGFP condition are shown.

[0432] Figure 57 : The HEK-TLR7 cells expressing the NF- K B-luciferase reporter gene were pretreated with 1 μM ( Figure 57 .A) or 200 nM ( Figure 57 .B) concentrations of the indicated oligonucleotides for approximately 60 minutes, and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF- K B-luciferase values were reported relative to the R848 condition. All oligonucleotide conditions were co-stimulated with R848. SEM and one-way ANOVA as well as multiple comparisons to the R848 condition are shown. All internucleotide linkages were phosphorothioates.

[0433] Figure 58 : The HEK-TLR7 cells expressing the NF- K B-luciferase reporter gene were pretreated with 5 μM ( Figure 64 .A) or 200 nM (Figure 64 .B) or 1 μM ( Figure 64 .C) or 50 nM ( Figure 64 .D) The indicated oligonucleotides at the indicated concentrations were pre-treated for approximately 60 minutes, and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates, except for ( Figure 64 .C) GUC-v1. Background correction was performed relative to the NT control, and then the NF- K B-luciferase values were reported relative to the R848 condition. All oligonucleotide conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons of the R848 condition are shown. All internucleotide linkages were phosphorothioates, except for GUC-v49 and GAG-v21 using PMO.

[0434] Figure 59 : HEK-TLR7 cells expressing the NF- K B-luciferase reporter gene were pre-treated with the indicated oligonucleotides at a concentration of 2 μM for approximately 60 minutes, and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF- K B-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons of the R848 condition are shown. All internucleotide linkages were phosphorothioates.

[0435] Figure 60 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pre-treated with 1 μM ( Figure 66 .A) or 200 nM ( Figure 66 .B) oligonucleotides for approximately 60 minutes, and then stimulated with R848 (0.125 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM is shown.

[0436] Figure 61 : RAW-ELAM stable cells expressing the ELAM-luciferase reporter gene were pre-treated with 5 μM ( Figure 67 .A) or 1 μM ( Figure 67.B) The oligonucleotides were pre-treated for approximately 60 minutes and then stimulated with R848 (0.125 ug / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the ELAM-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM is shown.

[0437] Figure 62 : HEK-TLR8 cells expressing the NF- K B-luciferase reporter gene were pre-treated with 1 μM ( Figure 62 .A and B) or 200 nM ( Figure 28 .C and D) of the indicated oligonucleotides for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF- K B-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons to the R848 condition are shown. All internucleotide linkages were phosphorothioates.

[0438] Figure 63 : HEK-TLR8 cells expressing the NF- K B-luciferase reporter gene were pre-treated with 5 μM ( Figure 63 .A) or 1 μM ( Figure 63 .B) of the indicated oligonucleotides for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates except for GAG-v1 in ( Figure 63 .B). Background correction was performed relative to the NT control, and then the NF- K B-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA and multiple comparisons to the R848 condition are shown. All internucleotide linkages were phosphorothioates.

[0439] Figure 64 : HEK-TLR8 cells expressing the NF- K B-luciferase reporter gene were pre-treated with 5 μM of the indicated oligonucleotides for approximately 60 minutes and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF- KB-luciferase values are reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA with multiple comparisons to the R848 condition are shown. All internucleotide linkages are phosphorothioates.

[0440] Figure 65 : Splenocytes from WT mice and mice expressing the functional human TLR8 receptor and non-functional TLR7 (B-hTLR8 / hTLR7) were pretreated or not with 5 μM of oligonucleotide 38-2 (mG*dC*dC) for 1 hour and then 1 μg / mL R848 was added overnight. TNF levels in the supernatant were measured by ELISA and expressed as fold increase relative to R848 alone. n = 3 mice ± SEM shown by one-way ANOVA.

[0441] Figure 66 : Healthy skin punch biopsies (3 mm x 3 mm) inserted into Transwell filters with the epidermis facing up at the air-liquid interface and the dermis suspended in medium were pretreated or not with 5 μM of oligonucleotide 38-2 (mG*dC*dC) for 30 minutes and then 600 nM motolimod was added for 24 hours. IL-8 levels in the supernatant were measured by ELISA and reported as fold increase relative to motolimod alone. Data shown are from 3 independent punch biopsies from 1 human donor ± SD.

[0442] Figure 67 : WT 129X1 / SvJ mice were injected i.v. with LNPs containing either FLuc mRNA alone or FLuc mRNA and GGC-v1. A, B, Bioluminescence imaging of luciferase expression was measured at 6 hours (A) and 24 hours (B) post-injection. C, Luciferase activity in liver homogenates at 24 hours post-injection. n = 5 mice / treatment group and 3 naive mice ± SEM shown by one-way ANOVA. Data are from 1 experiment.

[0443] Figure 68 : WT 129X1 / SvJ mice were injected i.v. with LNPs containing either FLuc mRNA alone or FLuc mRNA and GGC-v1. Serum levels of A, IFN-α, B, IL-6, C, IFN-γ, D, RANTES were measured by (A) ELISA or (B-D) Bio-Plex. n = 5 mice / treatment group and 3 naive mice ± SEM shown by one-way ANOVA. Data are from 1 experiment.

[0444] Figure 69 : Expressing NF- KHEK-TLR7 (A) and HEK-TLR8 (B) cells with B-luciferase reporter gene were pretreated with the indicated oligonucleotides at a concentration of 1 μM or 5 μM for about 60 minutes, and then stimulated with R848 (1 μg / ml) overnight. The data shown are the mean of 2 independent experiments in biological triplicates. Background correction was performed relative to the NT control, and then the NF- K B-luciferase values were reported relative to the R848 condition. All trimer conditions were co-stimulated with R848. SEM and one-way ANOVA as well as multiple comparisons to the R848 condition were shown. Detailed Description

[0445] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or apparent in the text or drawings. All such different combinations constitute various alternative aspects of the invention.

[0446] Definitions

[0447] For the purpose of interpreting this specification, the terms used in the singular form will also include the plural form, and vice versa.

[0448] As used herein, unless the context requires otherwise, the term "comprise" and variations of said term, such as "comprising", "comprises" and "comprised" are not intended to exclude additional additives, components, integers or steps.

[0449] As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of the oligonucleotides described herein to alleviate or eliminate at least one symptom of a disease, disorder or condition. Treating a subject includes delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, reducing exacerbation, improving, enhancing or affecting a disease or condition, the signs or symptoms of a disease or condition or the risk (or susceptibility) of a disease or condition. The term "treatment" refers to any measure of successful treatment or improvement of an injury, pathology or condition, including any objective or subjective parameter, such as alleviation; relief; reduced rate of exacerbation; reduced severity of a disease; stabilization, diminution of signs or symptoms or making an injury, pathology or condition more tolerable to an individual; slowing the rate of exacerbation or decline; making the terminal stage of exacerbation less debilitating.

[0450] In a particularly preferred embodiment, the method of the invention can prevent the signs or symptoms of a disease or condition as described herein, or reduce the severity of said signs or symptoms or inhibit the progression of said signs or symptoms or minimize said progression. Thus, the method of the invention can be used for treatment as well as prevention.

[0451] As used herein, the terms "preventing", "prevent" or "prevention" include administering a therapeutically effective amount of the oligonucleotides described herein, sufficient to prevent or impede the development of at least one symptom of a disease, disorder or condition. As used herein, "prevention" is intended to refer to at least reducing the likelihood of acquiring a disease or disorder (or susceptibility thereto) (i.e., preventing at least one of the clinical signs or symptoms of the disease from occurring in an individual who may be exposed to or is predisposed to the disease but has not yet experienced the disease or shown signs or symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known to physicians.

[0452] As used herein, the terms "subject", "individual" or "patient" may be used interchangeably with one another. The term "subject" refers to an animal that can be treated by the oligonucleotides and / or methods. In one example, the animal is a vertebrate. For example, the animal can be a mammal, avian, chordate, amphibian or reptile. Exemplary subjects include, but are not limited to, humans, primates, domestic animals (e.g., sheep, cattle, chickens, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g., foxes, deer). In one example, the mammal is a human.

[0453] When referring to measurable values such as amounts, durations, etc., as used herein, "about" is intended to cover variations of ±20% or ±10% relative to the specified value, in some instances ±5%, in some instances ±1% and in some instances ±0.1%, since such variations are appropriate for carrying out the disclosed methods.

[0454] Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as the individual values within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, for example 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This applies regardless of the width of the range.

[0455] The terms "reduce" or "inhibit" can generally refer to the ability of one or more of the oligonucleotides described herein to "decrease" a related physiological or cellular response (such as the symptoms of a disease or disorder described herein), as measured according to conventional techniques in the field of diagnosis. The related physiological or cellular response (in vivo or in vitro) will be apparent to those skilled in the art and can include alleviation of the symptoms or pathology of a disease. The "reduction" of the response can be statistically significant compared to the response produced by an oligonucleotide-free or control composition and can include at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% reduction, including all integers therebetween.

[0456] As used herein, the phrase "inhibit TLR7 activity" or variants thereof means that after administration of the oligonucleotides of the invention to a subject, the subject is unable to mount a TLR7-based immune response, or is only able to mount a reduced or partially TLR7-based immune response, such as an immune response to a pathogen or damaged endogenous nucleic acid. In one embodiment, the TLR7-based immune response is less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the response in the absence of the oligonucleotide. In one embodiment, the oligonucleotides of the invention inhibit or reduce TLR7 activity by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0457] As used herein, the phrase "inhibit TLR8 activity" or variants thereof means that after administration of the oligonucleotides of the invention to a subject, the subject is unable to mount a TLR8-based immune response, or is only able to mount a reduced or partially TLR8-based immune response, such as an immune response to a pathogen or damaged endogenous nucleic acid. In one embodiment, the TLR8-based immune response is less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the response in the absence of the oligonucleotide. In one embodiment, the oligonucleotides of the invention inhibit or reduce TLR8 activity by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0458] As used herein, the phrase "substantially does not reduce the translation of therapeutic RNA" or variations thereof means that the level of translation of therapeutic RNA in a subject is comparable in the presence or absence of the oligonucleotides of the invention. In the presence of the oligonucleotides of the invention, the level of translation of therapeutic RNA is about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% or 80% of the level of translation of therapeutic RNA in the absence of the oligonucleotides of the invention. In the absence of the oligonucleotides of the invention, the level of translation of therapeutic RNA may be referred to as the reference level of translation of therapeutic RNA. Those skilled in the art will be familiar with methods for obtaining the reference level of oligonucleotide translation. For example, the method may include obtaining data from a plurality of individuals to develop an appropriate reference data set. Alternatively, the reference level may be generated from the same individual at different time points, such as before administration of the therapeutic RNA, after administration of the therapeutic RNA, before administration of the oligonucleotides of the invention, after administration of the oligonucleotides of the invention, or combinations thereof.

[0459] The term "enhance" refers to an increase in a functional property relative to a control condition. The term "enhance" generally relates to the ability of one or more of the oligonucleotides described herein to "increase" the potency or potential of an existing immune response in a subject. For example, an increase in potency and efficacy can be achieved by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0460] As used herein, the phrase "increase or enhance TLR8 activity" or variations thereof means that after administration of the oligonucleotides of the invention to a subject, the subject is capable of eliciting only a TLR8-based immune response, or is capable of eliciting an increased or elevated TLR8-based immune response, such as an immune response to a pathogen, damaged endogenous nucleic acid, or exogenous TLR8 ligand. The enhancement of TLR8 activity can be greater than about 100%, such as about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 20-fold or about 50-fold. Preferably, the level of TLR8 enhancement is between about 2-fold and 50-fold, between about 2-fold and 20-fold, and / or between about 5-fold and greater than 20-fold.

[0461] As used herein, the terms "disease", "disorder" or "condition" refer to any unhealthy or abnormal state.

[0462] The term "disease, disorder, or condition of a subject responsive to TLR7 inhibition" includes diseases, disorders, and conditions for which inhibition of TLR7 provides a therapeutic benefit. This includes diseases, disorders, or conditions associated with increased TLR7 signaling. This also includes diseases, disorders, or conditions in which TLR7 signaling exacerbates an abnormal autoimmune response. Diseases, disorders, or conditions responsive to TLR7 inhibition include inflammation-related diseases, allergic diseases, infections, cancers, and autoimmune diseases.

[0463] The term "disease, disorder, or condition of a subject responsive to TLR8 inhibition" includes diseases, disorders, and conditions for which inhibition of TLR8 provides a therapeutic benefit. This includes diseases, disorders, or conditions associated with increased TLR8 signaling. This also includes diseases, disorders, or conditions in which TLR8 signaling exacerbates an abnormal autoimmune response. Diseases, disorders, or conditions responsive to TLR8 inhibition include inflammation-related diseases, allergic diseases, infections, cancers, and autoimmune diseases.

[0464] The term "disease, disorder, or condition of a subject responsive to increased TLR8 signaling" includes diseases, disorders, and conditions for which activation of TLR8 provides a therapeutic benefit, such as cancers, chronic viral (e.g., HBV) and bacterial infections.

[0465] The term "TLR8 agonist" refers to an agent capable of eliciting a signaling response through the TLR8 signaling pathway, either as a direct ligand or indirectly as a ligand by generating an endogenous or exogenous ligand. Such natural or synthetic TLR8 agonists can be used as alternative or additional adjuvants. For example, TLR8 agonists capable of eliciting a signaling response through TLR8 are single-stranded RNA (ssRNA), imidazoquinoline molecules with antiviral activity, such as resiquimod (R848). Other TLR-8 agonists that can be used include those described in WO 2004 / 071459 and WO2021 / 232099.

[0466] The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, and / or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0467] The terms "therapeutically effective amount" and "effective amount" describe an amount of a particular agent, such as an oligonucleotide of the present invention, sufficient to achieve a desired effect in a subject or cell treated with or exposed to the agent. For example, this can be an amount of a composition comprising an agent that inhibits the activity of one or more nucleic acid sensors described herein (e.g., TLR7 or TLR8), which amount is necessary to reduce, alleviate, and / or prevent a disease, disorder, or condition. In some embodiments, a "therapeutically effective amount" is sufficient to alleviate or eliminate the symptoms of a disease, disorder, or condition. In other embodiments, a "therapeutically effective amount" or "effective amount" refers to an amount sufficient to achieve a desired biological effect, e.g., an amount that is effective to reduce or prevent an age-related disease, disorder, or condition or to inhibit or prevent cellular senescence.

[0468] Ideally, the therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing substantial cytotoxic effects in the subject. The effective amount of an agent for reducing, alleviating, and / or preventing a disease, disorder, or condition will depend on the subject to be treated, the type and severity of the relevant symptoms, and the mode of administration of the therapeutic composition.

[0469] A sub-therapeutic dose is a dose that fails to achieve a therapeutic goal. The goal can be, for example, reducing inflammation, minimizing an allergic response, reducing an infection, reducing tumor size, reducing an increase or decrease in cancer biomarker expression, arresting tumor growth, or reducing, alleviating, or eliminating the symptoms of an autoimmune disease. Preferably, a sub-therapeutic dose is a dose that does not cause significant adverse side effects in the subject.

[0470] As used herein, the term "alkyl" refers to, in some embodiments, a single-bonded chain of hydrocarbons in the range of 1-20 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms, and any range therebetween. The terms "C1-C2 alkyl", "C1-C3 alkyl", and "C1-C6 alkyl" refer to alkyl as defined herein and containing at least 1, and at most 2, 4, or 6 carbon atoms, respectively, or any range therebetween (e.g., an alkyl containing 2-5 carbon atoms is also within the range of C1-C6). Examples of alkyl as used herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, 2,2-dimethylbutyl. Preferably, the alkyl is C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C1 alkyl. Preferably, the alkyl is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl.

[0471] As used herein, the term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group having one or more double bonds and, unless otherwise specified, containing from about 2 to about 20 carbon atoms, and in some embodiments in the range of about 2 to about 10 carbon atoms, and in some embodiments in the range of about 2 to about 8 carbon atoms, and in some embodiments in the range of about 2 to about 6 carbon atoms. Examples of alkenyl include vinyl, allyl, 1,4-butadienyl, isopropenyl, and the like. Preferably, the alkenyl is C2-C 20 alkenyl, C2-C 10 alkenyl, C2-C8 alkenyl, C2-C6 alkenyl. Preferably, the alkenyl is C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl.

[0472] As used herein, the term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon group having one or more triple bonds and, unless otherwise specified, containing from about 2 to about 20 carbon atoms, and in some embodiments in the range of about 2 to about 10 carbon atoms, and in some embodiments in the range of about 2 to about 8 carbon atoms, and in some embodiments in the range of about 2 to about 6 carbon atoms. Examples of alkynyl include ethynyl, propynyl, butynyl, and the like. Preferably, the alkenyl is C2-C 20 alkynyl, C2-C 10 alkynyl, C2-C8 alkynyl, C2-C6 alkynyl. Preferably, the alkynyl is C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl.

[0473] As used herein, the term "amino" or "amine" refers to the group -NH2.

[0474] As used herein, the term "hydroxy" or "hydroxyl" refers to the group -OH.

[0475] As used herein, the term "oxo" refers to an oxygen substituent bonded to the attached carbon.

[0476] As used herein, the term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I), and the term "halo" refers to the halogen groups fluoro (-F), chloro (-Cl), bromo (-Br), and iodo (-I). Preferably, 'halo' is fluorine, chlorine, or bromine.

[0477] As used herein, the term "substituted" means that any one or more hydrogens on the specified atom are replaced by a selection from the indicated substituents, provided that the normal valence of the specified atom is not exceeded and the substitution results in a stable compound, i.e., a compound that can be isolated, characterized, and tested for biological activity.

[0478] The terms "substituted hydrocarbyl", "substituted alkyl", "substituted alkenyl", "substituted alkynyl" refer to any of the above hydrocarbyls, including "alkyl", "alkenyl", "alkynyl", further carrying one or more substituents selected from the following: hydroxy, hydrocarbyloxy, substituted hydrocarbyloxy, alkylthio, substituted alkylthio, arylthio, substituted arylthio, amino, alkylamino, substituted alkylamino, carboxy, -C(S)SR, -C(O)SR, -C(S)NR2, -OR, where each R is independently hydrogen, alkyl or substituted alkyl, nitro, cyano, halogen, -SO3M or -OSO3M, where M is H, Na, K, Zn, Ca or meglumine, guanidino, substituted guanidino, hydrocarbyl, substituted hydrocarbyl, hydrocarbylcarbonyl, substituted hydrocarbylcarbonyl, hydrocarbyloxycarbonyl, substituted hydrocarbyloxycarbonyl, hydrocarbylcarbonyloxy, substituted hydrocarbylcarbonyloxy, acyl, acyloxy, heterocycle, substituted heterocycle, heteroaryl, substituted heteroaryl, heteroaryl-carbonyl, substituted heteroarylcarbonyl, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, carbamate, dithiocarbamate, aroyl, substituted aroyl, organic sulfonyl, substituted organic sulfonyl, organic sulfinyl, substituted alkanesulfinyl, alkylsulfonylamino, substituted alkylsulfonylamino, arylsulfonylamino, substituted arylsulfonylamino, sulfonamido, sulfonyl, etc., including two or more of the above groups connected to the hydrocarbyl moiety through a linker / spacer moiety such as -O-, -S-, -NR-, where R is hydrogen, alkyl or substituted alkyl, -C(O)-, -C(S)-, C(=NR')-, -C(=CR'2)-,wherein R' is alkyl or substituted alkyl, -O-C(O)-, -O-C(O)-O-, -O-C(O)-NR-(or -NR-C(O)-O-), -NR-C(O)-, -NR-C(O)-NR-, -S-C(O)-, -S-C(O)-O-, -S-C(O)-NR-, -O-S(O)2-, -O-S(O)2-O-, -O-S(O)2-NR-, -O-S(O)-, -O-S(O)-O-, -O-S(O)-NR-, -O-NR-C(O)-, -O-NR-C(O)-O-, -O-NR-C(O)-NR-, -NR-O-C(O)-, -NR-O-C(O)-O-, -NR-O-C(O)-NR-, -O-NR-C(S)-, -O-NR-C(S)-O-, -O-NR-C(S)-NR-, -NR-O-C(S)-, -NR-O-C(S)-O-, -NR-O-C(S)-NR-, -O-C(S)-, -O-C(S)-O-, -O-C(S)-NR-(or -NR-C(S)-O-), NR-C(S)-, -NR-C(S)-NR-, -S-S(O), 2- -S-S(O)2-O-, -S-S(O)2-NR-, -NR-O-S(O)-, -NR-O-S(O)-O-, -NR-O-S(O)-NR-, -NR-O-S(O)2, -NR-O-S(O)2-O-, -NR-O-S(O)2-NR-, -O-NR-S(O)-, -O-NR-S(O)-O-, -O-NR-S(O)-NR-, -O-NR-S(O)2-O-, -O-NR-S(O)2-NR-, -O-NR-S(O)-, -O-P(O)R2, -S-P(O)R2 or -NR-P(O)R2, where each R is independently hydrogen, alkyl or substituted alkyl, etc. Preferably, one or more substituents are selected from the group consisting of: hydroxy, carboxy, amino, thio, halo and -OR, where R is alkyl, alkenyl or alkynyl. Even more preferably, "substituted alkyl" is "substituted C1-C6 alkyl", "substituted alkenyl" is "substituted C2-C 20 alkenyl", and "substituted alkynyl" is "substituted C2-C 20 alkynyl".

[0479] As used herein, a hydrophobic lipid refers to an amphiphilic molecule comprising a polar head group and a hydrophobic tail. The hydrophobic tail may comprise a hydrocarbon chain selected from the group consisting of: C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, substituted C1-C20 alkyl, substituted C2-C 20 alkenyl, and substituted C2-C 20 alkynyl. Hydrophobic lipids include, but are not limited to, phospholipids, cholesterol, cholesterol derivatives, and tocopherols.

[0480] As used herein, polyethylene glycol (PEG) refers to a polyether compound. Preferably, PEG comprises H(OCH2CH2) n OH, where n is from 1 to 20, inclusive of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Preferably, n is from 1 to 6.

[0481] The stereochemical definitions and conventions used herein generally follow those in the McGraw-Hill Dictionary of Chemical Terms, edited by S.P. Parker (McGraw-Hill Book Company, New York, 1984); and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds" (John Wiley & Sons, Inc., New York, 1994). The oligonucleotides of the present invention may contain asymmetric or chiral centers and may thus exist in different stereoisomeric forms. The term "stereoisomer" refers to oligonucleotides having the same chemical composition but different arrangements of atoms or groups in space. As used herein, the term "stereoisomer" includes, but is not limited to, diastereoisomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemic mixtures.

[0482] Oligonucleotide

[0483] In the context of the present invention, the term "oligonucleotide" refers to a ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA) oligomer or polymer, where the polymer or oligomer of nucleotide monomers contains any combination of nucleotides (referred to herein and in the art simply as "bases"), modified nucleotides, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages").

[0484] As used herein, a "target", such as a "target polynucleotide", refers to a molecule on which the oligonucleotides of the present invention act directly or indirectly. Generally, the oligonucleotides of the present invention or portions thereof interact or bind to the target under physiological conditions, thereby modulating the function of the target. In a first aspect, the target is TLR7. In second and third aspects, the target is TLR8.

[0485] As used herein, the term "nucleotide" includes all naturally occurring nucleotides, including all forms of nucleobases found in nature. The base rings most commonly found in naturally occurring nucleotides are the purine ring and the pyrimidine ring. Naturally occurring purine rings include, for example, adenine, guanine, and N 6 -methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, pseudouracil. For example, naturally occurring nucleotides include, but are not limited to, ribose, 2'-O-methyl or 2'-deoxyribose derivatives of adenosine, guanosine, thymidine, uridine, inosine, 7-methylguanosine or pseudouridine.

[0486] As used herein, the term "modifying group" or "modified" refers to any chemical moiety that can be attached to an oligonucleotide at a position including, but not limited to, the sugar, nucleobase, triphosphate bridge, and / or internucleotide phosphate.

[0487] As used herein, the terms "nucleotide analog", "modified nucleotide", or "nucleotide derivative" include the synthetic nucleotides described herein. Nucleotide derivatives also include nucleotides having modified bases and / or sugar moieties, with or without protecting groups, and include, for example, 2'-deoxy-2'-fluorouridine, 5-fluorouridine, etc. Other nucleotide derivatives that can be used in the present invention include, for example, LNA nucleotides, halogen-substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N 6 -ethyladenine, N 4 -(alkyl)-cytosine, 5-ethylcytosine, etc.

[0488] Generally, the oligonucleotides of the present invention will be synthesized in vitro.

[0489] Base

[0490] RNA nucleotides (rX) contain 2'-OH. DNA nucleotides (dX) contain 2'-H.

[0491] Modified nucleotides can include nucleotides having modified bases and / or sugar moieties.

[0492] As used herein, "mX" refers to a nucleotide comprising a 2'-methoxy and / or 3'-methoxy (2'-OMe and / or 3'-OMe) modification. The mX nucleotide at the terminal position of the sequence can be 2'-OMe or 3'-OMe, preferably 2'-OMe. The mX nucleotide at a non-terminal position of the sequence can be 2'-OMe. In a preferred embodiment, mX is 2'-OMe.

[0493] As used herein, "moX" refers to a nucleotide comprising a 2'-methoxyethoxy and / or 3'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE and / or (3'-O-CH2CH2OCH3, also known as 3'-O-(2-methoxyethyl) or 3'-MOE) modification. The moX nucleotide at the terminal position of the sequence can be 2'-OMe or 3'-MOE, preferably 2'-MOE. The moX nucleotide at a non-terminal position of the sequence can be 2'-MOE. In a preferred embodiment, moX is 2'-MOE.

[0494] As used herein, "fX" refers to a nucleotide comprising a 2'-fluoro and / or 3'-fluoro modification. The fX nucleotide at the terminal position of the sequence can be 2'-fluoro or 3'-fluoro, preferably 2'-fluoro. The fX nucleotide at a non-terminal position of the sequence can be 2'-fluoro. In a preferred embodiment, fX is 2'-fluoro.

[0495] As used herein, "LX" refers to a nucleotide comprising a locked nucleic acid (LNA) modification. LNA is a nucleic acid in which the 2'-hydroxyl is linked to the 3'- or 4'-carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In one embodiment, the bond is a methylene group (-CH2-) bridging the 2'-oxygen atom and the 4'-carbon atom n group, where n is 1 or 2, preferably 1.

[0496] As used herein, "morpholino-X" refers to a nucleotide comprising a 1-oxa-4-azacyclohexane ribose ring (also known as a morpholine ring). The morpholine ring carries a methylene group bound to a modified phosphate ester, where the anionic oxygen is replaced by a non-ionic dimethylamino group. The substituted phosphate ester is bound to the nitrogen atom of another morpholine ring by a phosphorus-nitrogen bond. A standard DNA or RNA nucleobase (adenine, guanine, cytosine, thymine or uracil) is bound to each morpholine ring.

[0497] As used herein, "modified nucleotide", including "modified dX", "modified rX", "modified mX", "modified moX", "modified fX", "modified LX", refers to a nucleotide that includes at least one modification or substitution. The modification or substitution can be of rX or dX, or can be an additional modification or substitution of a nucleotide that is already modified (such as mX, moX, fX or LX). Modified nucleotides can include modifications or substitutions at the base and / or sugar positions. For example, modified dX refers to a DNA base that includes at least one modification or substitution at one or more positions of the base and / or sugar. Modified rX refers to an RNA base that includes at least one modification or substitution at one or more positions of the base and / or sugar. Modified mX refers to a nucleotide that includes 2'-OMe and / or 3'-OMe modifications, preferably 2'-OMe modification, and at least one additional modification or substitution at one or more positions of the base and / or sugar. Modified moX refers to a nucleotide that includes 2'-MOE and / or 3'-MOE modifications, preferably 2'-MOE modification, and at least one additional modification or substitution at one or more positions of the base and / or sugar. Modified fX refers to a nucleotide that includes 2'-fluoro and / or 3'-fluoro modifications, preferably 2'-fluoro modification, and at least one additional modification or substitution at one or more positions of the base and / or sugar. Modified LX refers to a nucleotide that includes LNA modification, and at least one additional modification or substitution at one or more positions of the base and / or sugar.

[0498] Preferably, the modification or substitution is selected from the group consisting of: pseudouridine, 3'-deoxy, hydroxy, deamino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0499] In one embodiment, the modification or substitution is at one or more positions of the sugar. The modification or substitution of the sugar can be selected from the group consisting of: 3'-deoxy, hydroxy, amino, thio, halo, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0500] In one embodiment, the modification or substitution is at one or more positions of the base. The modification or substitution of the base can be selected from the group consisting of: pseudouridine, hydroxy, deamino, amino, thio, halo, oxo, aza, deaza, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, and combinations thereof.

[0501] In one embodiment, the modification or substitution is located at one or more positions of the base and at one or more positions of the sugar.

[0502] Modified nucleotides include, for example, 2'-OMe-2,6-diaminopurine (referred to herein as mG1), 2'-OMe-I (2'-O-methylinosine, which may be interchangeably referred to herein as mI or mG2), 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine, referred to herein as mU1), 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine, referred to herein as mU2), N3-Me-U (3-methyluridine, referred to herein as mU3), 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine, referred to herein as mC1), N7-methylated guanosine (referred to herein as m7 G), 5-methyl-substituted deoxycytidine (referred to herein as 5-Me-dC), 5-bromo-substituted deoxycytidine (referred to herein as 5-Br-dC), 5-hydroxymethyl-substituted deoxycytidine (referred to herein as 5-CH2OH-dC), 2'-deoxy-3'-deoxycytidine (referred to herein as ddC), 5-propynyl-substituted deoxycytidine (referred to herein as pdC), 3-methyl deoxycytidine (referred to herein as N3-Me-dC), 5-iodo deoxycytidine (referred to herein as 5-I-dC), deoxyinosine (referred to herein as dI), 8-bromo deoxyguanosine (referred to herein as 8-Br-dG), 7-deaza deoxyguanosine (referred to herein as 7-deaza-dG), 8-bromo deoxyadenosine (referred to herein as 8-Br-dA), 8-oxo deoxyadenosine (referred to herein as 8-oxo-dA), O6-methyl deoxyguanosine (referred to herein as O6-Me-dG), 8-amino deoxyguanosine (referred to herein as 8-NH2-dG), 2'-amino-2'-deoxyuridine (referred to herein as 2'-NH2-U), 2'-amino-2'-deoxycytidine (referred to herein as 2'-NH2-C), cytarabine (referred to herein as ara-C).

[0503] As used herein, modified dG includes, but is not limited to, 2,6-diaminopurine and inosine. Modified mG includes, but is not limited to: mG1, mI, and m7 G, where mG1 is 2'-OMe-2,6-diaminopurine, mI or mG2 is 2'-OMe-I (2'-O-methylinosine), and m7 G is 3'-OMe-N7-methylated guanosine. Preferably, the modified mG includes mG1 and mI.

[0504] The oligonucleotides of the present invention include nucleotides modified with at least one 2'-OMe or 3'-OMe, preferably nucleotides modified with 2'-OMe.

[0505] References to A, T, G, U or C can mean a naturally occurring base or a modified version thereof, unless stated to the contrary.

[0506] Backbone

[0507] The oligonucleotides of the present disclosure include oligonucleotides having a modified backbone or unnatural internucleoside linkages.

[0508] As used herein, the term "internucleoside linkage" refers to a linkage that connects two nucleotides of an oligonucleotide or nucleic acid and can be a natural phosphodiester linkage or a modified linkage. Internucleoside linkages include, but are not limited to: diphosphate esters, triphosphate esters, phosphorothioate esters, phosphodiesters, phosphoramidothioates, phosphodiamides, methylphosphonates, and guanidinopropylaminophosphates. In one embodiment, each internucleoside linkage is independently selected from the group consisting of diphosphate esters, triphosphate esters, phosphorothioate esters, and phosphodiesters. Preferably, each internucleoside linkage is independently selected from the group consisting of triphosphate esters, phosphorothioate esters, and phosphodiesters. More preferably, each internucleoside linkage is independently selected from phosphorothioate esters and phosphodiesters. Even more preferably, each internucleoside linkage is a phosphorothioate ester.

[0509] Each internucleoside linkage can be the same or different. In a preferred embodiment, each internucleoside linkage is the same, preferably a phosphorothioate ester.

[0510] A nucleotide internucleoside bond can be introduced at the 2'-end, 3'-end or 5'-end of a nucleotide. Each nucleotide internucleoside bond can be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond, 5'-2'-bond. Preferably, each nucleotide internucleoside bond is selected from the group consisting of: 3'-5'-bond and 5'-5'-bond. For example, a 3'-5'-thiolate phosphate nucleotide internucleoside bond can be formed by coupling the 3'-phosphate of a first nucleotide and the 5'-hydroxyl of a second nucleotide, where the non-bridging oxygen is replaced by sulfur. A 3'-5'-phosphodiester nucleotide internucleoside bond can be formed by coupling the 3'-hydroxyl of a first nucleotide and the 5'-phosphate of a second nucleotide. A 5'-5'-triphosphate nucleotide internucleoside bond can be formed by an additional phosphate group by coupling the 5'-phosphate of a first nucleotide and the 3'-phosphate of a second nucleotide. In one embodiment, the sequence includes a 5'-5'-bond. Preferably, the 5'-5'-bond connects the nucleotides at the first and second positions of the sequence. In one embodiment, the sequence includes a 5'-5'-bond and a 3'-5'-bond. Preferably, the 5'-5'-bond connects the nucleotides at the first and second positions of the sequence, and the 3'-5'-bond connects the nucleotides at the second and third positions of the sequence. In a preferred embodiment, each nucleotide internucleoside bond is a 3'-5'-bond.

[0511] In a particularly preferred embodiment, each nucleotide internucleoside bond is a 3'-5'-thiolate phosphate bond.

[0512] Each nucleotide internucleoside bond can contain an asymmetric or chiral center and can thus exist in different stereoisomeric forms. The oligonucleotides of the present invention can contain a mixture of different oligonucleotide stereoisomers. Due to the chirality introduced by the two sulfur atoms in the PS nucleotide internucleoside bond, a trimer oligonucleotide of the present invention containing a thiolate phosphate nucleotide internucleoside bond can contain a mixture of up to 4 different oligonucleotide thiolate phosphate stereoisomers. For example, 5'-[mX / modified mX]* y X A * z X B -3' can contain up to 4 different stereoisomers generated by different thiolate phosphate stereochemical configurations at each nucleotide internucleoside bond, where * y and * z each represent a thiolate phosphate nucleotide internucleoside bond, where * y and * z each have an R configuration (RR), * y and * z each have an S configuration (SS), * yin the R configuration and * z in the S configuration (RS), and * y in the S configuration and * z in the R configuration (SR). The oligonucleotides of the invention can comprise a single phosphorothioate stereoisomer, or a mixture of 2 to 4 different oligonucleotide phosphorothioate stereoisomers, preferably a 1:1:1:1 mixture of 4 different oligonucleotide phosphorothioate stereoisomers. The oligonucleotides can comprise additional chiral centers, such as on one or more riboses, and can thus comprise a mixture of stereoisomers or a single stereoisomer at the additional chiral centers.

[0513] Functionalized sequence

[0514] In one embodiment, the sequence can be functionalized. Preferably, the functionalized sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclic group, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclic group, substituted arylalkyl, and hydrophobic lipid. Preferably, the compound is selected from the group consisting of: polyethylene glycol, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, heterocyclic group, arylalkyl, branched C1-C 20 alkyl, branched C2-C 20 alkenyl, branched C2-C 20 alkynyl, substituted C1-C 20 alkyl, substituted C2-C 20 alkenyl, substituted C2-C 20 alkynyl, substituted heterocyclic group, substituted arylalkyl, and hydrophobic lipid. Preferably, the hydrophobic lipid is selected from cholesterol and tocopherol. Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, and tocopherol.

[0515] In one embodiment, the compound is directly conjugated to the sequence. In another embodiment, the compound is conjugated to the sequence through a linker. The linker can be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0516] Preferably, the compound is conjugated to the terminal nucleotide of the sequence, preferably the terminal 3'-nucleotide. Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'-position.

[0517] The functionalized sequence may comprise functionalized nucleotides selected from the group consisting of: dX-TEG, dX-Chol, and dX-Toco, where dX-TEG is a DNA base having triethylene glycol covalently linked to the 3'-position via a monophosphate group, dX-Chol is a DNA base having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position via a monophosphate group, and dX-Toco is a DNA base having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position via a monophosphate group. Preferably, the functionalized sequence may comprise functionalized nucleotides selected from the group consisting of: dC-TEG, dC-Chol, dC-Toco, where dC-TEG is deoxycytidine having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position via a monophosphate group, dC-Chol is deoxycytidine having triethylene glycol covalently linked to the 3'-position via a monophosphate group, and dC-Toco is deoxycytidine having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position via a monophosphate group.

[0518] TLR7 Inhibitory Oligonucleotide

[0519] In a first aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0520] [mX / modified mX]* y X A * z X B

[0521] wherein:

[0522] * y and * z each independently represents an internucleotide bond, where * y and * z at least one of them is not a phosphorodiamidate;

[0523] X A and X B each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0524] where mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification, morpholino-X is a nucleotide containing a morpholine ring; and

[0525] where when [mX / modified mX] is mX, X A and X B at least one of which is not mX;

[0526] where when [mX / modified mX] is 3'-OMe N7-methylated guanosine, * y is not a 5'-5'-triphosphate nucleotide internucleotide bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleotide bond;

[0527] wherein the sequence is optionally functionalized.

[0528] Preferably, * y and * z are both not phosphorodiamides.

[0529] Preferably, X A is independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX; and X B is independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX and modified morpholino-X.

[0530] In one embodiment of the first aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0531] [mX / modified mX]* y X A * z X B

[0532] wherein:

[0533] * y and * z each independently represents an internucleotide bond;

[0534] X A and X BEach is independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, and modified fX;

[0535] wherein mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing a 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, and fX is a nucleotide containing a 2'-fluoro and / or 3'-fluoro modification; and

[0536] wherein when [mX / modified mX] is mX, at least one of A X B is not mX;

[0537] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleoside bond;

[0538] wherein the sequence is optionally functionalized.

[0539] Preferably, at least one of * y and * z is not a phosphorodiamidate. More preferably, neither * y nor * z is a phosphorodiamidate.

[0540] Any oligonucleotide of the first aspect inhibits TLR7 activity, preferably human TLR7 activity. In a preferred embodiment, the oligonucleotide of the first aspect does not enhance TLR8 activity, preferably human TLR8 activity. In a particularly preferred embodiment, the oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity. In an alternative preferred embodiment, the oligonucleotide of the first aspect enhances TLR8 activity, preferably human TLR8 activity.

[0541] Each internucleoside bond can be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond, and 5'-2'-bond. Preferably, each internucleoside bond can be selected from: 3'-5'-bond and 5'-5'-bond. Preferably, each internucleoside bond is a 3'-5'-bond.

[0542] In a preferred embodiment of the first aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0543] 5'-[mX / modified mX]* y X A * z X B -3'

[0544] Wherein:

[0545] * y and * z each independently represents an internucleotide bond, wherein * y and * z at least one of them is not a phosphorodiamidate;

[0546] X A and X B each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0547] Wherein mX is a nucleotide containing 2'-OMe and / or 3'-OMe modifications, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modifications, LX is a base modified with LNA, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modifications, and morpholino-X is a nucleotide containing a morpholine ring; and

[0548] Wherein when [mX / modified mX] is mX, at least one of X A and X B is not mX;

[0549] Wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate internucleotide bond, and * z is not a 3'-5'-phosphodiester internucleotide bond;

[0550] Wherein the sequence is optionally functionalized.

[0551] In a particularly preferred embodiment, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0552] 5'-[mX / modified mX]* y X A * z X B-3'

[0553] Wherein:

[0554] * y and * z each independently represents an internucleotide bond;

[0555] X A and X B each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, and modified fX;

[0556] wherein mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing a 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, and fX is a nucleotide containing a 2'-fluoro and / or 3'-fluoro modification; and

[0557] where when [mX / modified mX] is mX, at least one of A X B and X

[0558] is not mX;

[0559] Preferably, each internucleotide bond is a 3'-5' bond.

[0560] Preferably, each internucleotide bond is independently selected from the group consisting of diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is phosphorothioate.

[0561] In a particularly preferred embodiment, each internucleotide bond is a 3'-5'-phosphorothioate bond.

[0562] In one embodiment, the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers. In another embodiment, the oligonucleotide of the first aspect comprises a single phosphorothioate stereoisomer, preferably wherein * y is in the S configuration.

[0563] Preferably, mX is a nucleotide containing a 2'-OMe modification.

[0564] Preferably, moX is a nucleotide containing a 2'-MOE modification.

[0565] Preferably, fX is a nucleotide containing a 2'-fluoro modification.

[0566] Modified dX, modified rX, and modified morpholino contain at least one modification or substitution at the position of the base and / or sugar. Modified mX, modified moX, modified LX, and modified fX contain at least one additional modification or substitution at an additional position of the base and / or sugar. Preferably, the modification or substitution is selected from the group consisting of: pseudouridine, 3'-deoxy, hydroxyl, deamination, amino, thio, halogen, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0567] Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mU3, mC1, and m7 G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mU3 is N3-Me-U (3-methyluridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), and m7 G is 3'-OMe-N7-methylated guanosine. Preferably, the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, and mC1. Most preferably, the modified mX is mC1.

[0568] Exemplary modified dX includes but is not limited to: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, PSU, N3-Me-dC, 5-I-dC, dI, 8-Br-dG, 7-deaza-dG, 8-Br-dA, 8-oxo-dA, O6-Me-dG, 8-NH2-dG, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, PSU is pseudouridine, N3-Me-dC is 3-methyl deoxycytidine, 5-I-dC is 5-iodo-deoxycytidine, dI is deoxyinosine, 8-Br-dG is 8-bromo-deoxyguanosine, 7-deaza-dG is 7-deaza-deoxyguanosine, 8-Br-dA is 8-bromo-deoxyadenosine, 8-oxo-dA is 8-oxo-deoxyadenosine, O6-Me-dG is O6-methyl deoxyguanosine, and 8-NH2-dG is 8-amino-deoxyguanosine.

[0569] Exemplary modified rXs include, but are not limited to, PSU, 2'-NH2-rX, and ara-rX, where 2'-NH2-rX is an RNA base modified with 2'-amino, and ara-rX is an RNA base modified with arabinose. Exemplary 2'-NH2-rXs include, but are not limited to, 2'-NH2-U and 2'-NH2-C, where 2'-NH2-U is 2'-NH2-uridine, and 2'-NH2-C is 2'-NH2-cytidine. Exemplary ara-rX is ara-C (cytarabine).

[0570] In one embodiment, [mX / modified mX] is selected from the group consisting of: mG, mI, mG1, and mU. Preferably, [mX / modified mX] is mG or mI. In one embodiment, [mX / modified mX] is a modified mX. Preferably, the modified mX is mI or mG1, preferably mI. Preferably, the modified mX is not 2'-OMe-N1-Me-G (2'-O-methyl-N1-methylguanosine). In another embodiment, [mX / modified mX] is mX. Preferably, mX is mG or mU, preferably mG.

[0571] In a preferred embodiment, [mX / modified mX] is [mG / modified mG]. Preferably, the modified mG is not 2'-OMe-N1-Me-G (2'-O-methyl-N1-methylguanosine). Modified mGs include, but are not limited to: mG1 and mI, where mG1 is 2'-OMe-2,6-diaminopurine, and mI is 2'-OMe-I (2'-O-methylinosine). Preferably, [mG / modified mG] is [mG / mI]. In one embodiment, [mG / modified mG] is mG. In another embodiment, [mG / modified mG] is mI.

[0572] In a preferred embodiment, X A and X B are each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, and morpholino-X. In a particularly preferred embodiment, X A and X B are each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, and modified dX.

[0573] In one embodiment, X A is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified rX. Preferably, XA is selected from the group consisting of: mX, dX, rX, modified mX, modified dX, and modified rX. Preferably, X A is selected from the group consisting of: mU, mU1, mU2, mU3, PSU, mG, mA, mC, dT, dG, dA, dC, rU, 2'-NH2-rU, 8-Br-dA, and 8-oxo-dA. In one embodiment, X A is selected from the group consisting of: mX, dX, rX, and modified mX. Preferably, X A is selected from the group consisting of: mU, mU1, mU2, PSU, mG, mA, mC, dT, dG, dA, dC, and rU. More preferably, X A is mU.

[0574] In one embodiment, X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, and morpholino-X. Preferably, X B is selected from the group consisting of: dA, dC, dG, dT, mC, mC1, mG, rC, moC, LC, LA, LT, LG, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, N3-Me-dC, 5-I-dC, 2'-NH2-C, ara-C, morpholino-C, N3-Me-mU, dI, 8-Br-dG, 7-deaza-dG, O6-Me-dG, and 8-NH2-dG. In one embodiment, X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, and modified dX. Preferably, X B is selected from the group consisting of: dA, dC, dG, dT, mC, mC1, mG, rC, moC, LC, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, and pdC. In a preferred embodiment, X B is selected from the group consisting of: mX, dX, LX, modified mX, modified dX, and modified rX. Preferably, X B is selected from the group consisting of: LC, dC, 5-Me-dC, 5-Br-dC, mC, mC1, ara-C. In a particularly preferred embodiment, X B is selected from the group consisting of: LX, modified mX, modified dX, and modified rX. Preferably, X B is selected from the group consisting of: LC, 5-Me-dC, 5-Br-dC, and mC1. In an even more preferred embodiment, XB is LX, preferably LC.

[0575] In one embodiment, X A and X B at least one of which is LX. In one embodiment, X A and X B are independently LX. In another embodiment, X A and X B one of which is LX. Preferably, X B is LX. Preferably, X B is LX, and X A is mX.

[0576] In one embodiment, X A and X B at least one of which is dX. In one embodiment, X A and X B are independently dX. In another embodiment, X A and X B one of which is dX. Preferably, X B is dX. Preferably, X B is dX, and X A is mX. More preferably, X B is dX, and X A is mU.

[0577] In one embodiment, X A and X B at least one of which is rX. In one embodiment, X A and X B are independently rX. In another embodiment, X A and X B one of which is rX. Preferably, X A is rX. Preferably, X B is mX or rX, and X A is rX. More preferably, X B is mX, and X A is rU; X A is rA, and X B is rA; or X A is rU, and X B is rC. Preferably, when X A and X B at least one of which is rX, each internucleotide bond is a 3'-5'-thiophosphate bond.

[0578] In a particularly preferred embodiment, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0579] 5'-mG*mU*X B -3'

[0580] wherein:

[0581] *each independently represents a 3'-5'-phosphorothioate bond;

[0582] X B is selected from the group consisting of: dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0583] where dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide containing a morpholine ring; and

[0584] wherein the sequence is optionally functionalized.

[0585] In one embodiment, the sequence can be functionalized. Preferably, the functionalized sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclic group, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclic group, substituted arylalkyl, and hydrophobic lipid. Preferably, the hydrophobic lipid is selected from cholesterol and tocopherol. Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, and tocopherol.

[0586] In one embodiment, the compound is directly conjugated to the sequence. In another embodiment, the compound is conjugated to the sequence via a linker. The linker can be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0587] Preferably, the compound is conjugated to the terminal nucleotide of the sequence, preferably the terminal 3'-nucleotide. Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'-position.

[0588] The functionalized sequence may comprise functionalized nucleotides selected from the group consisting of dX-TEG, dX-Chol, and dX-Toco, where dX-TEG is a DNA base having triethylene glycol covalently linked to the 3'-position via a monophosphate group, dX-Chol is a DNA base having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position via a monophosphate group, and dX-Toco is a DNA base having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position via a monophosphate group. Preferably, the functionalized sequence may comprise functionalized nucleotides selected from the group consisting of dC-TEG, dC-Chol, dC-Toco, where dC-TEG is deoxycytidine having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position via a monophosphate group, dC-Chol is deoxycytidine having triethylene glycol covalently linked to the 3'-position via a monophosphate group, and dC-Toco is deoxycytidine having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position via a monophosphate group.

[0589] Preferably, the oligonucleotide of the first aspect is selected from the group of oligonucleotides in Table 1.

[0590] Table 1: The trimer oligonucleotides are inhibitors of TLR7 sensing. "m" indicates a 2'-OMe base, * indicates a phosphorothioate backbone, "d" indicates a DNA base, "r" indicates an unmodified RNA base, "mo" indicates 2'-MOE, "L" indicates LNA, "f" indicates 2'-fluoro.

[0591]

[0592]

[0593]

[0594] In one embodiment, [mX / modified mX] is [mG / mI]; X A is mU; and X B is selected from the group consisting of mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified rX, and modified dX, where the sequence is optionally functionalized. Preferably, X B is selected from the group consisting of LX, mX, dX, modified mX, modified rX, and modified dX. More preferably, X BSelected from the group consisting of: LC, mC, ara-C, dC, mC1, and modified dC. Preferably, the modified dC is selected from the group consisting of: 5-Me-dC, 5-Br-dC, and 5-I-dC. Preferably, the sequence is selected from the group consisting of: mG*mU*LC, mI*mU*LC, mG*mU*mC1, mG*mU*5-Me-dC, mG*mU*5-Br-dC, mG*mU*dC, mG*mU*dC-TEG, mI*mU*mC, mG*mU*dC-Chol, mG*mU*dC-Toco, mG*mU*ara-C, and mG*mU*5-I-dC.

[0595] In another preferred embodiment, [mX / modified mX] is [mG / mI]; X A is mU; and X B is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified rX, and modified dX. Preferably, X B is selected from the group consisting of: LC, mC1, dC, mC, and modified dC. Preferably, the modified dC is selected from the group consisting of: 5-Me-dC, 5-Br-dC, and 5-I-dC. Preferably, the sequence is selected from the group consisting of: mG*mU*LC, mI*mU*LC, mG*mU*mC1, mG*mU*5-Me-dC, mG*mU*5-Br-dC, and mG*mU*5-I-dC.

[0596] In another preferred embodiment, [modified mX] is [mG / mI]; and X A and X B is rX. Preferably, the sequence is selected from the group consisting of: mG*rA*rA, mG*rU*rC, mG*rG*rA, mG*rU*rA, mG*rU*rU, mG*rA*rG, mG*rG*rC, mG*rA*rU, mG*rG*rG. More preferably, the sequence is selected from: mG*rA*rA, mG*rU*rC, and mG*rG*rA.

[0597] In another particularly preferred embodiment, the oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity. Preferably, the oligonucleotide that further inhibits TLR8 activity comprises the sequence mI*mU*mC or mI*mA*dG or consists of the same.

[0598] In one embodiment, the oligonucleotide consists of a sequence.

[0599] In another embodiment, the oligonucleotide comprises a sequence. Preferably, the oligonucleotide comprising the sequence is no more than 20 bases in length, preferably 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 bases in length. Preferably, the sequence is located at the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In a preferred embodiment, the oligonucleotide comprises the sequence 5'-mG*mU*X B -3', where X B is dX, preferably X B is dC. Even more preferably, the oligonucleotide comprises the sequence 5'-mG*mU*dC*dC*dC*dC-3'.

[0600] In another embodiment of the first aspect, a method of modifying the TLR7 activity of an oligonucleotide is provided, the method comprising modifying the oligonucleotide by adding a sequence to the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In one embodiment, the method reduces the TLR7 enhancing activity of the oligonucleotide. In another embodiment, the method increases the TLR7 inhibitory activity of the oligonucleotide.

[0601] TLR8 Inhibitory Oligonucleotide

[0602] In a second aspect, an oligonucleotide is provided that comprises a sequence consisting of or consisting of the following:

[0603] X C * y X D * z X E

[0604] where:

[0605] *y and * z each independently represents an internucleotide bond;

[0606] X C is selected from the group consisting of: mX, modified mX, dG, and morpholino-X;

[0607] X D and X E are each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0608] wherein mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide comprising a 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, fX is a nucleotide comprising a 2'-fluoro and / or 3'-fluoro modification, morpholino-X is a nucleotide comprising a morpholine ring; and

[0609] wherein:

[0610] when X C is mX, at least one of X D and X E is not mX;

[0611] when X C is dG, at least one of X D and X E is not dX;

[0612] when X C is mG and when:

[0613] X D is dG, X E is not dA or dC;

[0614] X D is dT or mU, X E is not dC or dT;

[0615] X D is mC, X E is not dT, dG or dC; and

[0616] X D is mG or dC, X E is not dX; or

[0617] when X C is dG, X E is not mG;

[0618] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate internucleotide linkage, and * z is not a 3'-5'-phosphodiester internucleotide linkage;

[0619] wherein the sequence is optionally functionalized.

[0620] Preferably, X C is selected from the group consisting of: mX, modified mX, dG; X DSelected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX; and X E Selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X.

[0621] In one embodiment of the second aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0622] X C * y X D * z X E

[0623] Wherein:

[0624] * y And * z Each independently represents an internucleotide bond;

[0625] X C Selected from the group consisting of: mX, modified mX, and dG;

[0626] X D And X E Each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, and modified fX;

[0627] Where mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, fX is a nucleotide containing 2'-fluoro and / or 3'-fluoro modification; and

[0628] Wherein:

[0629] When X C Is mX, at least one of X D And X E Is not mX;

[0630] When X C Is dG, at least one of X D And X E Is not dX;

[0631] When XC is mG and when:

[0632] X D is dG, X E is not dA or dC;

[0633] X D is dT or mU, X E is not dC or dT;

[0634] X D is mC, X E is not dT, dG or dC; and

[0635] X D is mG or dC, X E is not dX; or

[0636] When X C is dG, X E is not mG;

[0637] wherein when [mX / modified mX] is 3'-OMe N7-methylated guanosine, * y is not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleoside bond;

[0638] wherein the sequence is optionally functionalized.

[0639] Any oligonucleotide of the second aspect inhibits TLR8 activity, preferably human TLR8 activity. In a preferred embodiment, the oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activity. In an alternative preferred embodiment, the oligonucleotide of the second aspect substantially does not inhibit TLR7 activity, preferably human TLR7 activity.

[0640] Each nucleotide internucleoside bond can be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond and 5'-2'-bond. Preferably, each nucleotide internucleoside bond can be selected from: 3'-5'-bond and 5'-5'-bond. Preferably, each nucleotide internucleoside bond is a 3'-5'-bond.

[0641] In a particularly preferred embodiment of the second aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0642] 5'-X C * y X D * z XE -3'

[0643] Wherein:

[0644] * y and * z each independently represents an internucleotide bond;

[0645] X C is selected from the group consisting of: mX, modified mX, dG, and morpholino-X;

[0646] X D and X E each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X;

[0647] wherein mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide containing a 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, fX is a nucleotide containing a 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide containing a morpholine ring; and

[0648] wherein:

[0649] when X C is mX, at least one of X D and X E is not mX;

[0650] when X C is dG, at least one of X D and X E is not dX;

[0651] when X C is mG and when:

[0652] X D is dG, X E is not dA or dC;

[0653] X D is dT or mU, X E is not dC or dT;

[0654] X D is mC, X E is not dT, dG, or dC; and

[0655] X D is mG or dC, XE not dX; or

[0656] When X C is dG, X E is not mG;

[0657] wherein when [mX / modified mX] is 3'-OMe N7-methylated guanosine, * y is not a 5'-5'-triphosphate nucleotide internucleotide bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleotide bond;

[0658] wherein the sequence is optionally functionalized.

[0659] In a particularly preferred embodiment of the second aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of:

[0660] 5'-X C * y X D * z X E -3'

[0661] wherein:

[0662] * y and * z each independently represents an internucleotide bond;

[0663] X C is selected from the group consisting of mX, modified mX, and dG;

[0664] X D and X E each independently is selected from the group consisting of mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, modified moX, modified LX, and modified fX;

[0665] wherein mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide comprising a 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, and fX is a nucleotide comprising a 2'-fluoro and / or 3'-fluoro modification; and

[0666] wherein:

[0667] When X C is mX, at least one of X D and X E is not mX;

[0668] When XC When X is dG, D and X E at least one of which is not dX;

[0669] When X C is mG and when:

[0670] X D is dG, X E is not dA or dC;

[0671] X D is dT or mU, X E is not dC or dT;

[0672] X D is mC, X E is not dT, dG or dC; and

[0673] X D is mG or dC, X E is not dX; or

[0674] When X C is dG, X E is not mG;

[0675] wherein said sequence is optionally functionalized.

[0676] Preferably, each internucleotide bond is a 3'-5' bond.

[0677] Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, phosphodiester, aminophosphate, and phosphorodiamidate. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is phosphorothioate.

[0678] In a particularly preferred embodiment, each internucleotide bond is a 3'-5'-phosphorothioate bond.

[0679] In one embodiment, the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers. In another embodiment, the oligonucleotide comprises a single stereoisomer.

[0680] Preferably, mX is a nucleotide comprising a 2'-OMe modification.

[0681] Preferably, moX is a nucleotide comprising a 2'-MOE modification.

[0682] Preferably, fX is a nucleotide comprising a 2'-fluoro modification.

[0683] Modified dX, modified rX, and modified morpholino-X contain at least one modification or substitution at a base and / or sugar position. Modified mX, modified moX, modified LX, and modified fX contain at least one additional modification or substitution at an additional position of the base and / or sugar. Preferably, the modification or substitution is selected from the group consisting of: pseudouridine, 3'-deoxy, hydroxy, deamino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0684] Exemplary modified mX include, but are not limited to: mG1, mI, mU1, mU2, mU3, mC1, and m7 G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mU3 is N3-Me-U (3-methyluridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), and m7 G is N7-methylated guanosine. Preferably, the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, and mC1.

[0685] Exemplary modified dX include, but are not limited to: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, PSU, dI, 8-Br-dG, N1-Me-dG, 7-deaza-dG, 8-Br-dA, 8-oxo-dA, O6-Me-dG, and 8-NH2-dG, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, PSU is pseudouridine, dI is deoxyinosine, 8-Br-dG is 8-bromodeoxyguanosine, N1-Me-dG is 1-methyldeoxyguanosine, 7-deaza-dG is 7-deaza-deoxyguanosine, 8-Br-dA is 8-bromodeoxyadenosine, 8-oxo-dA is 8-oxodeoxyadenosine, O6-Me-dG is O6-methyldeoxyguanosine, and 8-NH2-dG is 8-aminodeoxyguanosine.

[0686] Exemplary modified rXs include, but are not limited to, PSU, 2'-NH2-rX, and ara-rX, where 2'-NH2-rX is an RNA base modified with 2'-amino, and ara-rX is an RNA base modified with arabinose. Exemplary 2'-NH2-rXs include, but are not limited to, 2'-NH2-U and 2'-NH2-C, where 2'-NH2-U is 2'-NH2-uridine, and 2'-NH2-C is 2'-NH2-cytidine. Exemplary ara-rX is ara-C (cytarabine).

[0687] In one embodiment, X C is selected from the group consisting of: mG, mU, mC, mI, mG1, and dG.

[0688] In one embodiment, X C is selected from the group consisting of: mX and modified mX. Preferably, mX is selected from the group consisting of: mG, mC, and mU, more preferably mG; and modified mX is mI. In a preferred embodiment, XC is selected from the group consisting of: mG and mI. In a particularly preferred embodiment, X C is mI.

[0689] In one embodiment, X D is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified rX. Preferably, X D is selected from the group consisting of: mA, mU, mC, dA, dT, dG, mU1, mU2, mU3, PSU, 8-Br-dA, 8-oxo-dA, rA, rG, rU, and 2'-NH2-rU. In one embodiment, XD is selected from the group consisting of: mX, dX, LX, modified mX, and modified dX. Preferably, XD is selected from the group consisting of: mX, dX, modified mX, and modified dX. Preferably, XD is selected from the group consisting of: mA, mU, mC, dA, dT, dG, mU1, mU2, and PSU. More preferably, XD is selected from the group consisting of: mA, mU, dA, dT, and dG.

[0690] In one embodiment, X E is selected from the group consisting of: mX, dX, rX, morpholino-X, moX, LX, fX, rX, modified mX, modified dX, and modified rX. Preferably, X ESelected from the group consisting of: dA, dC, dG, dT, rG, mC, mC1, mG, mU3, moC, LA, LC, LG, LT, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, dI, 8-Br-dG, N1-Me-dG, 7-deaza-dG, O6-Me-dG, 8-NH2-dG, morpholino-G, rA, rG, rU, rC, N3-Me-dC, 5-I-dC, 2'-NH2-C, ara-C and morpholino-C. In one embodiment, XE is selected from the group consisting of: mX, dX, moX, LX, fX, rX, modified mX and modified dX. Preferably, XE is selected from the group consisting of: dA, dC, dG, dT, rG, mC, mC1, mG, moC, LA, LC, LG, LT, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC and pdC. More preferably, XE is selected from the group consisting of: dA, dC, dG, dT, rG, mC, mC1, mG, moC, LA, LC, LG, LT, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC and pdC. In one embodiment, X E Selected from the group consisting of: mX, dX, rX and LX. Preferably, XE is selected from the group consisting of: dA, dC, dG, dT, rG, mC, LA, LC, LG and LT. Even more preferably, XE is selected from the group consisting of: mX and dX. Preferably, X E Selected from the group consisting of: dC, dG, dT and mC.

[0691] In one embodiment, X D and X E At least one of is LX. In one embodiment, X D and X E Are independently LX. In another embodiment, one of XD and XE is LX. Preferably, X E Is LX. Preferably, X E Is LX, and X D Is mX.

[0692] In one embodiment, X D and X E At least one of is dX. In one embodiment, X D and X E Are independently dX. In another embodiment, one of XD and XE is dX. Preferably, X E Is dX. Preferably, X E Is dX, and X D Is mX.

[0693] In one embodiment, X D and X E at least one of which is rX. In one embodiment, one of XD and XE is rX. In another embodiment, X D and X E are each independently rX. Preferably, X D is selected from rA and rG, and X E is selected from rA, rG and rC. Preferably, X D is rA, and X E is rA; X D is rG, and X E is rA; X D is rA, and X E is rG; X D is rA, and X E is rC. Preferably, when at least one of X A and X B is rX, each internucleotide bond is a 3'-5'-thiophosphate bond.

[0694] In one embodiment, the sequence can be functionalized. Preferably, the functionalized sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclic group, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclic group, substituted arylalkyl and hydrophobic lipid. Preferably, the hydrophobic lipid is selected from cholesterol and tocopherol. Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol.

[0695] In one embodiment, the compound is directly conjugated to the sequence. In another embodiment, the compound is conjugated to the sequence via a linker. The linker can be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0696] Preferably, the compound is conjugated to the terminal nucleotide of the sequence, preferably the terminal 3'-nucleotide. Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'-position.

[0697] The functionalized sequence may comprise functionalized nucleotides selected from the group consisting of dX-TEG, dX-Chol, and dX-Toco, where dX-TEG is a DNA base having a triethylene glycol covalently linked to the 3'-position via a monophosphate group, dX-Chol is a DNA base having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position via a monophosphate group, and dX-Toco is a DNA base having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position via a monophosphate group.

[0698] Preferably, the oligonucleotides of the second aspect are selected from the group of oligonucleotides in Table 2.

[0699] Table 2: The trimer oligonucleotides are inhibitors of TLR8 sensing. "m" indicates a 2'-OMe base, * indicates a phosphorothioate backbone, and "d" indicates a DNA base.

[0700]

[0701]

[0702]

[0703] Preferably, the sequences are selected from the group consisting of: mI*mA*dG, mI*mU*mC, mG*dA*dG, mG*mA*dT, mG*mA*dG, mG*mA*dC, mG*mA*LG, mG*mA*rG, mG*mA*LT, mG*mA*LC, mU*dT*dC, mU*dA*dC, mG*mA*LA, mU*dA*dG, mC*dA*dG, mU*dT*dT, mU*dA*dT, mU*dA*dA, mC*dT*dA, mU*dG*dT, mC*dT*dC, mC*dA*dT, mU*dG*dG, mC*dT*dT, mC*dT*dG, mU*dT*dA, mU*dT*dG, mG*mA*O6-Me-dG, mG*rA*rA, mG*rG*rA, mG*rA*rG, and mG*rA*rU.

[0704] More preferably, the sequences are selected from the group consisting of: mI*mA*dG, mI*mU*mC, mG*dA*dG, mG*mA*dT, mG*mA*dG, mG*mA*dC, mG*rA*rA, and mG*rG*rA. Even more preferably, the sequences are selected from the group consisting of: mI*mA*dG and mI*mU*mC.

[0705] In another particularly preferred embodiment, the oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activity. Preferably, the oligonucleotide that further inhibits TLR7 activity comprises or consists of the sequence mI*mU*mC or mI*mA*dG.

[0706] In one embodiment, the oligonucleotide consists of a sequence.

[0707] In another embodiment, the oligonucleotide comprises a sequence. Preferably, the oligonucleotide comprising the sequence has a length of no more than 20 bases, preferably a length of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 bases. Preferably, the sequence is located at the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end.

[0708] In another embodiment of the second aspect, a method of modifying the TLR8 activity of an oligonucleotide is provided, the method comprising modifying the oligonucleotide by adding a sequence to the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In one embodiment, the method reduces the TLR8 enhancing activity of the oligonucleotide. In another embodiment, the method increases the TLR8 inhibitory activity of the oligonucleotide.

[0709] TLR8 Potentiating Oligonucleotide

[0710] In a third aspect, an oligonucleotide is provided that comprises or consists of a sequence consisting of:

[0711] [mX / modified mX]* y X F * z X G

[0712] Wherein:

[0713] * y And * z Each independently represents an internucleotide bond;

[0714] X F And X G Each independently is selected from the group consisting of mX, dX, rX, LX, modified mX, modified dX, and modified LX; wherein X F And X G At least one of which is dX, LX, rX, modified dX, or modified LX;

[0715] wherein mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0716] when mX is mC and X F is dG, X G is not mG or dG; or

[0717] when mX is mG and when:

[0718] X F is mU, mC or dG, X G is not dT, dA, dG;

[0719] X F is dT, dA or mA, X G is not dX;

[0720] X F is dG, X G is not dA, dG, dT;

[0721] wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleoside bond;

[0722] wherein the sequence is optionally functionalized.

[0723] In an embodiment of the third aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0724] mX* y X F * z X G

[0725] wherein:

[0726] * y and * z each independently represents a nucleotide internucleoside bond;

[0727] X F and X G each independently is selected from the group consisting of: mX, dX, LX, modified mX, modified dX, and modified LX; wherein X F and X G at least one of which is dX, LX, modified dX or modified LX;

[0728] where mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0729] When mX is mC and X F is dG, X G is not mG or dG; or

[0730] When mX is mG and when:

[0731] X F is mU, mC or dG, X G is not dT, dA, dG;

[0732] X F is dT, dA or mA, X G is not dX;

[0733] X F is dG, X G is not dA, dG, dT;

[0734] where when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate internucleotide bond, and * z is not a 3'-5'-phosphodiester internucleotide bond;

[0735] wherein the sequence is optionally functionalized.

[0736] Any oligonucleotide of the third aspect enhances TLR8 activity, preferably human TLR8 activity. In a preferred embodiment, the oligonucleotide of the third aspect substantially does not inhibit TLR7 activity, preferably human TLR7 activity. In an alternative preferred embodiment, the oligonucleotide of the third aspect inhibits TLR7 activity, preferably human TLR7 activity.

[0737] Each internucleotide bond can be selected from the group consisting of: 3'-5'-bond, 5'-5'-bond, 5'-3'-bond, 3'-3'-bond, 3'-2'-bond, 2'-3'-bond, 2'-2'-bond, 2'-5'-bond and 5'-2'-bond. Preferably, each internucleotide bond can be selected from: 3'-5'-bond and 5'-5'-bond. Preferably, each internucleotide bond is a 3'-5'-bond.

[0738] In a particularly preferred embodiment of the third aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0739] 5'-[mX / modified mX]* y X F *z X G -3'

[0740] Wherein:

[0741] * y and * z each independently represents an internucleotide linkage;

[0742] X F and X G each independently is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified LX; wherein X F and X G at least one of which is dX, rX, LX, modified dX, or modified LX;

[0743] wherein mX is a nucleotide containing 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0744] When mX is mC and X F is dG, X G is not mG or dG; or

[0745] When mX is mG and when:

[0746] X F is mU, mC, or dG, X G is not dT, dA, dG;

[0747] X F is dT, dA, or mA, X G is not dX;

[0748] X F is dG, X G is not dA, dG, dT;

[0749] wherein the sequence is optionally functionalized.

[0750] In a particularly preferred embodiment of the third aspect, there is provided an oligonucleotide comprising a sequence consisting of or consisting of the following:

[0751] 5'-mX* y X F * z X G -3'

[0752] Wherein:

[0753] * y and * z each independently represents an internucleotide linkage;

[0754] X F and X G each independently selected from the group consisting of: mX, dX, LX, modified mX, modified dX, and modified LX; wherein X F and X G at least one of

[0755] wherein mX is a nucleotide containing a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA;

[0756] when mX is mC and X F is dG, X G is not mG or dG; or

[0757] when mX is mG and when:

[0758] X F is mU, mC, or dG, X G is not dT, dA, dG;

[0759] X F is dT, dA, or mA, X G is not dX;

[0760] X F is dG, X G is not dA, dG, dT;

[0761] wherein the sequence is optionally functionalized.

[0762] Preferably, each internucleotide bond is a 3'-5' bond.

[0763] Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is phosphorothioate.

[0764] In a particularly preferred embodiment, each internucleotide bond is a 3'-5'-phosphorothioate bond.

[0765] In one embodiment, the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers. In another embodiment, the oligonucleotide of the third aspect comprises a single phosphorothioate stereoisomer, preferably wherein* z is in the R configuration.

[0766] Preferably, mX is a nucleotide containing a 2'-OMe modification.

[0767] The modified dX and the modified rX contain at least one modification or substitution at the position of the base and / or the sugar. The modified mX, the modified moX, the modified LX, and the modified fX contain at least one additional modification or substitution at an additional position of the base and / or the sugar. Preferably, the modification or substitution is selected from the group consisting of: pseudouridine, 3'-deoxy, hydroxy, deamino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.

[0768] Exemplary modified mX includes, but is not limited to: mG1, mI, mU1, mU2, mC1, m7 G, and N1-Me-G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), m7 G is 3'-OMe-N7-methylated guanosine, and N1-Me-G (1-methylguanosine). Preferably, the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, and mC1.

[0769] Exemplary modified dX includes, but is not limited to: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, and PSU, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, and PSU is pseudouridine.

[0770] In one embodiment, X F and X G each independently is selected from the group consisting of: mX, dX, and LX; wherein at least one of X F and X G is dX or LX.

[0771] In one embodiment, mX is selected from the group consisting of: mG, mC, and mU. In a preferred embodiment, mX is mG. In another preferred embodiment, mX is mC. In yet another preferred embodiment, mX is mU.

[0772] In one embodiment, X F is selected from the group consisting of: mX and dX. Preferably, XF is selected from the group consisting of: dC, dG, dA, dT, mG, mC, and mU. Preferably, XF is selected from the group consisting of: dC, dG, and mG. In a preferred embodiment, X F is dX, preferably dC.

[0773] In one embodiment, X G is selected from the group consisting of: dX and LX. Preferably, X G is selected from the group consisting of: dC, dT, dA, dG, LG, LC, LT, and LA. More preferably, XG is selected from the group consisting of: dX and LG, preferably, dX.

[0774] In one embodiment, X F and X G at least one of which is dX. In one embodiment, one of XF and XG is dX. In a preferred embodiment, XF and XG are independently dX.

[0775] In one embodiment, X F is selected from the group consisting of: dX and mX; and X G is dX. In another embodiment, X F is mX; and X G is selected from the group consisting of: dX and LX. In yet another embodiment, when X F is dC or mG, X F is dX or LX.

[0776] In one embodiment, the sequence can be functionalized. Preferably, the functionalized sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclic group, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclic group, substituted arylalkyl, and hydrophobic lipid. Preferably, the hydrophobic lipid is selected from cholesterol and tocopherol. Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, and tocopherol.

[0777] In one embodiment, the compound is directly conjugated to the sequence. In another embodiment, the compound is conjugated to the sequence through a linker. The linker can be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0778] Preferably, the compound is conjugated to the terminal nucleotide of the sequence, preferably the terminal 3'-nucleotide. Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'-position.

[0779] The functionalized sequence may comprise functionalized nucleotides selected from the group consisting of dX-TEG, dX-Chol, and dX-Toco, where dX-TEG is a DNA base having triethylene glycol covalently linked to the 3'-position via a monophosphate group, dX-Chol is a DNA base having (N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl covalently linked to the 3'-position via a monophosphate group, and dX-Toco is a DNA base having [(9-DL-α-tocopherolyl)-triethylene glycol-1-yl]-glyceryl covalently linked to the 3'-position via a monophosphate group.

[0780] Preferably, the oligonucleotide is selected from the group of oligonucleotides in Table 3.

[0781] Table 3: Trimers of oligonucleotides are synergists for TLR8 sensing. "m" indicates 2'-OMe bases, * indicates phosphorothioate backbone, and "d" indicates DNA bases.

[0782] Name Sequence GCC-V4 mG*dC*dC CCT-V4 mC*dC*dT GCA-V4 mG*dC*dA GCG-V4 mG*dC*dG CCC-V4 mC*dC*dC UCC-V4 mU*dC*dC CGC-V4 mC*dG*dC GCT-V4 mG*dC*dT GGA-V1 mG*mG*dA UCG-V4 mU*dC*dG UGG-V8 mU*mG*LG UCA-V4 mU*dC*dA UCT-V4 mU*dC*dT CCA-V4 mC*dC*dA CCG-V4 mC*dC*dG CAC-V4 mC*dA*dC CGT-V4 mC*dG*dT CTC-V4 mC*dT*dC CGA-V4 mC*dG*dA GCC-V1 mG*mC*dC GGC-V1 mG*mG*dC GUC-V1 mG*mU*dC GGG-V1 mG*mG*dG CGG-V1 mC*mG*dG UCC-V8 mU*mC*LC GGG-V8 mG*mG*LG UCA-V8 mU*mC*LA UCG-V8 mU*mC*LG UCT-V8 mU*mC*LT UGA-V8 mU*mG*LA UUG-V8 mU*mU*LG UGC-V8 mU*mG*LC UGT-V8 mU*mG*LT GGT-V1 mG*mG*dT GGC-V4 mG*dG*dC GUC-V41 N1-Me-mG*mU*dC GUC-V54 mG*rG*rU

[0783] Preferably, the sequence is selected from the group consisting of mG*dC*dC, mC*dC*dT, mG*dC*dA, mG*dC*dG, mC*dC*dC, mU*dC*dC, mC*dG*dC, mG*dC*dT, mG*mG*dA, mU*dC*dG, mU*mG*LG, mU*dC*dA, and mU*dC*dT.

[0784] More preferably, the sequence is selected from the group consisting of mG*dC*dC, mC*dC*dT, mU*mG*LG, mC*dC*dC, mU*dC*dC, mG*dC*dA, mG*dC*dG, and mG*dC*dT. Even more preferably, the oligonucleotide is mG*dC*dC.

[0785] In one embodiment, the oligonucleotide consists of a sequence.

[0786] In another embodiment, the oligonucleotide comprises a sequence. Preferably, the oligonucleotide comprising the sequence has a length of no more than 20 bases, preferably a length of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 bases. Preferably, the sequence is located at the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In a preferred embodiment, the oligonucleotide comprises the sequence 5'-mG*mU*dC-3'. Even more preferably, the oligonucleotide comprises the sequence 5'-mG*mU*dC*dC*dC*dC-3'.

[0787] In another embodiment of the third aspect, a method of modifying the TLR8 activity of an oligonucleotide is provided, the method comprising modifying the oligonucleotide by adding a sequence to the 5'-end and / or 3'-end of the oligonucleotide, preferably the 5'-end. In one embodiment, the method increases the TLR8 enhancing activity of the oligonucleotide. In another embodiment, the method decreases the TLR8 inhibitory activity of the oligonucleotide.

[0788] Fusion Oligonucleotide

[0789] On the other hand, a fusion oligonucleotide is provided, which comprises:

[0790] A-[Y-A] n

[0791] wherein

[0792] each A independently represents an oligonucleotide according to that described herein, and each A can be the same or different;

[0793] Y represents a cleavable linker, and each Y can be the same or different; and

[0794] n is equal to or greater than 1.

[0795] In one embodiment, each A independently represents an oligonucleotide according to the first aspect. In another embodiment, each A independently represents an oligonucleotide according to the second aspect. In another embodiment, each A independently represents an oligonucleotide according to the third aspect. In another embodiment, at least one A is an oligonucleotide according to the first aspect, and at least one other A is an oligonucleotide according to the second aspect.

[0796] A cleavable linker can be linked to the 5' and / or 3' ends of an oligonucleotide, wherein the fusion oligonucleotide comprises 5'-A-3'-Y-3'-A-5', 5'-A-3'-Y-5'-A-3' or 3'-A-5'-Y-5'-A-3'. Preferably, the fusion oligonucleotide comprises 5'-A-3'-Y-3'-A-5'.

[0797] Preferably, Y is enzymatically cleavable. Preferably, Y is selected from the group consisting of: TEG linker, carbon spacer (such as C3, C6, C9, C 12 ), glycerol, and PolydT. More preferably, Y is a TEG linker.

[0798] The term "TEG linker" refers to a triethylene glycol linker.

[0799] Preferably, each A is independently bound to Y by an internucleotide bond (*). Preferably, each internucleotide bond is independently selected from the group consisting of: diphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide bond can be the same or different. In a preferred embodiment, each internucleotide bond is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide bond is a phosphorothioate.

[0800] In one embodiment, the fusion oligonucleotide comprises or consists of the sequence 5'-mG*mU*dC-3'-*TEG*-3'-dC*mU*mG-5'.

[0801] On the other hand, a modified oligonucleotide is provided, which comprises an agent linked to the oligonucleotide or fusion oligonucleotide described herein through a linker. The agent can be a therapeutic agent and / or a diagnostic agent. Suitable therapeutic agents include but are not limited to: monoclonal antibodies, oligonucleotides, small molecules, cholesterol, radiotherapeutic agents. Suitable diagnostic reagents include but are not limited to: radiolabels, dyes. Preferably, the agent is a therapeutic agent, more preferably a therapeutic RNA. The therapeutic RNA can be a synthetic oligonucleotide sequence or a naturally occurring oligonucleotide sequence. A synthetic oligonucleotide sequence refers to an oligonucleotide sequence lacking a corresponding naturally occurring sequence. The therapeutic RNA can be synthesized in vitro. However, in some cases where modified bases and backbones are not required, it can be expressed in vitro or in vivo in a suitable system, such as through a recombinant virus or cell. Therapeutic RNAs include but are not limited to: DNA, RNA, mRNA, siRNA, RNA aptamer, antisense oligonucleotide, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. In a preferred embodiment, the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. The linker can be cleavable or non-cleavable. Preferably, the linker is a cleavable linker.

[0802] Test for Inhibiting TLR7 Activity

[0803] Some embodiments of the methods of the present invention relate to testing for inhibition of TLR7 activity, which can be determined using any method known in the art. In some embodiments, TLR7 activity in cells can be measured by the expression and / or secretion of one or more pro-inflammatory cytokines (e.g., TNFα), and / or the activation or expression of transcription factors (e.g., NF-κB).

[0804] The ability of an oligonucleotide to inhibit TLR7 activity can be analyzed, for example, by incubating cells expressing TLR7 with the oligonucleotide and then stimulating the cells with a TLR7 agonist (e.g., R848, guanosine, or immunostimulatory ssRNA such as B-406-AS-1), and analyzing the overall TLR7 response in the cell population, or the proportion of cells with TLR7-positive activity after a defined period of time.

[0805] In such instances, inhibition of TLR7 activity can be identified by observing an overall reduced TLR7 response in the cell population, or a lower proportion of cells with TLR7-positive activity compared to a positive control condition in which the cells are treated with a TLR7 agonist in the absence of the oligonucleotide (or in the presence of a suitable control inhibitor). In one example, 293XLhTLR7 (referred to as HEK-TLR7) cells are transfected with the pNF-κB-Luc4 reporter gene, incubated with the oligonucleotide, and then stimulated with R848. TLR7 activity can be determined by a luciferase assay that measures activated NF-κB by luminescence. TLR7 activity can also be analyzed by measuring cytokine levels, e.g., by ELISA.

[0806] Test for Inhibiting TLR8 Activity

[0807] Some embodiments of the methods of the present invention relate to testing for inhibition of TLR8 activity, which can be determined using any method known in the art. In some embodiments, TLR8 activity in cells can be measured by the expression and / or secretion of one or more pro-inflammatory cytokines (e.g., IL-6, TNFα, IP-10), and / or the activation or expression of transcription factors (e.g., NF-κB).

[0808] The ability of an oligonucleotide to inhibit TLR8 activity can be analyzed, for example, by incubating cells expressing TLR8 with the oligonucleotide and then stimulating the cells with a TLR8 agonist, and analyzing the overall TLR8 response in the cell population, or the proportion of cells with TLR8-positive activity after a defined period of time.

[0809] In such instances, inhibition of TLR8 activity can be identified by observing an overall reduced TLR8 response of a cell population, or a lower proportion of cells with TLR8 positive activity compared to a positive control condition, in which cells are treated with a TLR8 agonist in the absence of the oligonucleotide (or in the presence of a suitable control inhibitor). In one example, 293XLhTLR8 (referred to as HEK-TLR8) cells are transfected with the pNF-κB-Luc4 reporter gene, incubated with the oligonucleotide, and then stimulated with R848. TLR8 activity can be determined by a luciferase assay that measures activated NF-κB by luminescence. TLR8 activity can also be analyzed by measuring cytokine levels, such as by ELISA.

[0810] Test for Potentiating TLR8 Activity

[0811] Some embodiments of the methods of the invention relate to testing for enhancement of TLR8 activity, which can be determined using any method known in the art. In some embodiments, TLR8 activity in cells can be measured by the expression and / or secretion of one or more pro-inflammatory cytokines (e.g., IL-6, TNFα, IP-10), and / or the activation or expression of a transcription factor (e.g., NF-κB).

[0812] The ability of an oligonucleotide to enhance TLR8 activity can be analyzed, for example, by incubating cells expressing TLR8 with the oligonucleotide, then stimulating the cells with a TLR8 agonist, and analyzing the overall TLR8 response in the cell population, or the proportion of cells with TLR8 positive activity after a defined period of time.

[0813] In such instances, enhancement of TLR8 activity can be identified by observing an overall increased TLR8 response of a cell population, or a higher proportion of cells with TLR8 positive activity compared to a negative control condition, in which cells are treated with a TLR8 agonist in the absence of the oligonucleotide (or in the presence of a suitable control non-enhancer). In one example, 293XLhTLR8 (referred to as HEK-TLR8) cells are transfected with the pNF-κB-Luc4 reporter gene, incubated with the oligonucleotide, and then stimulated with R848. TLR8 activity can be determined by a luciferase assay that measures activated NF-κB by luminescence. TLR8 activity can also be analyzed by measuring cytokine levels, such as by ELISA.

[0814] Use

[0815] The oligonucleotides of the present invention are designed for administration to animals. For this purpose, the oligonucleotides can be administered in combination with another molecule, such as another nucleic acid (e.g., mRNA molecule, short interfering RNA, antisense oligonucleotide, CRISPR guide RNA, etc.), peptide, carrier agent, therapeutic agent, etc. In one embodiment, the oligonucleotides can be conjugated to other molecules.

[0816] Generally, oligonucleotides are used to modify the traits of animals, and more commonly for treating or preventing diseases or conditions. In a preferred embodiment, the disease or condition would benefit from an animal that does not produce a TLR7 and / or TLR8 response after administration of the oligonucleotide. In an alternative embodiment, the disease or condition would benefit from an animal that produces an increased TLR8 response after administration of the oligonucleotide.

[0817] In one embodiment of the first aspect, a method of inhibiting TLR7 activity in a cell is provided, the method comprising contacting the cell with an oligonucleotide, fusion oligonucleotide or composition according to the first aspect, thereby inhibiting TLR7 activity in the cell.

[0818] In one embodiment of the first aspect, a method of inhibiting TLR7 activity in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the first aspect, thereby inhibiting TLR7 activity in the subject.

[0819] In one embodiment of the first aspect, a method of treating or preventing a disease, disorder or condition in a subject responsive to TLR7 inhibition is provided, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the first aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0820] In one embodiment of the second aspect, a method of inhibiting TLR8 activity in a cell is provided, the method comprising contacting the cell with an oligonucleotide, fusion oligonucleotide or composition according to the second aspect, thereby inhibiting TLR8 activity in the cell.

[0821] In one embodiment of the second aspect, a method of inhibiting TLR8 activity in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the second aspect, thereby inhibiting TLR8 activity in the subject.

[0822] In one embodiment of the second aspect, there is provided a method of treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, fusion oligonucleotide or composition according to the second aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0823] Diseases, disorders or conditions responsive to TLR7 and / or TLR8 inhibition include immune-inflammatory related diseases, allergic diseases, infections, cancers and autoantibody-dependent autoimmune diseases.

[0824] Examples of immune-inflammatory related diseases can include diseases of the connective tissue and musculoskeletal system (such as systemic lupus erythematosus, cutaneous and subcutaneous lupus, rheumatoid arthritis, juvenile idiopathic arthritis, adult-onset Still's disease, ankylosing spondylitis, systemic sclerosis, polymyositis, dermatomyositis, psoriatic arthritis, fibromyalgia, osteoarthritis, mixed connective tissue disease, Guillain-Barre syndrome, and muscular dystrophy), the hematological system (such as autoimmune hemolytic anemia, aplastic anemia, and idiopathic thrombocytopenic purpura), the digestive system (such as Crohn's disease, ulcerative colitis, and ileitis), the hepatobiliary and pancreatic system and the endocrine system (such as autoimmune hepatitis, viral hepatitis, alcoholic hepatitis, non-alcoholic fatty liver disease, primary sclerosing cholangitis, celiac disease, fatty liver, inflammatory bowel disease, pancreatitis, primary biliary cirrhosis, Sjogren's syndrome, autoimmune thyroiditis, Graves' disease, and Hashimoto's thyroiditis), the respiratory system (such as chronic obstructive pulmonary disease, cystic fibrosis, bronchitis, and interstitial pneumonia), the cranio-nervous system (such as multiple sclerosis, myasthenia gravis, meningitis, encephalomyelitis, and autoimmune encephalitis, Parkinson's disease, Alzheimer's disease, Huntington's disease), the visual system (such as uveitis, trachoma, and endophthalmitis), the cardiovascular system (such as vasculitis syndromes, granulomatosis with polyangiitis, Wegener's granulomatosis, myocarditis, ischemic heart disease, hypertension, stroke, and atherosclerosis), the cutaneous and epidermal system (such as psoriasis, pemphigus, vitiligo, contact dermatitis, and eczema), the renal system (such as glomerulonephritis, diabetic nephropathy, IgA nephropathy, purpuric nephritis, nephrosis, and interstitial cystitis), and the endocrine system (such as type 1 diabetes, type 2 diabetes, autoimmune thyroiditis, Graves' disease, and Hashimoto's thyroiditis) and systemic inflammation (such as Behcet's disease, antiphospholipid antibody syndrome, IgG4-related diseases, sepsis, hemorrhage, hypersensitivity, transplant rejection, and shock symptoms caused by, for example, cancer chemotherapy). Preferably, the immune-inflammatory related diseases are selected from: systemic lupus erythematosus, cutaneous lupus, and psoriasis.

[0825] Examples of allergic diseases can include atopic dermatitis, hay fever, asthma, anaphylaxis, anaphylactoid reactions, food allergies, rhinitis, otitis media, drug reactions, insect bite reactions, plant reactions, latex allergy, conjunctivitis, and urticaria.

[0826] Examples of infections can include diseases caused by: viruses (such as single-stranded RNA viruses, double-stranded RNA viruses, single-stranded DNA viruses, and double-stranded DNA viruses), bacteria (such as Gram-negative bacteria, Gram-positive bacteria, acid-fast bacteria, actinomycetes, spirochetes, spirilla, Rickettsia, Chlamydia, and Mycoplasma), fungi (such as Trichophyton, Candida, Cryptococcus, Aspergillus, Pneumocystis, and Malassezia), and parasites (such as filariae, trematodes, cestodes, Distoma, Echinococcus, Entamoeba histolytica, fleas, lice, mites, roundworms, and oxyurids).

[0827] Examples of cancer treatments can include the treatment of: blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesothelioma, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, skin cancers (including melanoma), leukemias or lymphoid malignancies, lung cancers (including small cell lung cancer (SGLG), non-small cell lung cancer (NSGLG), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancers, hepatocellular carcinomas, gastric / stomach cancers (including gastrointestinal cancers), pancreatic cancers, glioblastomas, ovarian cancers, liver cancers, bladder cancers, liver cancers, breast cancers (including metastatic breast cancers), colon cancers, rectal cancers, colorectal cancers, salivary gland cancers, kidney / renal cancers, prostate cancers, thyroid cancers, liver cancers, anal cancers, penile cancers, testicular cancers, esophageal cancers, biliary tract tumors, and head and neck cancers.

[0828] The present invention also provides methods of alleviating or minimizing symptoms associated with diseases, disorders, and conditions responsive to TLR7 and / or TLR8 inhibition. Symptoms associated with diseases, disorders, and conditions responsive to TLR7 and / or TLR8 inhibition include inflammation, fever, muscle pain, and fatigue. In a preferred embodiment, the condition responsive to TLR7 and / or TLR8 inhibition is mRNA administration.

[0829] In one embodiment of the third aspect, a method of enhancing TLR8 activity in a cell is provided, the method comprising contacting the cell with an oligonucleotide, a fusion oligonucleotide or a composition according to the third aspect, thereby enhancing TLR8 activity in a subject.

[0830] In one embodiment of the third aspect, a method of enhancing TLR8 activity in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide or a composition according to the third aspect, thereby enhancing TLR8 activity in the subject.

[0831] In one embodiment of the third aspect, a method of treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signaling is provided, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide or a composition according to the third aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0832] Diseases, disorders or conditions associated with reduced TLR8 signaling include cancer, viral and bacterial infections.

[0833] Examples of cancers include blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas and islet cell carcinomas), mesothelioma, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, skin cancers (including basal cell carcinoma, melanoma), leukemias or lymphoid malignancies, lung cancers (including small cell lung cancer (SGLG), non-small cell lung cancer (NSGLG), lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancers (including gastrointestinal cancers), pancreatic cancer, glioblastoma, ovarian cancer, cervical cancer, liver cancer, bladder cancer, liver cancer, breast cancers (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, head and neck cancers, and cancers associated with viral infections.

[0834] In another form, the oligonucleotides of the first and second aspects are used in a method for preventing or suppressing inflammation associated with the administration of a therapeutic RNA (such as those known in the art) to a subject. Specifically, the oligonucleotides described herein can be used to prevent or suppress inflammation mediated by one or more nucleic acid sensors (e.g., TLR7, TLR8) during or after the administration of a therapeutic RNA. It is contemplated that the inflammation can involve or include any cell, tissue, or organ of the human body. In a specific embodiment, the inflammation is or includes liver inflammation. To this end, the therapeutic RNA can be conjugated to N-acetylgalactosamine (GalNAc), which can enhance the uptake of desialylated glycoprotein receptor (ASGR)-mediated liver hepatocytes (Nair et al., 2014), and thereby specifically target the liver.

[0835] In certain instances, the oligonucleotides of the first aspect, and more specifically the oligonucleotides exhibiting TLR7 inhibitory activity described herein, can be used to prevent or suppress TLR7-dependent inflammatory responses associated with the administration of a therapeutic RNA in vitro or in vivo, the therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. More specifically, the therapeutic RNA can be part of an RNA-based therapeutic agent, such as an mRNA vaccine. In this regard, the oligonucleotides can at least partially inhibit the engagement or sensing of these therapeutic RNA molecules by TLR7. Thus, the oligonucleotides of the first aspect can minimize the need to use modified bases (such as pseudouridine) and / or other modifications that reduce the immunogenicity of the mRNA molecule in order to include it in an mRNA vaccine composition.

[0836] In certain instances, the oligonucleotides of the second aspect, and more specifically the oligonucleotides exhibiting TLR8 inhibitory activity described herein, can be used to prevent or suppress TLR8-dependent inflammatory responses associated with the administration of a therapeutic RNA in vitro or in vivo, the therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. More specifically, the therapeutic RNA can be part of an RNA-based therapeutic agent, such as an mRNA vaccine. In this regard, the oligonucleotides can at least partially inhibit the engagement or sensing of these therapeutic RNA molecules by TLR8. Thus, the oligonucleotides of the second aspect can minimize the need to use modified bases (such as pseudouridine) and / or other modifications that reduce the immunogenicity of the mRNA molecule in order to include it in an mRNA vaccine composition.

[0837] Thus, the oligonucleotides of the first and second aspects can be components within or included in an immunogenic composition, such as an RNA or mRNA vaccine composition known in the art. The term "RNA vaccine" refers to a vaccine comprising RNA encoding one or more nucleotide sequences that encode an antigen capable of inducing an immune response in a mammal. For example, mRNA vaccines are described in International Patent Applications Nos. PCT / US2015 / 027400 and PCT / US2016 / 044918, which are incorporated herein by reference in their entirety.

[0838] In a particular form, the present invention provides an immunogenic composition (such as a vaccine composition) comprising a therapeutic RNA and an oligonucleotide or fusion oligonucleotide provided herein. Preferably, the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. Preferably, the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof. Suitably, the oligonucleotide of the immunogenic composition exhibits TLR7 and / or TLR8 inhibitory activity as described herein. In certain embodiments, the oligonucleotide of the immunogenic composition exhibits TLR7 inhibitory activity. In certain embodiments, the oligonucleotide of the immunogenic composition exhibits TLR8 inhibitory activity. In some embodiments, the oligonucleotide of the immunogenic composition exhibits both TLR7 and TLR8 inhibitory activity. The immunogenic composition is suitable for use in the following methods: (a) inducing an immune response in a subject; and / or (b) preventing, treating, or ameliorating an infection, disease, or condition in a subject in need thereof.

[0839] It should be understood that mRNA vaccines provide a unique therapeutic alternative to peptide- or DNA-based vaccines. When an mRNA vaccine is delivered to a cell, the mRNA will be processed by intracellular machinery into a polypeptide or peptide, which can then be processed by the intracellular machinery into immunogenic fragments capable of stimulating an immune response. To this end, the oligonucleotide can be included as a separate or discrete component and / or conjugated to the therapeutic RNA of the vaccine composition. Preferably, the therapeutic RNA is selected from RNA or mRNA. For such embodiments, the therapeutic RNA of the RNA vaccine can be unmodified or substantially unmodified (e.g., not including any modified bases). Alternatively, the therapeutic RNA can contain one or more modifications that generally enhance stability, such as modified nucleotides, modified sugar-phosphate backbones, and 5' and / or 3' untranslated regions (UTRs).

[0840] In addition, as is known in the art, therapeutic RNAs can be included or incorporated into delivery, transfer, or carrier systems of immunogenic compositions. For example, the therapeutic RNAs of immunogenic compositions can be encapsulated or complexed in nanoparticles, and more specifically, encapsulated or complexed in lipid nanoparticles. According to various embodiments, suitable nanoparticles include, but are not limited to, polymer-based carriers such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both exosomes of natural and synthetic origin, natural, synthetic, and semi-synthetic lamellar bodies, nanoparticles, calcium phosphate silicate nanoparticles, calcium phosphate nanoparticles, silica nanoparticles, nanocrystal particles, semiconductor nanoparticles, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, vesicles, multi-domain block polymers (vinyl polymers, polyacrylic acid polymers, dynamic poly-conjugates), and dry powder formulations.

[0841] In some embodiments, the oligonucleotides are included in an immunogenic composition separate from the carrier system. In other embodiments, the oligonucleotides are included or incorporated within the carrier system of the immunogenic composition, such as incorporated into lipid nanoparticles together with the therapeutic RNAs of an RNA vaccine.

[0842] In some embodiments, the oligonucleotides can be applied to the surface of an implantable biomaterial, such as a prosthesis.

[0843] In specific instances, a therapeutically effective amount of the therapeutic RNAs and oligonucleotides of the present invention can be administered simultaneously, in parallel, sequentially, successively, alternately, or separately in any particular combination and / or order.

[0844] Composition

[0845] The oligonucleotides of the present disclosure can be mixed, encapsulated, conjugated (such as fused), or otherwise associated with other molecules, molecular structures, or compounds at room temperature to produce, for example, liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations for assisting uptake, distribution, and / or absorption.

[0846] The oligonucleotides of the present disclosure can be administered with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be solid or liquid. Useful examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, excipients, suspending agents, buffers, lubricants, adjuvants, vehicles, emulsifiers, absorbents, dispersion media, coatings, stabilizers, protective colloids, binders, thickeners, thixotropic agents, permeants, chelating agents, isotonic agents that do not affect the activity of the active agents of the present disclosure, and absorption delaying agents.

[0847] In one embodiment, the pharmaceutical carrier is water for injection (WFI), and the pharmaceutical composition is adjusted to a pH of 7.4, 7.2 - 7.6. In one embodiment, the salt is a sodium salt or a potassium salt.

[0848] Oligonucleotides may contain chiral (asymmetric) centers, or the molecule as a whole may be chiral. Preferably, the oligonucleotide contains a chiral center at the phosphorothioate bond. Individual stereoisomers and mixtures thereof are within the scope of the present disclosure.

[0849] The oligonucleotides of the present disclosure may be pharmaceutically acceptable salts, esters or salts of esters, or any other compound which, upon administration, is capable of (directly or indirectly) providing a bioactive metabolite. As used herein, the term "pharmaceutically acceptable salt" refers to a physiological and pharmaceutically acceptable salt of an oligonucleotide which retains the desired biological activity of the parent compound and which does not produce an undesired toxicological effect upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860.

[0850] The oligonucleotides of the present disclosure may be prodrugs or pharmaceutically acceptable salts of prodrugs, or other bioequivalents. As used herein, the term "prodrug" refers to a therapeutic agent prepared in an inactive form which is converted to an active form (i.e., the drug) upon administration by the action of endogenous enzymes or other chemicals and / or conditions. Specifically, prodrug forms of the oligonucleotides of the present disclosure are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93 / 24510, WO 94 / 26764 and US 5,770,713.

[0851] Prodrugs can be converted, for example, in vivo, e.g., by hydrolysis in the blood, into an active form having a therapeutic effect. Pharmaceutically acceptable prodrugs are described in: T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series (1976); "Design of Prodrugs" by H. Bundgaard, edited by Elsevier, 1985; and Edward B. Roche, Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987. Those skilled in the art of organic chemistry will understand that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are called "solvates". For example, a complex with water is called a "hydrate".

[0852] In one embodiment, the oligonucleotides of the invention can be complexed with a complexing agent to increase cellular uptake of the oligonucleotides. Examples of complexing agents include cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells.

[0853] The term "cationic lipid" includes lipids and synthetic lipids having both polar and nonpolar domains and capable of being positively charged at or near physiological pH and of binding polyanions such as nucleic acids and facilitating delivery of nucleic acids to cells. Generally, cationic lipids include saturated and unsaturated alkyl groups of amines, amides or their derivatives and alicyclic ethers and esters. The straight-chain and branched-chain alkyl and alkenyl groups of cationic lipids can contain, for example, from 1 to about 25 carbon atoms. Preferred straight-chain or branched-chain alkyl or alkenyl groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with various counterions (anions), including Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite and nitrate.

[0854] Examples of cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE TM (e.g., LIPOFECTAMINE TM2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from: N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3.β-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; and dimethyloctadecylammonium bromide (DDAB). The oligonucleotide can also be complexed with, for example, poly(L-lysine) or avidin, and the lipid may or may not be included in this mixture, such as sterol-poly(L-lysine).

[0855] Cationic lipids have been used in the art to deliver oligonucleotides (and mRNA vaccines) to cells. Other lipid compositions that can be used to facilitate the uptake of the oligonucleotides of the present invention can be used in combination with the methods of the present invention. In addition to those listed above, other lipid compositions are known in the art and include, for example, those taught in US 4,235,871; US 4,501,728; 4,837,028; 4,737,323.

[0856] In one embodiment, the lipid composition can further comprise an agent, such as a viral protein, to enhance lipid-mediated transfection of the oligonucleotide. In another embodiment, N-substituted glycine oligonucleotides (peptoids) can be used to optimize the uptake of the oligonucleotide.

[0857] In another embodiment, the composition for delivering the oligonucleotides of the present invention comprises a peptide having from about one to about four basic residues. These basic residues can be located on the amino terminus, C terminus, or internal region of the peptide. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine (which can also be considered non-polar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Except for the basic amino acids, most or all of the other residues of the peptide can be selected from non-basic amino acids, such as amino acids other than lysine, arginine, or histidine. Preferably, neutral amino acids with long neutral side chains are used.

[0858] In one embodiment, the oligonucleotide is modified by linking a peptide sequence that transports the oligonucleotide into cells, herein referred to as a "transport peptide". In one embodiment, the composition includes an oligonucleotide complementary to a target nucleic acid molecule encoding a protein, and a covalently linked transport peptide.

[0859] In a further embodiment, the oligonucleotide is linked to a targeting moiety, such as N-acetylgalactosamine (GalNAc), an antibody, an antibody-like molecule, or an aptamer (see, for example, Toloue and Ford (2011) and Esposito et al. (2018)).

[0860] Administration

[0861] In one embodiment, the oligonucleotides of the present disclosure are administered systemically. In another embodiment, the oligonucleotides of the present disclosure are administered locally.

[0862] As used herein, "systemic administration" is an enteral or parenteral administration route.

[0863] As used herein, "enteral" refers to an administration form involving any part of the gastrointestinal tract and includes, for example, oral administration of the oligonucleotide in the form of tablets, capsules, or drops; nasogastric tube, duodenal tube, or gastrostomy; and rectal administration of the oligonucleotide in the form of suppositories or enemas.

[0864] As used herein, "parenteral" includes administration by injection or infusion. Examples include intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), subcutaneous (under the skin), intraosseous infusion (into the bone marrow), intradermal (into the skin itself), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical (infusion into the bladder), transdermal (diffusion through intact skin), transmucosal (diffusion through a mucous membrane), inhalation.

[0865] In one embodiment, administration of the pharmaceutical composition is subcutaneous administration.

[0866] Preferably, administration of the pharmaceutical composition is intravenous administration or topical administration, preferably intravenous administration.

[0867] The oligonucleotide can be administered as a single dose or periodically in repeated doses, for example, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every eight days, once every nine days, once every ten days, once every eleven days, once every twelve days, once every thirteen days or once every fourteen days, once a week, twice a week, three times a week, once every two weeks, once every three weeks, once a month, once every two months, once every three to six months or once every 12 months.

[0868] In one embodiment, the administration is 1 to 3 times per week, or once a week, once every two weeks, once every three weeks, once every four weeks or once every two months.

[0869] In one embodiment, the administration is once a week.

[0870] In one embodiment, a low dose is administered for 3 to 6 months, such as about 25 - 50 mg / week, for at least three to six months, and then for up to 12 months, and for long-term administration.

[0871] Exemplary doses range from about 10 mg to 5,000 mg. Exemplary doses include 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 1,000 mg, 2,000 mg. Exemplary doses include 1.5 mg / kg (about 50 mg to 100 mg) and 3 mg / kg (100 - 200 mg), 4.5 mg / kg (150 - 300 mg), 10 mg / kg, 20 mg / kg or 30 mg / kg. In one embodiment, the dose is administered once a week. Thus, in one embodiment, a low dose of about 10 mg to 30 mg, or 20 mg to 40 mg, or 20 mg to 28 mg can be administered to a subject having a body weight generally ranging from about 25 kg to 65 kg. In one embodiment, the oligonucleotide is administered at a dose of less than 50 mg / dose, or less than 30 mg / dose, or about 25 mg / dose to produce a therapeutic effect.

[0872] Example

[0873] Example 1 - Materials and Methods

[0874] Cell culture and stimulation

[0875] 293XL-hTLR7-HA and 293XL-hTLR8-HA, which stably express human TLR7 or TLR8, were purchased from Invivogen and maintained in Dulbecco's modified Eagle's medium supplemented with 1× antibiotic / antimycotic (Thermo Fisher Scientific), 10% heat-inactivated fetal bovine serum (referred to as complete DMEM), and 10 μg / ml Blasticidin (Invivogen) plus L-glutamine. Human acute myeloid leukemia THP-1 cells were grown in RPMI 1640 supplemented with 1× antibiotic / antimycotic and 10% heat-inactivated fetal bovine serum (referred to as complete RPMI) plus L-glutamine (Life Technologies). Unless otherwise stated, THP-1 cells were not differentiated with PMA in any experiment and were used in suspension. RAW264.7-ELAM macrophages, TLR7-deficient RAW264.7 cells, and immortalized wild-type BMDM (Ferrand et al., Frontiers in Cellular and Infection Microbiology, 2018; 8:87), as well as TLR7 / 8 double-deficient immortalized BMDM, were grown in complete DMEM. All cells were cultured at 37°C with 5% CO2. Cell lines were passaged 2-3 times per week, and mycoplasma contamination was detected by PCR or using Mycostrip (Invivogen) on a regular basis.

[0876] As indicated, the indicated cells were treated with the indicated concentration of oligonucleotides or empatoran (MedChemExpress) for 20 - 60 minutes and then treated with R848 (Invitrogen), uridine (Sigma), or motolimod (MedChemExpress). Desalted trimers and longer oligonucleotides were synthesized by Integrated DNA Technologies (IDT) or Syngenis Pty Ltd or Wuxi AppTec and resuspended in RNase-free TE buffer (pH 8.0) (Thermo Fisher Scientific). For in vivo experiments, the oligonucleotides were HPLC purified and confirmed endotoxin-free by the Lonza PYROGENT Plus Limulus Amebocyte Lysate gel-clot method. Sequences and modifications are provided in Tables 1 - 3. 2'-MOE is moX, 2'-MOE is mX, DNA is dX, and phosphorothioate internucleotide linkages are denoted by *.

[0877] Luciferase assay

[0878] According to the manufacturer's protocol, HEK293 cells stably expressing TLR8 or TLR7 were reverse transfected with the pNF-κB-Luc4 reporter gene (Clontech) and Lipofectamine 2000 (Thermo Fisher Scientific). Briefly, in each well of a 6-well plate, 500,000 - 700,000 cells were reverse transfected with 200 - 400 ng of the reporter gene and 1.2 μl of Lipofectamine 2000 and incubated at 37 °C with 5% CO2 for 3 - 24 hours. After transfection, cells were harvested from the 6 wells and aliquoted into 96 wells, followed by trimer and overnight TLR stimulation (as described above). Similarly, stably-expressed RAW264.7 cells and the ELAM-Luc reporter gene were treated overnight. The next day, the cells were lysed in 40 μl (for 96-well plates) of 1X Glo lysis buffer (Promega) for 10 minutes at room temperature. Then 15 μl of the lysate was subjected to a firefly luciferase assay using 40 μl of luciferase assay reagent (Promega). Luminescence was quantified using a Fluostar OPTIMA (BMG LABTECH) photometer.

[0879] RNA reverse transcription quantitative real-time PCR (RT-qPCR)

[0880] Total RNA was purified from cells using the RNeasy Mini Kit (Bioline). Random hexamer cDNA was synthesized from the isolated RNA using the High Capacity cDNA Archive Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RT-qPCR was performed on a QuantStudio 6 RT-PCR system (Thermo Fisher Scientific) using Power SYBR Green Master Mix (Thermo Fisher Scientific). Each PCR was performed in technical replicates, and human 18S was used as a reference gene. Each amplicon was gel purified and used to generate a standard curve for gene expression quantification (for each run). A melting curve was used in each run to confirm the specificity of amplification.

[0881] Detection of cytokines

[0882] Human IP-10, TNFα, and IL-6 levels were measured using supernatants from different cultures according to the manufacturer's protocol, and were quantified using IP-10 (BD Biosciences, cat. no. 550926), TNFα (BD Biosciences, cat. no. 555212), and IL-6 (BD Biosciences, cat. no. 555220) ELISA kits, respectively. 3,3',5,5'-Tetramethylbenzidine substrate (Thermo Fisher Scientific) was used to quantify cytokines on a Fluostar OPTIMA (BMG LABTECH) microplate reader.

[0883] Enhanced effect of TLR8 in humanized TLR8 / TLR7 mice

[0884] Spleens were harvested from 3 C57 / BL6 and 3 humanized C57BL / 6-Tlr8 tm1(TLR8) Tlr7 tm1(TLR7) / Bcgen (B-hTLR8 / hTLR7) mice, and splenocytes were isolated by passing the spleens through a 70 μm cell strainer. After erythrocyte lysis, the cells were washed and resuspended in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (ExCell), and then seeded at 3×10 5 cells / well in technical triplicates in 96-well flat-bottomed polystyrene TC-treated microplates. The cells were pretreated with 5 μM of TLR8 enhancer oligonucleotide 38-2 or vehicle control (TE buffer) for 1 hour, and then 1 μg / mL of resiquimod (R848) (MedChemExpress; HY-13740) was added overnight. The next day, the supernatants were harvested and used with LegendMax TMMouse TNF ELISA Kit (Biolegend, 430907) measures TNF levels by ELISA.

[0885] Enhancement of TLR8 in human skin grafts

[0886] Healthy human skin tissue was obtained from surgical residual sources with full ethical consent. Forty-eight full-thickness 3 mm × 3 mm punch biopsies (including epidermis, dermis, and subcutaneous tissue) obtained from 1 human donor were equilibrated overnight in basal skin medium. The next day, the biopsies were inserted into Transwell filters (Corning) in a 12-well culture plate, with the epidermis facing up at the air-liquid interface, and the dermis suspended in 1 mL of medium and incubated at 37 °C + 5% CO2. Then, the biopsies were pretreated with 5 μM TLR8-enhancing oligonucleotide, 38-2, or vehicle control (TE buffer) for 30 minutes, followed by the addition of 600 nM motolimod (Cambridge Bioscience; CAY22952) or vehicle control (DMSO) for 24 hours. The medium was collected to measure IL-8 levels by flow cytometry beads.

[0887] Co-encapsulation of GGC-v1 and mRNA within lipid nanoparticles

[0888] Using Nanoassemblr Ignite (Precision NanoSystems), 1 mg of FLuc mRNA (TriLink; L-7602) was premixed with 0.2 mg of GGC-v1 (5:1 weight ratio) either by pipetting or without pipetting, and then encapsulated in lipid nanoparticles (LNPs) with a composition of III-3:IVa PEG-lipid:DSPC:cholesterol = 47.4:10:40.9:1.7 (molar ratio). The concentration of the LNP-formulated mRNA sample was adjusted to 0.2 g / L. The LNP particle size, polydispersity index (PDI), ζ potential, and mRNA encapsulation rate were evaluated. LC-MS / MS and RT-qPCR were used to quantify GGC-v1 and mRNA in the LNPs, respectively.

[0889] Co-delivery of GGC-v1 and mRNA in vivo

[0890] Eight-week-old female 129X1 / SvJ mice (Jackson Laboratories) (approximately 25 g) were injected intravenously (i.v.) with approximately 20 μg of FLuc mRNA encapsulated in LNP with or without GGC-v1 (actual amounts were 20.349 μg and 20.484 μg) (see details above). Bioluminescence imaging was performed at 6 hours and 24 hours post-injection. Briefly, mice were anesthetized with 4% isoflurane and 3 mg / mouse of d-luciferin potassium salt was administered i.v. to quantify luminescence expression using images recorded within 3 minutes starting 5 minutes after luciferin injection. For bioluminescence image analysis, the IVIS Spectrum was used to define the region of interest encompassing the signal area, and the total number of photons / second [counts per second (cps)] was recorded. Blood samples were taken by submandibular bleeding at 6 hours post-injection and finally by cardiac puncture at 24 hours post-injection to serially quantify IFN-α by ELISA (Invitrogen; BMS 6027) and other inflammatory cytokines by Bio-Plex multiplex immunoassay (BIORAD; custom 8-Plex). Livers were also harvested at 24 hours and snap-frozen at -80 °C until analysis. To measure luciferase activity, livers were homogenized, sonicated, and vortexed in luciferase cell culture lysis reagent (Promega), followed by centrifugation at 4 °C and 15,000 rpm for 10 minutes. Then, the supernatant samples were mixed with luciferase assay reagent and luminescence was measured using a microplate reader within 5 minutes. Luciferase activity was quantified as relative light units (RLU). Bioluminescence imaging was performed at 6 hours and 24 hours post-injection. Briefly, mice were anesthetized with 4% isoflurane and 3 mg / mouse of d-luciferin potassium salt was administered i.v. to quantify luminescence expression using images recorded within 3 minutes starting 5 minutes after luciferin injection. For bioluminescence image analysis, the IVIS Spectrum was used to define the region of interest encompassing the signal area, and the total number of photons / second [counts per second (cps)] was recorded. Blood samples were taken by submandibular bleeding at 6 hours post-injection and finally by cardiac puncture at 24 hours post-injection to serially quantify IFN-α by ELISA (Invitrogen; BMS 6027) and other inflammatory cytokines by Bio-Plex multiplex immunoassay (BIORAD; custom 8-Plex). Livers were also harvested at 24 hours and snap-frozen at -80 °C until analysis. To measure luciferase activity, livers were homogenized, sonicated, and vortexed in luciferase cell culture lysis reagent (Promega), followed by centrifugation at 4 °C and 15,000 rpm for 10 minutes. Then, the supernatant samples were mixed with luciferase assay reagent and luminescence was measured using a microplate reader within 5 minutes. Luciferase activity was quantified as relative light units (RLU).

[0891] Statistical analysis

[0892] Statistical analysis was performed using Prism 9 (GraphPad Software Inc.). Each experiment was independently repeated at least twice (except for the trimer screening to validate the key trimer in independent experiments). When comparing groups of conditions, one-way analysis of variance (ANOVA) was used, while when comparing paired conditions, a two-tailed unpaired non-parametric Mann-Whitney U test or an unpaired two-tailed t-test was used. The symbols used were: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, and "ns" was not significant.

[0893] Example 2 - Structure-Activity Relationship of GUC Inhibition on Human TLR7

[0894] The C2Mut1-dC oligonucleotide containing 2'-OMe / DNA (mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC, where mX is a 2'-OMe modified base, dX is a DNA base and * is a phosphorothioate nucleotide internucleotide bond) has been shown to inhibit human TLR7 sensing. To determine the motif that exhibits the inhibitory effect of C2Mut1-dC, the inventors studied mutants of the 5'-end region of C2Mut1-dC, thereby generating 3'-end variants C2Mut1-dC2 and C2Mut1-dC3 carrying the mG*mG*mU*dA or mG*mG*mU*dC motif. Both C2Mut1-dC2 and C2Mut1-dC3 inhibit TLR7 sensing. In contrast, changing mG*mG*mU*dA to MG*MG*MU*dA or LG*LG*LU*dA (where M is MOE and L is LNA) significantly reduced the inhibitory effect of C2Mut1-dC2 ( Figure 1 ).

[0895] Based on these observations, the inventors directly tested the effects of the pentamer oligonucleotide mG*mG*mU*dA*dT (5-Short-Mut1-Hyb) and its related trimers mG*mG*mU, mG*mU*dA, and mU*dA*dT, as well as mG*mU*dC, to reflect the motif in C2Mut1-dC3 ( Figure 2 ). It was found that mG*mG*mU had only a moderate inhibitory effect (about 25% at 5 μM), while mG*mU*dA and mG*mU*dC were as potent as the pentamer oligonucleotide (it should also be noted that mU*dA*dT had only a marginal effect - Figure 2 ).

[0896] These observations confirmed that the trimers can retain inhibitory activity against TLR7 sensing, although they contain a DNA moiety at their 3rd base. To test whether other bases can tolerate DNA modification in the trimers, the inventors next evaluated the activities of 6 GUC variants incorporating a DNA moiety at different positions. As shown in Figure 3 , although the DNA modification at the 3rd position (GUCv1) had no effect on inhibition, it significantly reduced the activities at the 2nd base (GUCv2) and the 1st base (GUCv3). Interestingly, combining two DNA bases at the 2nd and 3rd bases (GUCv4) was more inhibitory than a single DNA modification at the 2nd base - without being bound by theory, this may be attributed to the fact that the DNA modification at the 3rd base is actually more inhibitory than the 2'-OMe base at this position. Thus, GUCv1 was found to be more inhibitory than the native intact 2'-OMe GUC trimer (see Figure 5)。Importantly, DNA substitution of the first base (as seen in GUCv3, v5, and v6) has the strongest negative effect on TLR7 inhibition.

[0897] Having shown that DNA modifications are compatible with trimeric TLR7 inhibition, the inventors next investigated whether RNA modifications and internucleotide modifications are also involved in the immunosuppressive effects of the trimers ( Figure 4 )。RNA bases on a phosphodiester (PO) or phosphorothioate (PS) backbone do not inhibit R848 sensing (see GUC-v7 and v9), but these trimers also do not activate any response in the absence of R848. In the GUC-v8 construct, replacement of mU with rU still has an inhibitory effect—although it is reduced compared to fully 2'-OMe GUC. mGrUrC (on a PO backbone) neither inhibits nor activates any response.

[0898] The inventors next tested whether chemical modifications of the 2'-OMe bases could be used to improve the inhibitory effects of the trimers. The first 5 variants of mG*mU*mC were tested, including two modifications of mG, two variants of mU, and one variant of mC.

[0899] As shown in Figure 5 , replacement of mG with 2'-OMe-2,6-diaminopurine or 2'-OMe-I, or replacement of mU with 2'-OMe-5-Me-U or 2'-OMe-5-Br-U, maintained strong inhibitory activity at 5 μM. However, replacement of mG with 2'-OMe-2,6-diaminopurine, or replacement of mU with 2'-OMe-5-Me-U or 2'-OMe-5-Br-U significantly reduced the inhibitory activity of GUC at 200 nM (whereas replacement with mG 2'-OMe-I inhibited GUC at this dose). Importantly, replacement of mC with 2'-OMe-5-Me-C significantly increased TLR7 inhibition at 200 nM and 50 nM (see GUC-v19) ( Figure 5 )。This indicates that modification of the cytosine base with an additional methyl group (as seen in GUC-v19) can improve TLR7 inhibition. Notably, GUC-v19 has approximately 50% inhibition at 50 nM, which is only seen for native 2'-OMe GUC at 200 nM. This is approximately a 4X improvement in IC 50 .

[0900] Combined with the previous finding that the deoxyribose of this base in GUC-v1 (with the dC variant) is also more potent, the inventors next tested a series of GUC variants focused on cytidine. The inventors tested the selective replacement of mC and dC in mG*mU*mC with LNA, 2'-MOE, 2'-fluoro, and RNA C (i.e., GUC-v11-14) or modified cytidines (i.e., GUC-v20, GUC-v21, GUC-v23, GUC-v24, GUC-v26 - with 5-methyl-modified deoxycytidine, 5-bromo-modified deoxycytidine, 5-hydroxymethyl-modified deoxycytidine, 2'-deoxy-3'-deoxycytidine, and 5-propynyl-modified deoxycytidine) on the PS backbone. These experiments showed that the modification of cytidine maintained strong TLR7 inhibition ( Figure 7 ).

[0901] These structure-activity relationship analyses together confirmed that the GUC trimer is a very potent inhibitor of TLR7. The inventors next compared the head-to-head dose responses of GUC-v19, GUC-v1, and the full 2'-OMe GUC trimer in HEK TLR7 cells ( Figure 8 ). The trimer is clearly a very potent inhibitor, with GUC-v19 being the most potent, and its IC 50 expected to be below 100 nM.

[0902] Importantly, the screening analysis of the 2'-OMe trimer showed that the GUX motif is the most potent, where X can be mC, mG, mU, or mA. The observation that both mG*mU*dA and mG*mU*dC retain inhibitory function indicates that mG*mU*dX (where dX is a DNA base) can also inhibit TLR7. The inventors next tested whether the dinucleotide mG*mU could also inhibit TLR7. As shown in Figure 9 A, although it is clearly able to inhibit TLR7 sensing at high doses (5 μM), mG*mU is a weaker inhibitor of TLR7 compared to the mG*mU*mC trimer, with most of its inhibitory activity lost below 5 μM ( Figure 9 B).

[0903] This finding confirmed the importance of the 3rd base pair in suppressing TLR7 - also consistent with the ability of the 3rd base to increase the inhibitory potency seen with GUC-v1 and GUC-v19. However, to define whether the optimal length of inhibition is 3, 4, or 5 bases, the inventors compared the inhibitory activities of mG*mU*mA, mG*mG*mU*mA, and mG*mG*mU*mA*mU against TLR7 inhibition. This compared the activities of mG*mU*dC with the pentamer mG*mG*mU*dC*dT, and two mG*mU*dC trimers linked with a triethylene glycol (TEG) linker in a 5'-3'-3'-5' orientation ( Figure 10 ).

[0904] Overall, these results confirmed the fact that while longer molecules can also inhibit TLR7 with similar potency at higher doses (e.g., the pentamer short-Mut-1 at 5 μM), the GUC-v1 trimer is the optimal length for maximal inhibition at a lower dose (500 nM). Importantly, the fusion of two GUC-v1 trimers with a TEG linker maintained strong inhibitory activity even at a concentration of 50 nM, at which a single GUC-v1 trimer was not inhibitory. This suggests that the TEG linker is cleaved to release two GUC-v1 molecules for each linked oligonucleotide molecule, which is the basis for its stronger activity. This demonstrates the potential of fusing trimers with linkers such as TEG (which can be further functionalized for specific cellular uptake) while maintaining TLR7 inhibitory activity.

[0905] Since trimers containing as few as a single 2'-OMe moiety in combination with two DNA moieties retained inhibitory activity against TLR7 (as seen with GUC-v4), the inventors next tested all 64 possible combinations of DNA trimers for TLR7 inhibition. A comparison of the inhibition at 2 μM showed that relative to 2'-OMe there were 26, and only 3 DNA trimers inhibited TLR7 by more than 20% (TCT, TTT, and TTG)( Figure 11 ). Similarly, the inventors tested the inhibition of 64 combinations of 2'-MOE trimers against TLR7. At 5 μM, no MOE trimer inhibited by more than 20%, with GAA being the most potent, which only inhibited 19% at this high dose( Figure 11 ).

[0906] To complement these studies, the inventors also tested 32 mG*X*X hybrid variants where the second and / or third base was a DNA base( Figure 12)。These assays performed at 5 μM and 500 nM confirmed the ability of the mG*mU*dX variants to strongly inhibit human TLR7 sensing - where mG*mU*dC was the most potent inhibitor, followed by mG*mU*dG, mG*mU*dA, mG*mG*dC, and mG*mU*dT (similarly observed in the fully 2'-OMe modified trimers). However, the inhibition of DNA modifications at both the second and third bases (as seen in the mG*dX*dX molecules) was lower than that of the trimers with a single DNA modification, which is consistent with Figure 3 the results of the GUC variants in

[0907] Example 3 - Trimeric Inhibition of Mouse TLR7

[0908] Unlike the potent inhibition of TLR7 sensing seen in human cells with 2'-OMe GUC, these trimers were weak inhibitors of mouse TLR7. Therefore, the inventors tested a set of 64 2'-OMe trimers for mTLR7 sensing in RAW 264.7 cells stably expressing the ELAM-luciferase reporter gene, which is activated by R848 sensing (Zamanian-Daryoush et al., Journal of Interferon and Cytokine Research, 2008; 28(4):221 - 33). This screening was performed at two doses of R848 (0.5 μg / ml and 0.125 μg / ml), and GGC, GAC, and GAG were identified as the most potent inhibitors of mouse TLR7 sensing ( Figure 13 ). The inventors also tested the set of 64 DNA trimers on mouse TLR7 sensing and observed that 8 trimers inhibited more than 20% at 5 μM with mild inhibition (the maximum inhibition of ACG was 27%). However, at this dose, 16 2'-OMe trimers inhibited mTLR7 by more than 20%, thus showing a clear advantage of the 2'-OMe trimers ( Figure 13 ).

[0909] To define whether, like human TLR7, mouse TLR7 inhibition could be increased after DNA substitution of the selected bases in the trimers, the inventors next tested a set of 6 variants of 2'-OMe GGC and GAG ( Figure 14 ). Similar to what was observed in the human 2'-OMe GUC variants, the v1 mutation of both trimers increased inhibition, while the DNA modification of the first mG abolished activity. Importantly, these experiments showed that for both GGC and GAG, two DNA base substitutions in the v4 trimer were well tolerated and resulted in a substantial improvement in inhibition compared to the intact 2'-OMe trimersFigure 14 ) These findings suggest that, although closely related to human TLR7 sensing, the inhibition of murine TLR7 by the trimers is slightly different - which is consistent with the better inhibition of murine TLR7 by GGC than by GUC.

[0910] The inventors also tested the head-to-head dose-dependent activities of GGC, GGC-v1, and GGC-v3 on murine TLR7 sensing. GGC-v1 was slightly more potent than GGC, and critically, GGC-v3 had no inhibitory activity ( Figure 15 ).

[0911] The inventors next tested whether the trimers could also inhibit TLR7 sensing driven by an immunostimulatory ssRNA (designated B-406-AS-1) (Sarvestani et al., Nucleic Acids Res, 2015; 43(2): 1177-88). As Figure 16 shown, in RAW-ELAM cells, GGC significantly inhibited ELAM-luc driven by transfection of B-406-AS-1.

[0912] It has been shown that trimers carrying 2 DNA bases (e.g., GAG-v4- Figure 14 ) remain very potent inhibitors of murine TLR7. The inventors next evaluated the inhibition of 64 DNA trimers. As shown in Figure 17 , the DNA trimers had only modest inhibitory activity, with only 8 oligonucleotides inhibiting more than 20% (the maximum inhibition by ACG at 5 μM was 28%). There was little overlap in the inhibition between the chemicals (trimers that inhibit TLR7 with 2'-OMe when composed of DNA bases had no inhibitory activity), and ACG was the only trimer that showed >20% inhibition for both DNA and 2'-OMe chemicals.

[0913] These observations suggest that at least one 2'-OMe base is crucial for the activity of trimers on murine TLR7. To define whether other combinations of mG*mX*dX or mG*dX*dX (where dX is DNA and mX is 2'-OMe) trimers could be superior in activity to mG*dA*dG or mG*mG*dC, the inventors next tested 32 possible mG*X*X trimers on murine TLR7. These results identified mG*mA*dC as the most potent inhibitor of TLR7, slightly superior in activity to GAG-v4 (mG*dA*dG) and GGC-v1 (mG*mG*dC) ( Figure 18 ).

[0914] Example 4 - Regulation of TLR8 Sensing by Trimers

[0915] To determine whether 2'-OMe trimers would enhance or inhibit TLR8 sensing, the inventors screened single doses of various trimers in HEK-TLR8 and THP-1 cells. As shown in Figure 19 , in each cell type, there was a significant correlation between the enhancing and inhibitory effects in response to two doses (slightly better in HEK-TLR8). In both cell types, 2'-OMe-CGG was the strongest enhancer of R848 or motolimod sensing. UCG was also a top enhancer in both cells. However, AGG was very potent in HEK-TLR8 cells but not in THP-1 cells, and UCA / CGC was strong in THP-1 cells but not in HEK-TLR8 cells. GAX was a strong inhibitor of TLR8 in both cell types, with GAG being the most potent in THP-1 cells at 1 μM.

[0916] Given the effect of the DNA moiety on TLR7 inhibition, the inventors next tested the effect of sequential DNA modifications of CGG and GAG according to the same protocol as GUCv1-v6. As shown in Figure 20 A and B, in both HEK-TLR8 and THP-1 cells, replacing the last base of 2'-OMe in CGG-v1 with a DNA base maintained the enhancing effect, but this was not as potent as the native 2'-OMe CGG sequence. DNA modifications at other positions dwarfed the enhancing effect.

[0917] Conversely, GAG-v1, v2, v4, and v5 maintained good TLR8 inhibitory effects across both cell models ( Figure 20 C and D). GAG-v3 and v6 had less TLR8 inhibitory effects in both cell types, indicating that DNA modification of the 5'-terminal G is very important for TLR8 inhibition.

[0918] Thus, the inhibition of TLR8 by trimers is very similar to what was observed for human and mouse TLR7 inhibition, where the inhibitory effect of the first 2'-OMe base is common, although the retention effect of GAG-v5 should be noted. GAG-v1 was slightly better than native 2'-OMe GAG in THP-1 and was thus continued in further experiments.

[0919] Having shown that GAG-v4 / 5 with two DNA bases retained some inhibitory activity against TLR8, the inventors next evaluated the immunomodulatory effects of 64 DNA and 64 2'-MOE trimers on TLR8 sensing in HEK-TLR8 cells, testing at 1 μm and 5 μM. As shown in Figure 21As shown, only one trimer (2'-MOE GGT) increased TLR8 sensing, and this was very modest compared to 2'-OMe CGG. In contrast, many trimers were partially inhibitory to both chemicals, yet a low correlation was noted between the two concentrations screened for 2'-MOE (indicating experimental differences rather than true inhibition). On the other hand, the inhibitory correlation for DNA trimers was better - where the AGT sequence inhibited TLR8 sensing the most ( Figure 21 ).

[0920] Based on this TLR8 inhibitory activity of DNA trimers and considering the strong inhibitory effect of GAG-v4 (mG*dA*dG), the inventors next tested whether other combinations of high-dose (5 μM) mG*mX*dX or mG*dX*dX (where dX is DNA and mX is 2'OMe) could inhibit human TLR8. These studies identified mG*dA*dG as the most potent inhibitor of TLR8, while mG*dC*dC was the strongest potentiator ( Figure 22 A). These observations were further verified in independent experiments with trimers at a low dose (500 nM), where mG*dA*dG was the most potent inhibitor of TLR8 ( Figure 22 B). Importantly, at this low dose (500 nM), mG*dC*dC was a stronger potentiator of TLR8 sensing than mC*mG*mG. These results together establish the ability to both inhibit and enhance the activity of trimers on TLR8 by using DNA substitutions of selected bases in 2'-OMe trimers.

[0921] Example 5 - Alternative Modifications of GUC Trimers and Inhibition of Human TLR7

[0922] The inventors' previous analysis demonstrated that inhibition of human TLR7 sensing by trimeric oligonucleotides could be significantly improved by modifying the 3'-cytidine of the 2'-OMe-modified GUC trimer, which was the most potent TLR7 inhibitor in the inventors' previous systematic analysis of all 64 combinations of 2'-OMe trimers. In this regard, modifying the 2'-position of the sugar, such as using 2'-deoxycytidine to replace 2'-OMe-cytidine in GUC-v1, or modifying the cytosine moiety in GUC-v19 ( Figure 5 and 6 ), could both be used to improve the inhibitory activity of the GUC trimer. Modification of the 2'-position of the cytidine in GUC-v11 / v12 / v13 / v14, removal of the 3'-hydroxyl of the cytidine in GUC-v24, or modification of the cytosine base in GUC-v20 / v21 / v23 / v26 has been shown to maintain activity at 5 μM ( Figure 7),The inventors next tested the activity of these trimers at 50 nM compared to the parental 2'-OMe modified GUC.

[0923] These assays identified three trimers that were significantly inhibitory at 50 nM: GUC-v13 (with an LNA modification of the sugar), GUC-v20, and GUC-v21 (both with a modification at the 5-position of the cytosine base) ( Figure 23 ). Binding Figure 5 findings and the results of GUC-v19 (which is a 2'-OMe variant of GUC-v20), these results directly revealed the positions of the cytosine base that can be used to increase TLR7 inhibition. Thus, 5-methyl or 5-bromo modification of cytosine (in GUC-v19 / 20 and GUC-v21) increased inhibition. However, further modification of this 5-position with hydroxymethyl in GUC-v23 or 5-propynyl in GUC-v26 abolished inhibition (structural details see Figure 6 ). Among these experiments, GUC-v13 containing an LNA modification of its sugar was the most potent. Dose-response analysis showed that its IC 50 for TLR7 inhibition was as low as 18 nM compared to 77 nM for the parental 2'-OMe modified GUC. Importantly, the activity of GUC-v13 was close to that of the small molecule inhibitor emapatran of TLR7 / 8 ( Figure 24 ).

[0924] However, when the inventors tested a small panel of fully modified LNA or MOE trimers, including LNA·GUC, these did not significantly inhibit TLR7 even when used at a high dose of 2 μM ( Figure 25 ). Overall, this indicates that although full LNA modification of the GU[X] trimers abolishes inhibitory activity, selected nucleotide LNA modifications can instead increase inhibition.

[0925] To define how selected LNA modifications of the 3'-terminal nucleotide of other 2'-OMe modified trimers affect their activity against TLR7, the inventors designed a set of 16 mG*mX*LX and mU*mX*LX trimers in which the first and second nucleotides were 2'-OMe modified and the third nucleotide was LNA modified, and tested this set on HEK TLR7 cells ( Figure 26 ).

[0926] Figure 26The experiments shown in A revealed that, in the context of LNA modification at the 3rd nucleotide, GUC-v13 (i.e., mG*mU*IC) remained the most potent trimer for inhibiting hTLR7. These studies also showed that trimers containing GU[X] and GA[X] were the most potent inhibitors. Importantly, a direct comparison of LNA-modified trimers at the 3rd base with intact 2'-OMe trimers at 400 nM confirmed a direct correlation in inhibition between these two classes of trimers (r = 0.6335, P < 0.0001)( Figure 26 B). Nevertheless, LNA modification at the 3rd base increased the potency of inhibition in a sequence-specific manner, where 17 / 32 of the trimers inhibited by more than 20% at 400 nM, as opposed to 5 / 32 of the intact 2'-OMe trimers (GU[X] and UUC). This was particularly evident in the mG*mA*LX trimers, which were stronger inhibitors of the LNA moiety. Importantly, most of the strongest inhibitors contained 5'-mG (where 14 / 16 of the mG*mX*LX trimers inhibited 20% or more), further emphasizing the importance of this base in human TLR7 inhibition.

[0927] It has been shown that extending GUC-v1 with a TEG linker and another 3'-5' GUC-v1 monomer (GUC-v1 linked 3'-5') significantly increased TLR7 inhibition( Figure 10 ), and the inventors next investigated whether a single TEG group appended to GUC-v1 could increase TLR7 inhibition. The inventors also tested the activity of cholesterol and tocopherol conjugated to the 3' end of GUC-v1 to determine whether these groups could be used later for delivering the naked trimers( Figure 27 ).

[0928] These experiments revealed that 3'-TEG conjugation did not significantly improve GUC-v1-driven TLR7 inhibition, suggesting that the increased activity of GUC-v1 linked containing two GUC moieties was due to a doubling of the endosomal concentration of the trimer rather than an increase in its resistance to nucleases( Figure 27 B). Similarly, adding 3'-cholesterol (GUC-28) or tocopherol (GUC-29) to GUC-v1 decreased its inhibitory activity, although significant inhibitory function was clearly maintained at 200 nM( Figure 27 A). Collectively, these results suggest that increased protection against 3'-5' end degradation may not be the reason for the increased inhibitory activity of LNA (in the context of GU[X / ]GA[X] trimers) against TLR7.

[0929] Previously, it has been shown that replacing 2'-OMe-guanosine with 2'-OMe-inosine in GUC-v16 was at least as good as the parental GUC and GUC-v1Figure 5 ), the present inventors decided to study the activity of GUC-v35 / v36 with various 2'-OMe-inosine modifications( Figure 6 ). The present inventors also tested whether replacing 2'-OMe-uridine with pseudouridine at the second position of GUC-v1 could be used to enhance further inhibition( Figure 28 ).

[0930] These studies revealed that DNA and RNA inosine modifications in GUC-v35 and v36 significantly inhibited the inhibitory activity of the parental 2'-OMeGUC or GUC-v16, and almost all inhibitory effects were lost even at a very high dose of 5 μM( Figure 28 A). Although GUC-v31 maintained its inhibitory function at high concentrations, its inhibitory effect at 1 μM was also much lower than that of GUC-v13 / 16 / 30, indicating that this modification limited the inhibition( Figure 28 B). Head-to-head dose-response analysis of GUC-v30(mI*mU*LC) and GUC-v13 showed that although both sequences exhibited equivalent inhibitory activity above 125 nM, GUC-v13 was more potent at lower doses (in these experiments, GUC-v13 IC50 = 40 nM versus GUC-v30 IC50 = 62 nM)( Figure 29 ). These results confirmed the strong activity of the 3rd base LNA modification in the GUC background, but also revealed that the 5'-terminal 2'OMe-inosine modification could not further improve TLR7 inhibition in this background. The results of GUC-31 showed that the pseudouridine modification instead reduced the activity of 2'-OMe uridine on TLR7 inhibition (thus confirming that 2'-OMe uridine is the most potent at this position)( Figure 28 and 29 ).

[0931] Finally, the present inventors tested a group of 32 trimers with DNA and 2'-OMe modifications to expand the discovery of mG*d[X]*d[X] trimers shown in Figure 12 , now using mU*d[X]*d[X] and mC*d[X]*d[X] trimers.

[0932] This screening at a high dose of 5 μM of the trimers confirmed that mGmUmC was more potent than any other oligonucleotide tested( Figure 30)。mUdTdC, mUdTdA, mUdAdA, mUdGdC, mUdTdT, and mUdGdT are the most potent inhibitors, but only achieve about 50 - 75% inhibition, which is rather weak at such very high doses. Interestingly, all trimers showing inhibitory activity here start with mU, and mUdTd[X] is over-represented. None of the mCd[X]d[X] trimers inhibit TLR7 sensing.

[0933] Example 6 - Alternative Trimers for Inhibiting Mouse TLR7

[0934] The inventors previously Figure 1 analysis of LNA-modified and MOE-modified 20-mer oligonucleotides in [reference] showed that these modifications inhibited TLR7 in human cells. To define whether this is also the case for murine TLR7, the inventors tested a set of Mut1-dC variants on RAW-ELAM cells.

[0935] To support the importance of the 5'-terminal region of C2Mut1-dC in its inhibitory effect on murine TLR7, both 3'-terminal variants C2Mut1-dC2 and C2Mut1-dC3 carrying the mG*mG*mU*dA or mG*mG*mU*dC motif inhibited murine TLR7 sensing ( Figure 31 ). In contrast, changing mG*mG*mU*dA to MG*MG*MT*dA or LG*LG*LT*dA (where M is MOE and L is LNA) significantly reduced the inhibition of C2Mut1-dC2 ( Figure 1 and 31 ).

[0936] Based on these observations, the inventors directly tested the effect of the 5-mer oligonucleotide mG*mG*mU*dA*dT (5-short-Mut1-Hyb) and its related trimers mG*mG*mU, mG*mU*dA, and mU*dA*dT (the inventors also included mG*mU*dC to reflect the motif in C2Mut1-dC3) on murine TLR7 sensing ( Figure 32 ). Consistent with previous screening analyses ( Figure 13 and 17 ), mG*mG*mU only had moderate inhibition (about 50% at 5 μM), while mG*mU*dA and mG*mU*dC were more potent (based on the screening of mGd[X]d[X] oligonucleotides in Figure 17 ). Interestingly, given that the parental mG*mU*mC had only very negligible inhibitory activity (about 18%) on murine TLR7 in the screening, mG*mU*dC was the most potent trimer oligonucleotide in these experiments ( Figure 32) This indicates that even in a less favorable GUC sequence context, 3'-end modifications can still be used to increase murine TLR7 inhibition. This observation prompted the inventors to analyze the broad impact of a panel of GUC variants on murine TLR7.

[0937] As expected, different from what was observed in human TLR7 where GUC is the optimal sequence context, most GUC variants had little activity in sensing murine TLR7 ( Figure 33 ). However, compared to the parental 2'-OMe-modified GUC, DNA modifications of cytidine in GUC-v1, GUC-v20, GUC-v21, and GUC-v26 were associated with increased inhibition. This indicates that modifications at the 2'-position of the sugar can improve the inhibitory activity against murine TLR7, which is consistent with the findings of GGC-v1 and GAG-v1 / v2 / v4 ( Figure 14 ). It should be noted that other 2'-modifications of cytidine in GUC-v11 - GUC-v14, including 2'-hydroxy (RNA), 2'-O-MOE, LNA-modified, and 2'-fluoro, did not show improvement compared to 2'-OMe cytidine. Additionally, the lack of activity in GUC-v24 with 2',3'-dideoxycytidine indicates that the terminal 3'-hydroxy is important for activity.

[0938] Modification of 2'-OMe-guanosine in GUC-v15 / v16 increased the inhibition against murine TLR7 ( Figure 33 ). The result of GUC-v15 where guanine was modified to 2,6-diaminopurine indicates tha...

Claims

1. An oligonucleotide comprising a sequence consisting of or consisting of: [mX / Modified mX]* y X A * z X B Wherein: * y and * z each independently represents an internucleotide bond, wherein * y and * z at least one of them is not a phosphorodiamidate; X A and X B each independently is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X; Where mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide comprising a 2'-MOE and / or 3'-MOE modification, LX is an LNA-modified base, fX is a nucleotide comprising a 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide comprising a morpholine ring; and wherein when [mX / modified mX] is mX, X A and X B at least one of wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z not a 3'-5'-phosphodiester nucleotide internucleoside bond; Wherein the sequence is optionally functionalized.

2. The oligonucleotide according to claim 1, wherein each internucleotide bond is a 3'-5'-thioester bond.

3. The oligonucleotide according to claim 2, wherein * y it has an S configuration.

4. The oligonucleotide according to any one of claims 1 to 3, wherein mX is a nucleotide comprising a 2'-OMe modification, moX is a nucleotide comprising a 2'-MOE modification, and fX is a nucleotide comprising a 2'-fluoro modification.

5. The oligonucleotide according to any one of claims 1 to 4, wherein the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, mU3, mC1 and m7 G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mU3 is N3-Me-U (3-methyluridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), and m7 G is 3'-OMe-N7-methylated guanosine.

6. The oligonucleotide according to any one of claims 1 to 5, wherein the modified dX is selected from the group consisting of: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, PSU, N3-Me-dC, 5-I-dC, dI, 8-Br-dG, 7-deaza-dG, 8-Br-dA, 8-oxo-dA, O6-Me-dG, 8-NH2-dG, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, PSU is pseudouridine, N3-Me-dC is 3-methyl deoxycytidine, 5-I-dC is 5-iodo-deoxycytidine, dI is deoxyinosine, 8-Br-dG is 8-bromo-deoxyguanosine, 7-deaza-dG is 7-deaza-deoxyguanosine, 8-Br-dA is 8-bromo-deoxyadenosine, 8-oxo-dA is 8-oxo-deoxyadenosine, O6-Me-dG is O6-methyl deoxyguanosine, and 8-NH2-dG is 8-amino-deoxyguanosine.

7. The oligonucleotide according to any one of claims 1 to 6, wherein the modified rX is selected from the group consisting of: PSU, 2'-NH2-U, 2'-NH2-C, and ara-C, where PSU is pseudouridine, 2'-NH2-U is 2'-amino uridine, and 2'-NH2-C is 2'-amino cytidine, and ara-rC is cytarabine.

8. The oligonucleotide according to any one of claims 1 to 7, wherein [mX / modified mX] is selected from the group consisting of: mG, mI, mG1, and mU.

9. The oligonucleotide according to any one of claims 1 to 8, wherein X A is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified rX.

10. The oligonucleotide according to claim 9, wherein X A is selected from the group consisting of: mU, mU1, mU2, PSU, mG, mA, mC, dT, dG, dA, dC, rU, 2'-NH2-rU, 8-Br-dA, and 8-oxo-dA.

11. The oligonucleotide according to any one of claims 1 to 10, wherein X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX, and morpholino-X.

12. The oligonucleotide according to claim 11, wherein X B is selected from the group consisting of: dA, dC, dG, dT, mC, mC1, mG, rC, moC, LC, LA, LT, LG, fC, 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, N3-Me-dC, 5-I-dC, 2'-NH2-C, ara-C, morpholino-C, N3-Me-mU, dI, 8-Br-dG, 7-deaza-dG, O6-Me-dG, and 8-NH2-dG.

13. The oligonucleotide according to any one of claims 1 to 12, wherein [mX / modified mX] is [mG / mI]; X A is mU; and X B is selected from the group consisting of: mX, dX, LX, modified mX, modified rX, and modified dX.

14. The oligonucleotide according to any one of claims 1 to 12, wherein the sequence is selected from the group consisting of:

15. The oligonucleotide according to claim 14, wherein the sequence is selected from the group consisting of: mG*mU*LC, mI*mU*LC, mG*mU*mC1, mG*mU*5-Me-dC, mG*mU*5-Br-dC, mG*mU*dC, mG*mU*dC-TEG, mI*mU*mC, mG*mU*dC-Chol, mG*mU*dC-Toco, mG*mU*ara-C, and mG*mU*5-I-dC.

16. The oligonucleotide according to any one of claims 1 to 15, wherein the oligonucleotide consists of the sequence.

17. A fusion oligonucleotide, comprising: A-[Y-A] n wherein each A independently represents an oligonucleotide according to any one of claims 1 to 16, and each A can be the same or different; Y represents a cleavable linker, and each Y can be the same or different; and n is equal to or greater than 1.

18. The fusion oligonucleotide according to claim 17, having the sequence 5'-mG*mU*dC-3'-*TEG*-3'-dC*mU*mG-5'.

19. A composition, comprising an oligonucleotide according to any one of claims 1 to 16 or a fusion oligonucleotide according to any one of claims 17 to 18.

20. An immunogenic composition, comprising an oligonucleotide according to any one of claims 1 to 16 or a fusion oligonucleotide according to any one of claims 17 to 18, and a therapeutic RNA.

21. A method for inhibiting TLR7 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 16, a fusion oligonucleotide according to any one of claims 17 to 18, or a composition according to claim 19, thereby inhibiting TLR7 activity in the subject.

22. A method for inhibiting TLR7 activity in a cell, the method comprising contacting the cell with an oligonucleotide according to any one of claims 1 to 16, a fusion oligonucleotide according to any one of claims 17 to 18, a composition according to claim 19, or an immunogenic composition according to claim 20, thereby inhibiting TLR7 activity in the cell.

23. A method for inhibiting TLR7 activation in a subject caused by a therapeutic RNA, the therapeutic RNA being selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 16, a fusion oligonucleotide according to any one of claims 17 to 18, a composition according to claim 19, or an immunogenic composition according to claim 20, thereby inhibiting TLR7 activation in the subject.

24. A method for treating or preventing a disease, disorder or condition in a subject responsive to TLR7 inhibition, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 16, a fusion oligonucleotide according to any one of claims 17 to 18, a composition according to claim 19 or an immunogenic composition according to claim 20, thereby treating or preventing the disease, disorder or condition in the subject.

25. The method according to claim 23 or 24, wherein the disease, disorder or condition is selected from the group consisting of: inflammation-related diseases, allergic diseases, infections, cancers and autoimmune diseases.

26. An oligonucleotide comprising a sequence consisting of or consisting of: X C * y X D * z X E Wherein: * y and * z each independently represents an internucleotide bond; X C selected from the group consisting of: mX, modified mX, dG, and morpholino-X; X D and X E each independently selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified dX, modified rX, modified moX, modified LX, modified fX, and modified morpholino-X; Where mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, rX is an RNA base, moX is a nucleotide comprising a 2'-MOE and / or 3'-MOE modification, LX is a base modified with LNA, fX is a nucleotide comprising a 2'-fluoro and / or 3'-fluoro modification, and morpholino-X is a nucleotide comprising a morpholine ring; and Wherein: When X C is mX, at least one of X D and X E is not mX; When X C is dG, at least one of X D and X E is not dX; When X C is mG and when: X D When it is dG, X E is not dA or dC; X D When it is dT or mU, X E is not dC or dT; X D When it is mC, X E is not dT, dG or dC; and X D When X is mG or dC E it is not dX; or When X C is dG, X E is not mG, wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleoside bond; Where the sequence is optionally functionalized.

27. The oligonucleotide according to claim 26, wherein each internucleotide bond is a 3'-5'-thio-phosphate bond.

28. The oligonucleotide according to any one of claims 26 to 27, wherein mX is a nucleotide comprising a 2'-OMe modification, moX is a nucleotide comprising a 2'-MOE modification, and fX is a nucleotide comprising a 2'-fluoro modification.

29. The oligonucleotide according to any one of claims 26 to 28, wherein the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, mU3, mC1, m7 G, wherein mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mU3 is N3-Me-U (3-methyluridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), and m7 G is 3'-OMe-N7-methylated guanosine.

30. The oligonucleotide according to any one of claims 26 to 29, wherein the modified dX is selected from the group consisting of: 5-Me-dC, 5-Br-dC, ddC, pdC, PSU, dI, 8-Br-dG, N1-Me-dG, 7-deaza-dG, 8-Br-dA, 8-oxo-dA, O6-Me-dG and 8-NH2-dG, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, PSU is pseudouridine, dI is deoxyinosine, 8-Br-dG is 8-bromodeoxyguanosine, N1-Me-dG is 1-methyl deoxyguanosine, 7-deaza-dG is 7-deaza-deoxyguanosine, 8-Br-dA is 8-bromodeoxyadenosine, 8-oxo-dA is 8-oxodeoxyadenosine, O6-Me-dG is O6-methyl deoxyguanosine, and 8-NH2-dG is 8-amino deoxyguanosine.

31. The oligonucleotide according to any one of claims 26 to 30, wherein the modified rX is selected from the group consisting of: PSU, 2'-NH2-U, and 2'-NH2-C, where PSU is pseudouridine, 2'-NH2-U is 2'-aminouridine, and 2'-NH2-C is 2'-aminocytidine.

32. The oligonucleotide according to any one of claims 26 to 31, wherein X C is selected from the group consisting of: mG, mU, mC, mI, mG1, and dG.

33. The oligonucleotide according to any one of claims 26 to 32, wherein X D is selected from the group consisting of: mX, dX, rX, modified mX, modified dX, and modified rX.

34. The oligonucleotide according to any one of claims 26 to 33, wherein X E is selected from the group consisting of: mX, dX, rX, morpholino-X, moX, LX, fX, rX, modified mX, modified dX, and modified rX.

35. The oligonucleotide according to any one of claims 26 to 34, wherein the sequence is selected from the group consisting of:

36. The oligonucleotide according to claim 35, wherein the sequence is selected from the group consisting of: mI*mA*dG, mI*mU*mC, mG*dA*dG, mG*mA*dT, mG*mA*dG, mG*mA*dC, mG*mA*LG, mG*mA*rG, mG*mA*LT, mG*mA*LC, mU*dT*dC, mU*dA*dC, mG*mA*LA, mU*dA*dG, mC*dA*dG, mU*dT*dT, mU*dA*dT, mU*dA*dA, mC*dT*dA, mU*dG*dT, mC*dT*dC, mC*dA*dT, mU*dG*dG, mC*dT*dT, mC*dT*dG, mU*dT*dA, mU*dT*dG, mG*mA*O6-Me-dG, mG*rA*rA, mG*rG*rA, mG*rA*rG, and mG*rA*rU.

37. The oligonucleotide according to claim 36, wherein the sequence is selected from the group consisting of: mI*mA*dG, mI*mU*mC, mG*dA*dG, mG*mA*dT, mG*mA*dG, mG*mA*dC, mG*rA*rA, and mG*rG*rA.

38. The oligonucleotide according to any one of claims 26 to 37, wherein the oligonucleotide consists of the sequence.

39. A fusion oligonucleotide, comprising: A-[Y-A] n wherein each A independently represents an oligonucleotide according to any one of claims 26 to 38, and each A can be the same or different; Y represents a cleavable linker, and each Y can be the same or different; and n is equal to or greater than 1.

40. A composition comprising an oligonucleotide according to any one of claims 26 to 38 or a fusion oligonucleotide according to claim 39.

41. An immunogenic composition comprising an oligonucleotide according to any one of claims 26 to 38 or a fusion oligonucleotide according to claim 39, and a therapeutic RNA.

42. A method of inhibiting TLR8 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 26 to 38, a fusion oligonucleotide according to claim 39, or a composition according to claim 40, thereby inhibiting TLR8 activity in the subject.

43. A method of inhibiting TLR8 activity in a cell, the method comprising contacting the cell with an oligonucleotide according to any one of claims 26 to 38, a fusion oligonucleotide according to claim 39, or a composition according to claim 40, thereby inhibiting TLR8 activity in the cell.

44. A method of inhibiting TLR8 activation in a subject caused by a therapeutic RNA, the therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 26 to 38, a fusion oligonucleotide according to claim 39, a composition according to claim 40, or an immunogenic composition according to claim 41, thereby inhibiting TLR8 activation in the subject.

45. A method of treating or preventing a disease, disorder, or condition in a subject responsive to TLR8 inhibition, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 26 to 38, a fusion oligonucleotide according to claim 39, a composition according to claim 40, or an immunogenic composition according to claim 41, thereby treating or preventing the disease, disorder, or condition in the subject.

46. The method according to claim 45, wherein the disease, disorder, or condition is selected from the group consisting of: inflammation-related diseases, allergic diseases, infections, cancers, and autoimmune diseases.

47. An oligonucleotide comprising a sequence consisting of or consisting of: [mX / Modified mX]* y X F * z X G Wherein: * y and * z each independently represents an internucleotide bond; X F and X G each independently is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified LX; wherein F and X G at least one of Where mX is a nucleotide comprising a 2'-OMe and / or 3'-OMe modification, dX is a DNA base, and LX is a base modified with LNA; When mX is mC, X F is dG, X G is not mG or dG; or When mX is mG and when: X F When X is mU, mC or dG G it is not dT, dA, dG; X F When X is dT, dA, dG or mA, G it is not dX; X F When it is dG, X G is not dA, dG, or dT; wherein when [mX / modified mX] is 3'-OMe N7-methylguanosine, * y is not a 5'-5'-triphosphate nucleotide internucleoside bond, and * z is not a 3'-5'-phosphodiester nucleotide internucleoside bond; Where the sequence is optionally functionalized.

48. The oligonucleotide according to claim 47, wherein each internucleotide bond is a 3'-5'-thio-phosphate bond.

49. The oligonucleotide according to claim 48, wherein * z it is in the R configuration.

50. The oligonucleotide according to any one of 47 to 49, wherein mX is a nucleotide comprising a 2'-OMe modification.

51. The oligonucleotide according to any one of claims 47 to 50, wherein the modified mX is selected from the group consisting of: mG1, mI, mU1, mU2, mC1, m7 G, and N1-Me-G, where mG1 is 2'-OMe-2,6-diaminopurine, mI is 2'-OMe-I (2'-O-methylinosine), mU1 is 2'-OMe-5-Me-U (2'-O-methyl-5-methyluridine), mU2 is 2'-OMe-5-Br-U (2'-O-methyl-5-bromouridine), mC1 is 2'-OMe-5-Me-C (2'-O-methyl-5-methylcytidine), m7 G is 3'-OMe-N7-methylated guanosine and N1-Me-G (1-methylguanosine).

52. The oligonucleotide according to any one of claims 47 to 51, wherein the modified dX is selected from the group consisting of: 5-Me-dC, 5-Br-dC, 5-CH2OH-dC, ddC, pdC, and PSU, where 5-Me-dC is 5-methyl-substituted deoxycytidine, 5-Br-dC is 5-bromo-substituted deoxycytidine, 5-CH2OH-dC is 5-hydroxymethyl-substituted deoxycytidine, ddC is 2'-deoxy-3'-deoxycytidine, pdC is 5-propynyl-substituted deoxycytidine, and PSU is pseudouridine.

53. The oligonucleotide according to any one of claims 47 to 52, wherein X F and X G are each independently selected from the group consisting of mX, dX, and LX; wherein X F and X G at least one of which is dX or LX.

54. The oligonucleotide according to any one of claims 47 to 53, wherein [mX / modified mX] is selected from the group consisting of: mG, mC, and mU.

55. The oligonucleotide according to any one of claims 47 to 54, wherein X F is selected from the group consisting of: mX and dX.

56. The oligonucleotide according to any one of claims 47 to 55, wherein X G is selected from the group consisting of: dX and LX.

57. The oligonucleotide according to any one of claims 47 to 56, wherein the sequence is selected from the group consisting of:

58. The oligonucleotide according to claim 57, wherein the sequence is selected from the group consisting of: mG*dC*dC, mG*dC*dT, mG*dC*dA, mG*dC*dG, mC*dC*dC, mU*dC*dC, mC*dG*dC, mG*dC*dT, mG*mG*dA, mU*dC*dG, mU*mG*LG, mU*dC*dA, and mU*dC*dT.

59. The oligonucleotide according to any one of claims 47 to 58, wherein the oligonucleotide consists of the sequence.

60. The oligonucleotide according to any one of claims 47 to 58, wherein the oligonucleotide containing the sequence has a length of no more than 20 bases.

61. The oligonucleotide according to claim 60, wherein the oligonucleotide contains the sequence 5'-mG*mU*dC*dC*dC*dC-3'.

62. A fusion oligonucleotide, comprising: A-[Y-A] n wherein each A independently represents an oligonucleotide according to any one of claims 47 to 61, and each A can be the same or different; Y represents a cleavable linker, and each Y can be the same or different; and n is equal to or greater than 1.

63. A composition, comprising an oligonucleotide according to any one of claims 47 to 61 or a fusion oligonucleotide according to claim 62.

64. An immunogenic composition, comprising an oligonucleotide according to any one of claims 47 to 61 or a fusion oligonucleotide according to claim 62, and a therapeutic RNA.

65. The immunogenic composition according to claim 64, comprising a modified oligonucleotide, wherein the modified oligonucleotide comprises the oligonucleotide or the fusion oligonucleotide linked to the therapeutic RNA through a cleavable linker.

66. A method for enhancing TLR8 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 47 to 61, a fusion oligonucleotide according to claim 62, or a composition according to claim 63, thereby enhancing TLR8 activity in the subject.

67. A method for enhancing TLR8 activation in a subject caused by a therapeutic RNA, the therapeutic RNA being selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamer, single guide RNA, self-amplifying RNA, circular RNA, and combinations thereof, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 47 to 61, a fusion oligonucleotide according to claim 62, or a composition according to claim 63, or an immunogenic composition according to claim 64 or 65, thereby enhancing TLR8 activation in the subject.

68. A method of treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signaling, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 47 to 61, or a fusion oligonucleotide according to claim 62, or a composition according to claim 63, thereby treating or preventing the disease, disorder or condition in the subject.

69. The method according to claim 68, wherein the disease, disorder or condition is selected from the group consisting of: cancer, viral and bacterial infections.

70. A fusion oligonucleotide comprising: A-[Y-A] n wherein each A independently represents an oligonucleotide according to any one of claims 1 to 15 or 26 to 38, each A may be the same or different, provided that at least one A represents an oligonucleotide according to any one of claims 1 to 15, and at least one additional A represents an oligonucleotide according to any one of claims 26 to 38; Y represents a cleavable linker, each Y may be the same or different; and n is equal to or greater than 1.

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