WO2024077351A1 - Oligonucléotides modifiés - Google Patents

Oligonucléotides modifiés Download PDF

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Publication number
WO2024077351A1
WO2024077351A1 PCT/AU2023/051007 AU2023051007W WO2024077351A1 WO 2024077351 A1 WO2024077351 A1 WO 2024077351A1 AU 2023051007 W AU2023051007 W AU 2023051007W WO 2024077351 A1 WO2024077351 A1 WO 2024077351A1
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Prior art keywords
modified
oligonucleotide
group
oligonucleotide according
ome
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PCT/AU2023/051007
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English (en)
Inventor
Michael Gantier
Olivier Laczka
Daniel WENHOLZ
Mary SPEIR
Sunil SAPKOTA
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Pharmorage Pty Limited
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Priority claimed from AU2022902992A external-priority patent/AU2022902992A0/en
Application filed by Pharmorage Pty Limited filed Critical Pharmorage Pty Limited
Publication of WO2024077351A1 publication Critical patent/WO2024077351A1/fr

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    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
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    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N2310/32—Chemical structure of the sugar
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
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    • C12N2310/32—Chemical structure of the sugar
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present invention relates to oligonucleotides that inhibit Toll-Like Receptor 7 (TLR7) and/or Toll-Like Receptor 8 (TLR8), or potentiate TLR8, and uses thereof.
  • TLR7 Toll-Like Receptor 7
  • TLR8 Toll-Like Receptor 8
  • TLR8 Toll-Like Receptor 8
  • RNA-targeting therapeutics based on synthetic oligonucleotides have been gaining a lot of interest, with several regulatory approvals in the US and European Union, and multi-billion license deals in recent years. To ensure their essential functions related to gene targeting activities, oligonucleotides-based therapeutics require both increased affinity for their targets and stabilisation against nuclease activities.
  • TLR Toll- Like Receptors
  • oligonucleotides are designed to evade activation of innate immune sensors and avoid strong off-target pro-inflammatory immune responses in patients. From this angle, chemical modifications may have the dual benefit of increasing the targeting efficacy of the oligonucleotides, while decreasing their immunostimulatory effects. [0005] Nonetheless, it has also been clear for some time that select PS-modified DNA oligonucleotides (ODN) have broad immunosuppressive effects. This is best exemplified with the “TTAGGG” containing PS-ODN A151, involved in the inhibition of TLR9, TLR7, Absent In Melanoma 2 (AIM2) and cyclic-GMP-AMP synthase (cGAS).
  • ODN PS-modified DNA oligonucleotides
  • TLR7 Toll-Like Receptor 7
  • TLR8 Toll-Like Receptor 8
  • an oligonucleotide comprising or consisting of a sequence consisting of: [mX/modified mX]* y X A * z X B wherein: * y and * z each independently represent an inter-nucleotide linkage, wherein at least one of * y and * z is not phosphorodiamidate;
  • X A and X B 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; 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
  • both * y and * z are not phosphorodiamidate.
  • 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.
  • an oligonucleotide comprising or consisting of a sequence consisting of: [mX/modified mX]* y X A * z X B wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • 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, modified moX, modified LX, and modified fX; 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 LNA modified base, fX is a nucleotide comprising
  • any oligonucleotide of the first aspect inhibits TLR7 activity, preferably human TLR7 activity.
  • an oligonucleotide of the first aspect does not potentiate TLR8 activity, preferably human TLR8 activity.
  • an oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity.
  • an oligonucleotide of the first aspect potentiates TLR8 activity, preferably human TLR8 activity.
  • Each internucleotide linkage may be selected from the group consisting of: 3′- 5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, and 5′-2′- linkage.
  • each internucleotide linkage may be selected from: 3′-5′- and 5′-5′- linkage.
  • each internucletodie linkage is a 3′-5′- linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-[mX/modified mX]* y X A * z X B -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage, wherein at least one of * y and * z is not phosphorodiamidate;
  • X A and X B 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; 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 nucleot
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-[mX/modified mX]* y X A * z X B -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • 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, modified moX, modified LX, and modified fX; 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 LNA modified base, fX is a nucle
  • each internucleotide linkage is a 3′-5′ linkage.
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester.
  • Each internucleotide linkage may be the same or different.
  • each internucleotide linkage is independently selected from phosphorothioate and phosphodiester.
  • each internucleotide linkage is phosphorothioate. 1004921453 [0019]
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers.
  • the oligonucleotide of the first aspect comprises a single phosphorothioate stereoisomer, preferably wherein * y is in the S configuration.
  • mX is a nucleotide comprising a 2′-OMe modification.
  • moX is a nucleotide comprising a 2′-MOE modification.
  • fX is a nucleotide comprising a 2′-fluor modification.
  • Modified dX, modified rX and modified morpholino comprise at least one modification or substitution at positions of the base and/or sugar.
  • Modified mX, modified moX, modified LX and modified fX comprise at least one additional modification or substitution at additional positions of the base and/or sugar.
  • the modification or substitution is selected from the group consisting of: pseudouridine, 3′-deoxy, hydroxyl, des-amino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl and combinations thereof.
  • Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mU3, mC1, and 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.
  • mG1 is 2′-OMe-2,6-Diaminopurine
  • mI is 2′-OMe-I (2′-O-
  • modified mX is selected from the group consisting of: mG1, mI, mU1, mU2 and mC1. Most preferably, modified mX is mC1.
  • 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, wherein 5-Me-dC is 5-methyl substituted deoxycytidine, 5-Br-dC is 5-bromo substituted deoxycytidine5-CH2OH-dC is 5-hydroxymethyl substituted deoxycytidine, ddC is 2′-deoxy-3′-deoxy cytidine, p
  • Exemplary modified rX includes but is not limited to PSU, 2′-NH2-rX, and ara- rX, wherein 2′-NH2-rX is a 2′-amino modified RNA base, and ara-rX is an arabinose modified RNA base.
  • Exemplary 2′-NH2-rX includes but is not limited to 2′-NH2-U and 2′- NH2-C, wherein 2′-NH2-U is 2′-NH2-uridine, and 2′-NH2-C is 2′-NH2-cytidine.
  • Exemplary ara-rX is ara-C (aracytidine).
  • [mX/modified mX] is selected from the group consisting of: mG, mI, mG1 and mU.
  • [mX/modified mX] is mG or mI.
  • [mX/modified mX] is modified mX.
  • modified mX is mI or mG1, preferably mI.
  • modified mX is not 2′-OMe-N1-Me-G (2′-O-methyl-N1- methylguanosine).
  • [mX/modified mX] is mX.
  • mX is mG or mU, preferably mG.
  • [mX/modified mX] is [mG/modified mG].
  • modified mG is not 2′-OMe-N1-Me-G (2′-O-methyl-N1-methylguanosine).
  • Modified mG includes but is not limited to: mG1 and mI, wherein mG1 is 2′-OMe-2,6- Diaminopurine, and mI is 2′-OMe-I (2′-O-methylinosine).
  • [mG/modified mG] is [mG/mI].
  • [mG/modified mG] is mG.
  • [mG/modified mG] is mI.
  • 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.
  • 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.
  • X A is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified rX.
  • X A is selected from the group consisting of: mX, dX, rX, modified mX, modified dX, and modified rX.
  • X A is selected from the group consisting of: mU, mU1, mU2, mU3, PSU, mG, mA, mC, 1004921453 dT, dG, dA, dC, rU, 2′-NH2-rU, 8-Br-dA, and 8-oxo-dA.
  • X A is selected from the group consisting of: mX, dX, rX, and modified mX.
  • 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.
  • X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX and morpholino-X.
  • 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.
  • X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX and modified dX.
  • 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.
  • X B is selected from the group consisting of: mX, dX, LX, modified mX, modified dX and modified rX.
  • X B is selected from the group consisting of: LC, dC, 5-Me-dC, 5-Br-dC, mC, mC1, ara-C.
  • X B is selected from the group consisting of: LX, modified mX, modified dX and modified rX.
  • X B is selected from the group consisting of: LC, 5-Me-dC, 5-Br-dC, and mC1.
  • X B is LX, preferably LC.
  • at least one of X A and X B is LX.
  • X A and X B are independently LX.
  • one of X A and X B is LX.
  • X B is LX.
  • X B is LX and X A is mX.
  • at least one of X A and X B is dX.
  • X A and X B are independently dX.
  • one of X A and X B is dX.
  • X B is dX.
  • X B is dX and X A is mX. More preferably, X B is dX and X A is mU.
  • at least one of X A and X B is rX.
  • X A and X B are independently rX. In another embodiment, one of X A and X B 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, where at least one of X A and X B is rX, each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-[mG/mI]*mU*X B -3′ wherein: * each independently represent a 3′-5′- phosphorothioate linkage;
  • 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; wherein 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 LNA modified base, fX is a nucleotide comprising a 2′-fluor and/or 3′-fluor modification, morpholino-X is a
  • X B is selected from the group consisting of: mX, dX, LX, modified mX, modified rX, and modified dX.
  • the sequence may be functionalised.
  • the functionalised sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocycyl, arylalkyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the hydrophobic lipid is selected from cholesterol and tocopherol.
  • the compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol.
  • the compound is conjugated directly to the sequence.
  • the compound is conjugated to the sequence via a linker.
  • the linker may be cleavable or non-cleavable.
  • the linker is a non-cleavable linker.
  • the compound is conjugated to a terminal nucleotide of the sequence, preferably the terminal 3′- nucleotide.
  • Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG, dX-Chol and dX-Toco, wherein dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group, dX-Chol is a DNA base with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol- glyceryl group covalently linked to the 3′-position via a monophosphate group, dX-Toco is a DNA base with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group
  • dX-Chol is a DNA base with an (N-choleste
  • functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dC-TEG, dC-Chol, dC-Toco, wherein dC-TEG is deoxycytidine with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl group covalently linked to the 3′-position via a monophosphate group, dC-Chol, is deoxycytidine with triethylene glycol covalently linked to the 3′-position via a monophosphate group, and dC-Toco is deoxycytidine with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dC-TEG is deoxycytidine with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl group covalently linked
  • the sequence is selected from the group consisting of: 1004921453 1004921453 1004921453 [0043]
  • [mX/modified mX] is [mG/mI]
  • X A is mU
  • X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, morpholino-X, modified mX, modified rX and modified dX, wherein the sequence is optionally functionalised.
  • X B is selected from the group consisting of: LX, mX, dX, modified mX, modified rX, and modified dX.
  • X B is selected from the group consisting of: LC, mC, ara-C, dC, mC1, and modified dC.
  • modified dC is selected from the group consisting of: 5-Me-dC, 5-Br-dC and 5-I-dC.
  • 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.
  • [mX/modified mX] is [mG/mI];
  • X A is mU;
  • X B is selected from the group consisting of: mX, dX, rX, LX, modified mX modified rX, and modified dX.
  • X B is selected from the group consisting of: LC, mC1, dC, mC, and modified dC.
  • modified dC is selected from the group consisting of: 5-Me-dC, 5-Br-dC and 5-I-dC.
  • 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.
  • [modified mX] is [mG/mI]; and X A and X B are rX.
  • 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.
  • the oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity.
  • the oligonucleotide that further inhibits TLR8 activity comprises or consists of the sequence mI*mU*mC or mI*mA*dG. [0047] In one embodiment, the oligonucleotide consists of the sequence. [0048] In another embodiment, the oligonucleotide comprises the sequence. Preferably, the oligonucleotide comprising the sequence is no more than 20 bases in 1004921453 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 at the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the oligonucleotide comprises the sequence 5′-mG*mU*X B -3′, wherein X B is dX, preferably X B is dC. Even more preferably, the oligonucleotide comprises the sequence 5′- mG*mU*dC*dC*dC-3′.
  • a method of modifying the TLR7 activity of an oligonucleotide comprising modifying the oligonucleotide by adding the sequence to the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the method reduces the TLR7 potentiating activity of the oligonucleotide. In another embodiment, the method increases the TLR7 inhibitory activity of the oligonucleotide. [0050] In another embodiment of the first aspect, there is provided a fusion oligonucleotide comprising two or more oligonucleotides according to the first aspect linked by a cleavable linker.
  • the fusion oligonucleotide comprises: A-[Y-A]n wherein each A independently represents an oligonucleotide according to the first aspect, each A may be the same or different; Y represents a cleavable linker, each Y may be the same or different; and n is equal to or greater than 1.
  • Y is cleavable by an enzyme.
  • Y is selected from the group consisting of: a TEG linker, carbon spacers (such as C3, C6, C9, C12), glycerol and PolydT. More preferably, Y is a TEG linker.
  • each A is independently bound to Y by an internucleotide linkage (*).
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester.
  • Each internucleotide linkage may be the same or different.
  • each 1004921453 internucleotide linkage is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide linkage is phosphorothioate.
  • Each internucleotide linkage may be selected from the group consisting of: 3′- 5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, and 5′-2′- linkage.
  • each internucleotide linkage may be selected from: 3′-5′- and 5′-5′- linkage.
  • each internucletodie linkage is a 3′-5′- linkage. More preferably, each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the fusion oligonucleotide comprises or consists of the sequence 5′-mG*mU*dC-3′-*TEG*-3′-dC*mU*mG-5′.
  • a modified oligonucleotide comprising an agent linked to an oligonucleotide or fusion oligonucleotide according to the first aspect by a linker.
  • the agent may be a therapeutic and/or diagnostic agent.
  • the agent is a therapeutic agent, more preferably, a therapeutic RNA selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamers, antisense oligonucleotides, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the linker may be cleavable or non-cleavable.
  • the linker is a cleavable linker.
  • composition comprising an oligonucleotide or fusion oligonucleotide according to the first aspect, and a pharmaceutically acceptable excipient.
  • the composition is an immunogenic composition comprising a therapeutic RNA and an oligonucleotide or fusion oligonucleotide according to the first aspect.
  • the therapeutic RNA is selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamers, antisense oligonucleotides, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the immunogenic composition comprises a modified oligonucleotide according to the first aspect.
  • the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA 1004921453 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the composition may comprise at least one additional active agent, including but not limited to, an anti-inflammatory agent.
  • a method of inhibiting TLR7 activity in a subject comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, thereby inhibiting TLR7 activity in the subject.
  • a method of inhibiting TLR7 activity in a cell comprising contacting the cell with an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, thereby inhibiting TLR7 activity in the cell.
  • an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect in the manufacture of a medicament for inhibiting TLR7 activity in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect for inhibiting TLR7 activity in a subject.
  • RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, thereby inhibiting TLR7 activation in the subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof
  • the method comprises administering an immunogenic composition comprising an oligonucleotide or fusion oligonucleotide according to the first aspect, and a therapeutic RNA selected from the group consisting 1004921453 of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • 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 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the oligonucleotide, the fusion oligonucleotide, or the composition does not substantially reduce translation of the therapeutic RNA.
  • the therapeutic RNA comprises pseudouridine.
  • the therapeutic RNA does not comprise pseudouridine.
  • an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect in the manufacture of a medicament for inhibiting TLR7 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, , self-amplifying RNAs, circular RNAs and combinations thereof, in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, , self-amplifying RNAs, circular RNAs and combinations thereof, in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • a first composition and a second composition in the manufacture of a medicament for inhibiting TLR7 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject, wherein the first composition comprises an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, and the second composition comprises the therapeutic RNA.
  • an immunogenic composition comprising 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 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, in the manufacture of a medicament for inhibiting TLR7 activation by the therapeutic RNA in a subject.
  • the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect for inhibiting TLR7 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • a therapeutically effective amount of an immunogenic composition comprising 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 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, for inhibiting TLR7 activation by the therapeutic RNA in a subject.
  • the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, for inhibiting TLR7 activation by the therapeutic RNA in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect for use in inhibiting TLR7 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • an immunogenic composition comprising a modified oligonucleotide according to the first aspect, wherein the therapeutic agent is a therapeutic selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, for use in inhibiting TLR7 activation by the therapeutic RNA in a subject.
  • the therapeutic agent is a therapeutic selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, for use in inhibiting TLR7 activation by the therapeutic RNA in a subject.
  • a method of treating or preventing a disease, disorder or condition in a subject responsive to TLR7 inhibition comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, thereby treating or preventing the disease, disorder or condition in the subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect thereby treating or preventing the disease, disorder or condition in the subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect for treating or preventing a disease, disorder or condition in a subject responsive to TLR7 inhibition.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect for use in the prevention or treatment of a disease, disorder or condition in a subject responsive to TLR7 inhibition.
  • an oligonucleotide comprising or consisting of a sequence consisting of: X C * y X D * z X E wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X C is selected from the group consisting of: mX, modified mX, dG, and morpholino-X;
  • 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; wherein mX is a nucleotide comprising
  • 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;
  • 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.
  • an oligonucleotide comprising or consisting of a sequence consisting of: X C * y X D * z X E wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X C is selected from the group consisting of: mX, modified mX, and dG; 1004921453 X D and X E are 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; 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,
  • any oligonucleotide of the second aspect inhibits TLR8 activity, preferably human TLR8 activity.
  • an oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activtiy.
  • an oligonucleotide of the second aspect does not substantially inhibit TLR7 activity, preferably human TLR7 activtiy.
  • Each internucleotide linkage may be selected from the group consisting of: 3′- 5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, and 5′-2′- linkage.
  • each internucleotide linkage may be selected from: 3′-5′- and 5′-5′- linkage.
  • each internucletodie linkage is a 3′-5′- linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-X C * y X D * z X E -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X C is selected from the group consisting of: mX, modified mX, dG, and morpholino-X;
  • 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; 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
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-X C * y X D * z X E -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X C is selected from the group consisting of: mX, modified mX, and dG;
  • X D and X E are 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; 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′-
  • each internucleotide linkage is a 3′-5′ linkage.
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, phosphodiester, phosphoramidate and phosphorodiamidate.
  • Each internucleotide linkage may be the same or different.
  • each internucleotide linkage is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide linkage is phosphorothioate.
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers. In another embodiment, the oligonucleotide comprises a single stereoisomer.
  • mX is a nucleotide comprising a 2′-OMe modification.
  • moX is a nucleotide comprising a 2′-MOE modification.
  • fX is a nucleotide comprising a 2′-fluor modification.
  • Modified dX, modified rX and modified morpholino-X comprise at least one modification or substitution at positions of the base and/or sugar.
  • Modified mX, modified moX, modified LX and modified fX comprise at least one additional modification or substitution at additional positions of the base and/or sugar.
  • the modification or substitution is selected from the group consisting of: pseudouridine, 3′-deoxy, hydroxyl, des-amino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl and combinations thereof.
  • Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mU3, mC1, and 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 N7-methylated guanosine.
  • mG1 is 2′-OMe-2,6-Diaminopurine
  • mI is 2′-OMe-I (2′-O- methylinosine
  • modified mX is selected from the group consisting of: mG1, mI, mU1, mU2 and mC1.
  • Exemplary modified dX includes but is 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, wherein 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′-deoxy cytidine, pdC is 5-propynyl substituted deoxycytidine, PSU
  • Exemplary modified rX includes but is not limited to PSU, 2′-NH2-rX, and ara- rX, wherein 2′-NH2-rX is a 2′-amino modified RNA base, and ara-rX is an arabinose modified RNA base.
  • Exemplary 2′-NH2-rX includes but is not limited to 2′-NH2-U and 2′- NH2-C, wherein 2′-NH2-U is 2′-NH2-uridine, and 2′-NH2-C is 2′-NH2-cytidine.
  • Exemplary ara-rX is ara-C (aracytidine).
  • X C is selected from the group consisting of: mG, mU, mC, mI, mG1, and dG.
  • X C is selected from the group consisting of: mX and modified mX.
  • mX is selected from the group consisting of: mG, mC and mU, more preferably mG; and modified mX is mI.
  • X C is selected from the group consisting of: mG and mI.
  • X C is mI.
  • X D is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX and modified rX.
  • X D is selected from the 1004921453 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.
  • X D is selected from the group consisting of: mX, dX, LX, modified mX, and modified dX.
  • X D is selected from the group consisting of: mX, dX, modified mX, and modified dX.
  • X D is selected from the group consisting of: mA, mU, mC, dA, dT, dG, mU1, mU2, and PSU. More preferably, X D is selected from the group consisting of: mA, mU, dA, dT, and dG.
  • 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.
  • X E is 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.
  • X E is selected from the group consisting of: mX, dX, moX, LX, fX, rX, modified mX and modified dX.
  • X E 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.
  • X E 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.
  • X E is selected from the group consisting of: mX, dX, rX and LX.
  • X E is selected from the group consisting of: dA, dC, dG, dT, rG, mC, LA, LC, LG, and LT.
  • X E is selected from the group consisting of: mX and dX.
  • X E is selected from the group consisting of: dC, dG, dT and mC.
  • at least one of X D and X E is LX.
  • X D and X E are independently LX.
  • one of X D and X E is LX.
  • X E is LX.
  • X E is LX and X D is mX.
  • at least one of X D and X E is dX.
  • X D and X E are independently dX.
  • one of X D and X E is dX.
  • X E is dX.
  • X E is dX and X D is mX.
  • at least one of X D and X E is rX.
  • one of X D and X E is rX.
  • X D and X E are each independently rX.
  • X D is selected from rA and rG
  • X E is selected from rA, rG and rC.
  • 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 1004921453 and X E is rC.
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the sequence may be functionalised.
  • the functionalised sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocycyl, arylalkyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the hydrophobic lipid is selected from cholesterol and tocopherol.
  • the compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol. [0102] In one embodiment, the compound is conjugated directly to the sequence.
  • the compound is conjugated to the sequence via a linker.
  • the linker may be cleavable or non-cleavable.
  • the linker is a non-cleavable linker.
  • the compound is conjugated to a terminal nucleotide of the sequence, preferably the terminal 3′- nucleotide.
  • the compound is conjugated to the terminal 3′-nucleotide at the 3′- position.
  • Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG, dX-Chol and dX-Toco, wherein dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group, dX-Chol is a DNA base with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol- glyceryl group covalently linked to the 3′-position via a monophosphate group, dX-Toco is a DNA base with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group
  • dX-Chol is a DNA base with an (N-choleste
  • the sequence is selected from the group consisting of: 1004921453 1004921453 [0106]
  • 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, 1004921453 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*dA, mU*dG*d
  • 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. Even more preferably, the sequence is selected from the group consisting of: mI*mA*dG and mI*mU*mC.
  • the oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activity.
  • the oligonucleotide that further inhibits TLR7 activity comprises or consists of the sequence of mI*mU*mC or mI*mA*dG. [0109] In one embodiment, the oligonucleotide consists of the sequence. [0110] In another embodiment, the oligonucleotide comprises the 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 at the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • a method of modifying the TLR8 activity of an oligonucleotide comprising modifying the oligonucleotide by adding the sequence to the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the method reduces the TLR8 potentiating activity of the oligonucleotide.
  • the method increases the TLR8 inhibitory activity of the oligonucleotide.
  • a fusion oligonucleotide comprising two or more oligonucleotides according to the second aspect linked by a cleavable linker.
  • the fusion oligonucleotide comprises: A-[Y-A]n wherein 1004921453 each A independently represents an oligonucleotide according to the second aspect, each A may be the same or different; Y represents a cleavable linker, each Y may be the same or different; and n is equal to or greater than 1.
  • Y is cleavable by an enzyme.
  • Y is selected from the group consisting of: a TEG linker, carbon spacers (such as C3, C6, C9, C12), glycerol and PolydT. More preferably Y is a TEG linker.
  • each A is independently bound to Y by an internucleotide linkage (*).
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide linkage may be the same or different. In a preferred embodiment, each internucleotide linkage is independently selected from phosphorothioate and phosphodiester.
  • each internucleotide linkage is phosphorothioate.
  • the agent may be a therapeutic and/or diagnostic agent.
  • the agent is a therapeutic agent, more preferably, a therapeutic RNA selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamers, antisense oligonucleotides, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the linker may be cleavable or non-cleavable.
  • the linker is a cleavable linker.
  • the composition is an immunogenic composition comprising a therapeutic RNA and an oligonucleotide or fusion oligonucleotide according to the second aspect.
  • the therapeutic RNA is selected from the 1004921453 group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamers, antisense oligonucleotides, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the immunogenic composition comprises a modified oligonucleotide according to the second aspect.
  • the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the composition may comprise at least one additional active agent including, but not limited to, an anti-inflammatory agent.
  • a method of inhibiting TLR8 activity in a cell comprising contacting the cell with an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect, thereby inhibiting TLR8 activity in the cell.
  • a method of inhibiting TLR8 activity in a subject comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect, thereby inhibiting TLR8 activity in the subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect thereby inhibiting TLR8 activity in the subject.
  • use of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect in the manufacture of a medicament for inhibiting TLR8 activity in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect for inhibiting TLR8 activity in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect for use in inhibiting TLR8 activity in a subject.
  • a method of inhibiting TLR8 activation by a therapeutic RNA selected from the group consisting of: 1004921453 RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNA, Circular RNA and combinations thereof in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect, thereby inhibiting TLR8 activation in the subject.
  • the method comprises administering an immunogenic composition comprising an oligonucleotide or fusion oligonucleotide according to the second aspect, and a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • 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 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof .
  • the oligonucleotide, the fusion oligonucleotide, or the composition does not substantially reduce translation of the therapeutic RNA.
  • the therapeutic RNA comprises pseudouridine.
  • the therapeutic RNA does not comprise pseudouridine.
  • an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect in the manufacture of a medicament for inhibiting TLR8 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • a first composition and a second composition in the manufacture of a medicament for inhibiting TLR8 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject, wherein the first composition comprises an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect, and the second composition comprises the therapeutic RNA.
  • an immunogenic composition comprising a 1004921453 modified oligonucleotide according to the second aspect, wherein the therapeutic agent is a therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, in the manufacture of a medicament for inhibiting TLR8 activation by the therapeutic RNA in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect for inhibiting TLR8 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • a therapeutically effective amount of an immunogenic composition comprising 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 aptamers, single guide RNA, self- amplifying RNAs, circular RNAs and combinations thereof, for inhibiting TLR8 activation by the therapeutic RNA in a subject.
  • the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self- amplifying RNAs, circular RNAs and combinations thereof, for inhibiting TLR8 activation by the therapeutic RNA in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self- amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • an immunogenic composition comprising 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 aptamers, single guide RNA, self- amplifying RNAs, circular RNAs and combinations thereof, for use in inhibiting TLR8 activation by the therapeutic RNA in a subject.
  • the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self- amplifying RNAs, circular RNAs and combinations thereof, for use in inhibiting TLR8 activation by the therapeutic RNA in a subject.
  • a method of treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to 1004921453 the second aspect, thereby treating or preventing the disease, disorder or condition in the subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to 1004921453 the second aspect thereby treating or preventing the disease, disorder or condition in the subject.
  • use of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect in the manufacture of a medicament for treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect for treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect for use in the prevention or treatment of a disease, disorder or condition in a subject responsive to TLR8 inhibition.
  • the disease, disorder or condition responsive to TLR8 inhibition is selected from the group consisting of: inflammation-related diseases, allergic diseases, infections, cancers and auto-immune diseases.
  • 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.
  • the fusion oligonucleotide comprises: A-[Y-A]n wherein each A independently represents an oligonucleotide according to the first or second aspect, each A may be the same or different, with the proviso that at least one A is an oligonucleotide according to the first aspect and at least one further A is an oligonucleotide according to the second aspect; Y represents a cleavable linker, each Y may be the same or different; and n is equal to or greater than 1. 1004921453 [0137]
  • Y is cleavable by an enzyme.
  • Y is selected from the group consisting of: a TEG linker, carbon spacers (such as C3, C6, C9, C12), glycerol and PolydT. More preferably Y is a TEG linker.
  • each A is independently bound to Y by an internucleotide linkage (*).
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide linkage may be the same or different. In a preferred embodiment, each internucleotide linkage is independently selected from phosphorothioate and phosphodiester.
  • each internucleotide linkage is phosphorothioate.
  • a modified oligonucleotide comprising 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 by one or more linkers.
  • a modified oligonucleotide comprising a synthetic oligonucleotide linked to a fusion oligonucleotide by 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 by a linker.
  • the synthetic oligonucleotide is a therapeutic and/or diagnostic oligonucleotide, more preferably a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the linker may be cleavable or non-cleavable.
  • the linker is a cleavable linker.
  • the fusion oligonucleotide and modified oligonucleotide comprising at least one first oligonucleotide according to the first aspect linked to at least one second oligonucleotide according to the second aspect may be used in the methods and uses of the first and second aspects described herein.
  • an oligonucleotide comprising or consisting of a sequence consisting of: [mX/modified mX]* y X F * z X G wherein: 1004921453 * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, LX, rX, modified dX or modified LX; wherein mX is a nucleotide comprising a 2′-OMe and/or 3′-OMe modification, dX is a DNA base, LX is a LNA modified base; when mX is mC, X F is dG, X G is not mG or dG; or when mX is
  • an oligonucleotide comprising or consisting of a sequence consisting of: mX* y X F * z X G wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, LX, modified dX or modified LX; wherein mX is a nucleotide comprising a 2′-OMe and/or 3′-OMe modification, dX is a DNA base, LX is a LNA modified base; 1004921453 when mX is mC, X F is dG, X G is not mG or dG; or when mX is mG and when: X F is mU,
  • Any oligonucleotide of the third aspect potentiates TLR8 activity, preferably human TLR8 activity.
  • an oligonucleotide of the third aspect does not substantially inhibit TLR7 activity, preferably human TLR7 activity.
  • an oligonucleotide of the third aspect inhibits TLR7 activity, preferably human TLR7 activity.
  • Each internucleotide linkage may be selected from the group consisting of: 3′- 5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, and 5′-2′- linkage.
  • each internucleotide linkage may be selected from: 3′-5′- and 5′-5′- linkage.
  • each internucletodie linkage is a 3′-5′- linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-[mX/modified mX]* y X F * z X G -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, rX, LX, modified dX or modified LX; 1004921453 wherein mX is a nucleotide comprising a 2′-OMe
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-mX* y X F * z X G -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, LX, modified dX or modified LX; wherein mX is a nucleotide comprising a 2′-OMe and/or 3′-OMe modification, dX is a DNA base, LX is a LNA modified base; when mX is mC, X F is dG, X G is not mG or dG; or when mX is mG and when: X F is
  • each internucleotide linkage is a 3′-5′ linkage.
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester.
  • Each internucleotide linkage may be the same or different.
  • each internucleotide linkage is independently selected from phosphorothioate and phosphodiester.
  • each internucleotide linkage is phosphorothioate.
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers.
  • the oligonucleotide of the third aspect comprises a single phosphorothioate stereoisomer, preferably wherein * z is in the R configuration.
  • mX is a nucleotide comprising a 2′-OMe modification.
  • Modified dX and modified rX comprise at least one modification or substitution at positions of the base and/or sugar.
  • Modified mX, modified moX, modified LX and modified fX comprise at least one additional modification or substitution at additional positions of the base and/or sugar.
  • the modification or substitution is selected from the group consisting of: pseudouridine, 3′-deoxy, hydroxyl, des-amino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl and combinations thereof.
  • Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mC1, m7 G, and N1-Me-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), 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).
  • modified mX is selected from the group consisting of: mG1, mI, mU1, mU2 and mC1.
  • Exemplary modified dX includes but is not limited to: 5-Me-dC, 5-Br-dC, 5- CH2OH-dC, ddC, pdC, and PSU, wherein 5-Me-dC is 5-methyl substituted deoxycytidine, 5-Br-dC is 5-bromo substituted deoxycytidine, 5-CH 2 OH-dC is 5- hydroxymethyl substituted deoxycytidine, ddC is 2′-deoxy-3′-deoxy cytidine, pdC is 5- propynyl substituted deoxycytidine, and PSU is pseudo uridine.
  • X F and X G are each independently selected from the group consisting of: mX, dX, and LX; wherein at least one of X F and X G is dX or LX.
  • mX is selected from the group consisting of: mG, mC, and mU. In one preferred embodiment, mX is mG. In another preferred embodiment, mX is mC. In yet another preferred embodiment, mX is mU.
  • X F is selected from the group consisting of: mX and dX.
  • X F is selected from the group consisting of: dC, dG, dA, dT, mG, mC, and mU.
  • X F is selected from the group consisting of: dC, dG and mG.
  • X F is dX, preferably dC.
  • X G is selected from the group consisting of: dX and LX.
  • 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.
  • At least one of X F and X G is dX. In one embodiment, one of X F and X G is dX. In a preferred embodiment, X F and X G are independently dX. [0160] 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. [0161] In one embodiment, the sequence may be functionalised.
  • the functionalised sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocycyl, arylalkyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the hydrophobic lipid is selected from cholesterol and tocopherol.
  • the 1004921453 compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol.
  • the compound is conjugated directly to the sequence.
  • the compound is conjugated to the sequence via a linker.
  • the linker may be cleavable or non-cleavable.
  • the linker is a non-cleavable linker.
  • the compound is conjugated to a terminal nucleotide of the sequence, preferably the terminal 3′- nucleotide.
  • the compound is conjugated to the terminal 3′-nucleotide at the 3′- position.
  • Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG, dX-Chol and dX-Toco, wherein dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group, dX-Chol is a DNA base with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol- glyceryl group covalently linked to the 3′-position via a monophosphate group, dX-Toco is a DNA base with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group
  • dX-Chol is a DNA base with an (N-choleste
  • the sequence is selected from the group consisting of: 1004921453 [0166]
  • 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.
  • 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.
  • the oligonucleotide is mG*dC*dC.
  • the oligonucleotide consists of the sequence.
  • the oligonucleotide comprises the sequence.
  • 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.
  • the sequence is at the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • 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′.
  • a method of modifying the TLR8 activity of an oligonucleotide comprising modifying the oligonucleotide by adding the sequence to the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the method increases the TLR8 potentiating activity of the oligonucleotide.
  • the method reduces the TLR8 inhibitory activity of the oligonucleotide.
  • a fusion oligonucleotide comprising two or more oligonucleotides according to the third aspect linked by a cleavable linker.
  • the fusion oligonucleotide comprises: A-[Y-A]n wherein each A independently represents an oligonucleotide according to the third aspect, each A may be the same or different; Y represents a cleavable linker, each Y may be the same or different; and n is equal to or greater than 1.
  • Y is cleavable by an enzyme.
  • Y is selected from the group consisting of: a TEG linker, carbon spacers (such as C3, C6, C9, C12), glycerol and PolydT. More preferably Y is a TEG linker.
  • each A is independently bound to Y by an internucleotide linkage (*).
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide linkage may be the same or different. In a preferred embodiment, each internucleotide linkage is independently selected from phosphorothioate and phosphodiester.
  • each internucleotide linkage is phosphorothioate.
  • the agent may be a therapeutic and/or diagnostic agent.
  • the agent is a therapeutic agent, more preferably, a therapeutic RNA selected from the group consisting of: DNA, RNA, mRNA, 1004921453 siRNA, RNA aptamers, antisense oligonucleotides, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the linker may be cleavable or non-cleavable.
  • the linker is a cleavable linker.
  • the composition is an immunogenic composition comprising a therapeutic RNA and an oligonucleotide or fusion oligonucleotide according to the third aspect.
  • the therapeutic RNA is selected from the group consisting of: DNA, RNA, mRNA, siRNA, RNA aptamers, antisense oligonucleotides, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the immunogenic composition comprises a modified oligonucleotide according to the third aspect.
  • the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the composition may comprise at least one additional active agent selected from: a gene targeting agent and a TLR8 agonist.
  • a method of potentiating TLR8 activity in a cell comprising contacting the cell with an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect, thereby potentiating TLR8 activity in the subject.
  • a method of potentiating TLR8 activity in a subject comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect, thereby potentiating TLR8 activity in the subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect thereby potentiating TLR8 activity in the subject.
  • use of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect in the manufacture of a medicament for potentiating TLR8 activity in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect for potentiating TLR8 activity in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect for use in potentiating TLR8 activity in a subject.
  • RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject
  • 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 potentiating TLR8 activation in the subject.
  • the method comprises 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 aptamers, single guide RNA, self- amplifying RNAs, circular RNAs and combinations thereof.
  • 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 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect in the manufacture of a medicament for potentiating TLR8 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject
  • the first composition comprises an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect
  • the second composition comprises the therapeutic RNA.
  • an immunogenic composition comprising 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 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, in the manufacture of a medicament for potentiating TLR8 activation by the therapeutic RNA in a subject.
  • the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, in the manufacture of a medicament for potentiating TLR8 activation by the therapeutic RNA in a subject.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect for potentiating TLR8 activation by a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • an immunogenic composition comprising 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 aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, for potentiating TLR8 activation by the therapeutic RNA in a subject.
  • the therapeutic agent is a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof, for potentiating TLR8 activation by the therapeutic RNA in a subject.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in a subject.
  • the subject has received, is receiving, or about to receive the therapeutic RNA.
  • an immunogenic composition comprising 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 aptamers, single guide RNA, self-amplifying RNAs, 1004921453 circular RNAs and combinations thereof for use in potentiating TLR8 activation by the therapeutic RNA in a subject.
  • the oligonucleotide, the fusion oligonucleotide, or the composition does not substantially increase translation of the therapeutic RNA.
  • the therapeutic RNA comprises pseudouridine.
  • the therapeutic RNA does not comprise pseudouridine.
  • a method of treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signalling 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.
  • an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect in the manufacture of a medicament for treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signalling.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect for treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signalling.
  • a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect for use in the prevention or treatment of a disease, disorder or condition in a subject responsive to increased TLR8 signalling.
  • the oligonucleotides, fusion oligonucleotides, or compositions according to the third aspect activate or increase TLR8 signalling.
  • the disease, disorder or condition responsive to increased TLR8 signalling is selected from the group consisting of: cancer, chronic viral (eg HBV) and bacterial infection.
  • the method or use further comprises administration of a TLR8 agonist.
  • the TLR8 agonist is administered at a sub-therapeutic dose.
  • Figure 1 HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 100 nM indicated oligos, prior to R848 (1ug/ml) stimulation overnight. Data shown are averaged from 3 independent experiments in biological triplicate.
  • NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM and One-way ANOVA with Dunnett’s multiple comparisons to C2Mut1-dC condition are shown. All internucleotide linkages are phosphorothioate (only the first 4 are indicated with a *).
  • Figure 2 HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 5 ⁇ M indicated oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 3 independent experiments in biological triplicate.
  • NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM and One-way ANOVA with Dunnett’s multiple comparisons to 5-Short-Mut1-Hyb condition are shown.
  • Figure 3 HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 5 ⁇ M indicated trimer, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control.
  • FIG. 5 HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated doses of trimers, prior to R848 (1 ⁇ g/ml) stimulation overnight. mG*mU*mC, in (A) was used at 500nM. Data shown are averaged from 1 independent experiment (A) or 3 independent experiments (B,C) in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM and One-way ANOVA with Dunnett’s multiple comparisons to GUC condition are shown.
  • Figure 6 Chemical structures of nucleotides, trimers and linked trimers used in the studies. Chemical structures of nucleotides that are modified compared to parent GUC (mG*mU*mC) or parental GAG (mG*mA*mA) are shown.
  • Figure 7 A and B) HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 5uM indicated trimer, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control.
  • Figure 9 A) HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 5 ⁇ M of oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. B) HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated doses of oligos, prior to R848 (1 ⁇ g/ml) stimulation 1004921453 overnight. Data shown are averaged from 2 independent experiments in biological triplicate.
  • NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM and One-way ANOVA with Dunnett’s multiple comparisons to R848 condition are shown.
  • Figure 10 A) HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 5 ⁇ M of oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with indicated doses of oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. A, B and C) All trimer conditions are with R848 co- stimulation. SEM and One-way ANOVA with Dunnett’s multiple comparisons to R848 (A, B) or NT (C) condition are shown.
  • FIG. 11 HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated doses of oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 3 biological triplicate for each screen. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. U is to be read as T for DNA trimers.
  • FIG. 12 HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated doses of oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 3 biological triplicate for each screen. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. [0209] Figure 13: RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with indicated doses of oligos, prior to R848 (indicated dose) stimulation overnight.
  • FIG. 14 RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 5uM oligos, prior to R848 (0.125 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The ELAM-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation.
  • FIG. 15 RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with indicated doses of oligos, prior to R848 (0.125ug/ml) stimulation overnight. Data shown are averaged from 3 independent experiments in biological triplicate. The ELAM-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEMs are shown.
  • FIG. 16 RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 500nM GGC or not (NT), prior to transfection with 500nM of B406AS1 ssRNA with DOTAP overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The ELAM-luciferase values are reported to the NT condition. SEMs and two tailed unpaired t-test are shown. [0213] Figure 17: RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 5uM of DNA or 2′-OMe trimers, prior to R848 (0.125 ⁇ g/ml) stimulation overnight.
  • FIG. 18 RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with indicated doses of oligos, prior to R848 (0.125 ⁇ g/ml) stimulation overnight. Data shown are averaged from 3 biological triplicate for each screen. The ELAM-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation.
  • FIG. 19 A) THP-1 were pre-treated ⁇ 60 min with indicated doses of oligos, prior to R848 (1 ⁇ g/mL) stimulation for 8hours and supernatants analysed by IP-10 ELISA.
  • B) HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated 1004921453 ⁇ 60 min with indicated doses of oligos, prior to Motolimod (600nM) stimulation overnight. Data shown are averaged from 3 biological triplicate for each screen. The NF- ⁇ B-luciferase values in B are reported to the Motolimod only condition after background correction to NT control.
  • FIG. 20 HEK-TLR8 cells (A, C) expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 5 ⁇ M indicated oligos, prior to Motolimod (Mo) (600nM) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the Motolimod condition. All trimer conditions are with Motolimod co-stimulation. SEM and One-way ANOVA with Dunnett’s multiple comparisons to Motolimod only condition are shown.
  • THP-1 cells (B, D) were pre-treated ⁇ 60 min with 5 ⁇ M, prior to R848 stimulation with 1 ⁇ g/ml overnight. Supernatants were collected and analysed for IP-10 production by ELISA. Data shown is averaged from 2 independent experiments in biological triplicate. SEM and One-way ANOVA with Dunnett’s multiple comparisons to R848 only (B) or NT (D) condition are shown.
  • Figure 21 HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated doses of oligos, prior to Motolimod (400 for MOE and 600nM for DNA) stimulation overnight. Data shown are averaged from 3 biological triplicate for each screen.
  • FIG. 22 A) HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 5 ⁇ M of oligos, prior to Motolimod (600nM) stimulation overnight. Data shown are averaged from 3 biological triplicate for each screen. B) HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 500nM of the selected oligos prior to Motolimod (600nM) stimulation overnight.
  • FIG. 23 HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 50 nM indicated oligos, prior to R848 (1 ⁇ g/ml) stimulation 1004921453 overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 2 ⁇ M indicated oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition (no background correction was applied here). All trimer conditions are with R848 co- stimulation. SEM are shown. [0222] Figure 26.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with either 5 ⁇ M or 400 nM concentration of 3rd base LNA modified trimers (A) or 400 nM of fully 2′-OMe or 3rd base LNA modified trimers (B), prior to R848 (1ug/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. All internucleotide linkages are phosphorothioate. [0223] Figure 27.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated amount of oligos (A, 200 nM and B, 100 nM), prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 3 independent experiments in biological triplicate except for GUC-v1 sequence in Fig 27.b is from 2 independent experiments in biological triplicate.
  • the NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer 1004921453 conditions are with R848 co-stimulation. SEM and One-way ANOVA with multiple comparisons R848 condition are shown. [0224] Figure 28.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated amount of oligos (A, is 5 ⁇ M and B, is 1 ⁇ M), prior to R848 (1 ⁇ g/ml) stimulation overnight.Data shown in (A) and (B) are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM and One-way ANOVA with multiple comparisons R848 condition are shown. [0225] Figure 29.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with various doses of indicated oligos or Enpatoran, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co- stimulation. SEM and One-way ANOVA with multiple comparisons R848 condition are shown. [0226] Figure 30.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated amount of oligos (5 ⁇ M), prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 1 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co- stimulation. SEM is shown. [0227] Figure 31. RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 100 nM doses of oligos, prior to R848 (0.125 ⁇ g/ml) stimulation overnight.
  • FIG. 34 The ELAM-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM and One-way ANOVA with multiple comparisons to R848 condition are shown (non-significant comparisons are not shown).
  • Figure 34 RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 500 nM of oligos, prior to R848 (0.125 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The ELAM-luciferase values are reported to the R848 condition after background correction to NT control.
  • RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 5 mM oligos, prior to R848 (0.125 ⁇ g/ml) stimulation overnight. Data shown are averaged from 1 experiment in biological triplicate. The ELAM-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM are shown. [0233] Figure 37. RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 5 ⁇ M of oligos, prior to R848 (0.125 ⁇ g/ml) stimulation 1004921453 overnight.
  • TLR7 kika/wt BMDMs were treated overnight with 5 ⁇ M of GGC-v1 or 200 nM Enpatoran and RNA analysed by RNAseq (using 3 mice per condition). Generally, for each sample, there were about 2.8 million counts across 18,000 genes. One non-treated sample from the Kika group was excluded from further analysis due to low counts. Statistical comparisons were made using the contrasts.fit function from the limma package (v3.48.3) and empirical Bayes moderated t-tests were performed, with p-values obtained using eBayes, and using non-treated TLR7 kika/wt BMDMs cells as reference. [0236] Figure 40.
  • mice 6 WT mice were treated per group with 20 ⁇ g trimer oligonucleotides dissolved in PBS with 30% Pluronic F-127 on the ear, or 60 ⁇ g trimer oligonucleotide on the back, prior to administration of Aldara cream, daily, for 4 days.
  • a Vaseline group (with no Aldara) was used as control group. Thickness of the ear was measured daily with callipers, and ear redness scored along with back scaliness. On the 5 th day, the mice were culled and RNA from the back skin of 4 mice was collected for each group.
  • FIG. 43 HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 5 ⁇ M of oligos, prior to Motolimod (600 nM) stimulation overnight. Data shown are averaged from 3 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the Motolimod condition after background correction to NT control. All trimer conditions are with Motolimod co-stimulation. SEM and One-way ANOVA with multiple comparisons to GUC condition are shown.
  • FIG. 44 A) HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 500 nM of oligos, prior to Motolimod (600nM) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the Motolimod condition after background correction to NT control. All trimer conditions are with Motolimod co- stimulation. SEM and One-way ANOVA with multiple comparisons to GAG-v4 condition are shown.
  • HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with indicated concentration of GUC-v16 oligos, prior to Motolimod (600 nM) stimulation overnight. Data shown are averaged from 3 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the Motolimod condition after background correction to NT control. All trimer conditions are with Motolimod co-stimulation. SEM is shown. [0241] Figure 45.
  • HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 1 (A) or 5 ⁇ M of oligos (A and B), prior to Motolimod (600nM) stimulation overnight. Data shown is from two independent screens conducted in biological triplicate. The NF- ⁇ B-luciferase values are reported to the Motolimod condition after background correction to NT control. All trimer conditions are with Motolimod co-stimulation. [0243] Figure 47. HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 1 or 5 ⁇ M of oligos, prior to Motolimod (600 nM) stimulation overnight.
  • NF- ⁇ B-luciferase values are reported to the Motolimod condition after background correction to NT control. All trimer conditions are with Motolimod co- stimulation. GAG-v1 (mGmAdG and mGdCdC) were used as positive controls for inhibition and potentiation of TLR8, respectively). The 5′-end of all the trimers is 2′-- OMe.
  • Figure 48 HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 1 ⁇ M of oligos, prior to Motolimod (600 nM) stimulation overnight.
  • HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 5 ⁇ M of oligos, prior to uridine (20mM) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B- luciferase values are reported to the uridine condition after background correction to NT control. All trimer conditions are with uridine co-stimulation. SEM and One-way ANOVA with multiple comparisons to uridine condition are shown. [0248] Figure 52.
  • HEK-TLR8 cells expressing an NF- ⁇ B-luciferase reporter were pre- treated ⁇ 60 min with 5 ⁇ M of mGdCdC or 1 ⁇ M dT20, DOTAP transfection with 5 ⁇ g total mouse RNA overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the NT control. SEM and unpaired t-test are shown. [0249] Figure 53. PMA-differentiated and IFN ⁇ primed THP-1 cells were transfected overnight with 750 nM of indicated oligos with DOTAP, and supernatants analysed by ELISA. Data are shown averaged from 2 independent experiments in biological triplicate.
  • HEK-TLR7 cells expressing an NF- ⁇ B-luciferase reporter were pre-treated ⁇ 60 min with 5 ⁇ M indicated oligos, without serum, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF- ⁇ B-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co- stimulation. SEM and One-way ANOVA with multiple comparisons R848 condition are shown.
  • HEK-TLR7 cells expressing an NF-KB-luciferase reporter were pre- treated ⁇ 60 min with 2 ⁇ M concentration of indicated oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF-KB-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co- stimulation. SEM and One-way ANOVA with multiple comparisons R848 condition are shown. All internucleotide linkages are phosphorothioate. [0256] Figure 60.
  • RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 1 ⁇ M (Fig 66. A) or 200 nM (Fig 66. B) oligos, prior to R848 (0.125 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The ELAM-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM are shown. [0257] Figure 61. RAW-ELAM stably cells expressing an ELAM-luciferase reporter were pre-treated ⁇ 60 min with 5 ⁇ M (Fig 67.
  • TNF levels were measured in the supernatant by ELISA and are expressed as a fold increase relative to R848 alone.
  • n 3 mice ⁇ SEM with one-way ANOVA shown.
  • Figure 66 Healthy skin punch biopsies (3 mm x 3mm) inserted into Transwell filters with the epidermis facing upwards at the air-liquid interface and the dermis suspended in the culture medium were pre-treated, or not, with 5 ⁇ M of oligo 38-2 (mG*dC*dC) for 30 min before addition 600 nM Motolimod for 24 h.
  • IL-8 levels were measured in the supernatant by ELISA and are reported as a fold increase relative to Motolimod alone.
  • HEK-TLR7 (A) and HEK-TLR8 (B) cells expressing an NF-KB- luciferase reporter were pre-treated ⁇ 60 min with 1 or 5 ⁇ M concentration of indicated oligos, prior to R848 (1 ⁇ g/ml) stimulation overnight. Data shown are averaged from 2 independent experiments in biological triplicate. The NF-KB-luciferase values are reported to the R848 condition after background correction to NT control. All trimer conditions are with R848 co-stimulation. SEM and One-way ANOVA with multiple comparisons R848 condition are shown.
  • the terms “treating”, “treat” or “treatment” include administering a therapeutically effective amount of a oligonucleotide(s) described herein sufficient to reduce or eliminate at least one symptom of a disease, disorder or condition.
  • the term “treating” a subject includes delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the sign or symptom of the disease or 1004921453 condition, or the risk of (or susceptibility to) the disease or condition.
  • treating refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of signs or symptoms or making the injury, pathology or condition more tolerable to the individual; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating.
  • the methods of the present invention can be to prevent or reduce the severity, or inhibit or minimise progression, of a sign or symptom of a disease or condition as described herein. As such, the methods of the present invention have utility as treatments as well as prophylaxes.
  • preventing include administering a therapeutically effective amount of a oligonucleotide(s) described herein sufficient to stop or hinder the development of at least one symptom of a disease, disorder or condition.
  • preventing is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical signs or symptoms of the disease not to develop in an individual that may be exposed to or predisposed to the disease but does not yet experience or display signs or symptoms of the disease).
  • Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians.
  • the term “subject”, “individual” or “patient” can be used interchangeably with each other.
  • the term “subject” refers to an animal that is treatable by the oligonucleotide and/or method, respectively.
  • the animal is a vertebrate.
  • the animal can be a mammal, avian, chordate, amphibian or reptile.
  • Exemplary subjects include but are not limited to human, primate, livestock (e.g. sheep, cow, chicken, horse, donkey, pig), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animal (e.g. fox, deer).
  • the mammal is a human.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, in some instances ⁇ 5%, in some instances ⁇ 1%, and in some instances ⁇ 0.1% 1004921453 from the specified value, as such variations are appropriate to perform the disclosed methods.
  • Ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention.
  • a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the terms “reduce” or “inhibit” may relate generally to the ability of one or more oligonucleotides described herein to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art, and may include reductions in the symptoms or pathology of a disease.
  • a “decrease” in a response may be statistically significant as compared to the response produced by no oligonucleotide or a control composition, and may include at least about a 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% decrease, including all integers in between.
  • the phrase “inhibits TLR7 activity” or variations thereof means that after administration of an oligonucleotide of the invention to a subject, the subject is not able to elicit a TLR7 based immune response or is only able to elicit a reduced or partial TLR7 based immune response, such as to a pathogen or a damaged endogenous nucleic acid.
  • 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.
  • an oligonucleotide of the invention inhibits or reduces TLR7 activity by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the phrase “inhibits TLR8 activity” or variations thereof means that after administration of an oligonucleotide of the invention to a subject, the subject is not able to elicit a TLR8 based immune response or is only able to elicit a reduced or partial TLR8 based immune response, such as to a pathogen or a damaged endogenous nucleic acid.
  • 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.
  • an oligonucleotide of the invention inhibits or reduces TLR8 activity by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the phrase “does not substantially reduce translation of the therapeutic RNA” or variations thereof means that the level of translation of the therapeutic RNA in a subject is comparable in the presence or absence of an oligonucleotide of the invention.
  • the level of translation of the therapeutic RNA in the presence of an oligonucleotide of the invention is about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, or 80% of the level of translation of the therapeutic RNA in the absence of the oligonucleotide of the invention.
  • the level of translation of the therapeutic RNA in the absence of an oligonucleotide of the invention may be referred to as a reference level of translation of the therapeutic RNA.
  • the skilled person will be familiar with methods for obtaining a reference level of oligonucleotide translation.
  • the method may include obtaining data from multiple individuals to develop an appropriate reference data set.
  • a reference level may be generated from the same individual, but at a different time-points for example before administration of the therapeutic RNA, after administration of the therapeutic RNA, before administration of the oligonucleotide of the invention, after administration of the oligonucleotide of the invention, or a combination thereof.
  • potentiate refers to an increase in a functional property relative to a control condition.
  • the term “potentiate” may relate generally to the ability of one or more oligonucleotides described herein to “increase” the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency may be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.
  • the phrase “increases or potentiates TLR8 activity” or variations thereof means that after administration of an oligonucleotide of the invention to a subject, the subject is only able to elicit a TLR8 based immune response or is able to elicit an increased or elevated TLR8 based immune response, such as to a pathogen, a damaged endogenous nucleic acid or an exogenous TLR8 ligand. Potentiation of TLR8 activity may be greater than about 100%, e.g.
  • the level of TLR8 potentiation is between about 2 fold and 50 fold, between about 2 fold and 20 fold, and/or between about 5 fold and 20 fold greater.
  • the terms “disease”, “disorder” or “condition” relate to any unhealthy or abnormal state.
  • the term “disease, disorder or condition in a subject responsive to TLR7 inhibition” includes diseases, conditions, and disorders in which the inhibition of TLR7 provides a therapeutic benefit.
  • diseases, disorders and conditions associated with increased TLR7 signalling This includes diseases, disorders and conditions associated with increased TLR7 signalling. This also includes diseases, disorders and conditions wherein TLR7 signalling exacerbates an aberrant autoimmune response.
  • Diseases, disorders and conditions responsive to TLR7 inhibition include inflammation- related diseases, allergic diseases, infections, cancers and auto-immune diseases.
  • the term “disease, disorder or condition in a subject responsive to TLR8 inhibition” includes diseases, conditions, and disorders in which the inhibition of TLR8 provides a therapeutic benefit. This includes diseases, disorders and conditions associated with increased TLR8 signalling. This also includes diseases, disorders and conditions wherein TLR8 signalling exacerbates an aberrant autoimmune response.
  • TLR8 agonist refers to an agent that is capable of causing a signalling response through a TLR8 signalling pathway, either as a direct ligand or indirectly through generation of endogenous or exogenous ligand. Such natural or synthetic TLR8 agonists can be used as alternative or additional adjuvants.
  • the TLR8 agonist capable of causing a signalling response through TLR8 is a single stranded RNA (ssRNA), an imidazoquinoline molecule with anti-viral activity, for example resiquimod (R848).
  • TLR-8 agonists which can be used include those described in WO 2004/071459 and WO2021/232099.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and/or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a “therapeutically effective amount” and “effective amount” describe a quantity of a specified agent, such as an oligonucleotide of the invention, sufficient to achieve a desired effect in a subject or cell being treated or contacted with that agent.
  • a specified agent such as an oligonucleotide of the invention
  • this can be the amount of a composition comprising one or more agents that inhibit the activity of one or more nucleic acid sensors (e.g., TLR7 or TLR8) described herein, necessary to reduce, alleviate and/or prevent a disease, disorder or condition.
  • a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of a disease, disorder or condition.
  • a “therapeutically effective amount” or “effective amount” is an amount sufficient to achieve a desired biological effect, for example, an amount that is effective to decrease or prevent a senescence-associated disease, disorder or condition or inhibit or prevent senescence in a cell.
  • a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject.
  • the effective amount of an agent useful for reducing, alleviating and/or preventing a disease, disorder or condition will be dependent on the subject being treated, the type and severity of any associated symptoms and the manner of administration of the therapeutic composition.
  • a sub-therapeutic dose is a dose that is unable to achieve the therapeutic goal.
  • That goal may be for example, reducing inflammation, minimising an allergic response, a reduction in infection, a reduction in tumour size, a reduction in increase or decrease of cancer biomarker expression, stasis of tumour growth, or a reduction, alleviation or abrogation of autoimmune disease symptoms.
  • a sub-therapeutic dose is one which does not cause significant adverse side effects in the subject.
  • alkyl refers to a single bond chain of hydrocarbons ranging, in some embodiments, from 1-20 carbon atoms, ie 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 therein.
  • C1-C2 alkyl refers to an alkyl group, as defined herein, containing at least 1, and at most 2, 4 or 6 carbon atoms respectively, or any range in between (eg alkyl groups containing 2-5 carbon atoms are also within the range of C1- C6.
  • alkyl examples include, but are not limited to, are methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl, 2,2-dimethylbutyl.
  • alkyl is C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C1 alkyl.
  • alkyl is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl.
  • alkenyl refers to a straight-chain or branched-chain hydrocarbyl, which has one or more double bonds and, unless otherwise specified, contains from about 2 to about 20 carbon atoms, and ranging in some embodiments from about 2 to about 10 carbon atoms, and ranging in some embodiments from about 2 to about 8 carbon atoms, and ranging in some embodiments from about 2 to about 6 carbon atoms.
  • alkenyl radicals include vinyl, allyl, 1,4-butadienyl, isopropenyl, and the like.
  • alkenyl is C2-C20 alkenyl, C2-C10 alkenyl, C2-C8 alkenyl, C2-C6 alkenyl.
  • alkenyl is C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl.
  • alkynyl refers to a straight-chain or branched- chain hydrocarbyl, which has one or more triple bonds and, unless otherwise specified, contains from about 2 to about 20 carbon atoms, and ranging in some embodiments from about 2 to about 10 carbon atoms, and ranging in some embodiments from about 2 to about 8 carbon atoms, and ranging in some embodiments from about 2 to about 6 carbon atoms.
  • alkynyl radicals include ethynyl, propynyl, butynyl, and the 1004921453 like.
  • alkenyl is C2-C20 alkynyl, C2-C10 alkynyl, C2-C8 alkynyl, C2-C6 alkynyl.
  • alkynyl is C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl.
  • amino or “amine” refers to the group -NH 2 .
  • hydroxy or “hydroxyl” refers to the group –OH.
  • oxo refers to an oxygen substituent doble bonded to the attached carbon.
  • halogen refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) and the term “halo” refers to the halogen radicals fluoro (-F), chloro (- Cl), bromo (-Br), and iodo (-I).
  • halo is fluoro, chloro or bromo.
  • substituted means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, ie, a compound that can be isolated, characterized and tested for biological activity.
  • substituted hydrocarbyl refers to any of the above referenced hydrocarbyl groups, including “alkyl”, “alkenyl”, “alkynyl”, further bearing one or more substituents selected from hydroxyl, 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, halo, -SO3M or -OSO3M, where M is H, Na, K, Zn, Ca, or meglumine, guanidinyl, substitute
  • the one or more substituents is selected from the group consisting of: hydroxyl, carboxyl, amino, thio, halo, and -OR, wherein R is alkyl, alkenyl or alkynyl.
  • R is alkyl, alkenyl or alkynyl.
  • substituted alkyl is “substituted C1-C6 alkyl”
  • substituted alkenyl is “substituted C2-C20 alkenyl”
  • substituted alkynyl is “substituted C2-C20 alkynyl”.
  • hydrophobic lipid refers to an amphiphilic molecule that comprises a polar head group and a hydrophobic tail.
  • the hydrophobic tail may comprise a hydrocarbon chain selected from the group consisting of: C1-C20 alkyl, C2- C20 alkenyl, C2-C20 alkynyl, substituted C1-C20 alkyl, substituted C2-C20 alkenyl, and substituted C2-C20 alkynyl.
  • Hydrophobic lipids include, but are not limited to, phospholipids, cholesterol, cholesterol derivatives, and tocopherol.
  • polyethylene glycol (PEG) refers to a polyether compound.
  • PEG comprises H(OCH2CH2)nOH wherein n is 1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 or 20.
  • n is 1 to 6.
  • Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.
  • the oligonucleotides of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms.
  • stereoisomers refers to oligonucleotides which have identical chemical constitution, but differ with regard to the arrangement of the atoms or 1004921453 groups in space.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), wherein the polymer or oligomer of nucleotide monomers contain any combination of nucleotides (referred to in the art and herein as simply as “base”), modified nucleotides, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as “internucleotide linkage”).
  • a “target” such as “target polynucleotide” refers to a molecule upon which an oligonucleotide of the invention directly or indirectly exerts its effects. Typically, the oligonucleotide of the invention or portion thereof and the target, interact or bind under physiological conditions thereby modulating the function of the target.
  • the target is TLR7. In the second and third aspects the target is TLR8.
  • the term “nucleotide” includes all naturally occurring nucleotides, including all forms of nucleotide bases found in nature. Base rings most commonly found in naturally occurring nucleotides are purine and pyrimidine rings.
  • 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.
  • Naturally occurring nucleotides for example include, but are not limited to, ribo, 2′-O-methyl or 2′-deoxyribose derivatives of adenosine, guanosine, thymidine, uridine, inosine, 7-methylguanosine or pseudouridine.
  • modification group refers to any chemical moiety that may be attached to the oligonucleotide at locations, which include, but are not limited to, the sugar, nucleoside base, triphosphate bridge, and/or internucleotide phosphate.
  • nucleotide analogs include synthetic nucleotides as described herein.
  • Nucleotide derivatives also include nucleotides having modified base and/or sugar moieties, with or without protecting groups and include, for example, 2′-deoxy-2′-fluorouridine, 5- 1004921453 fluorouridine and the like.
  • Other nucleotide derivatives that may be utilized with the present invention include, for example, LNA nucleotides, halogen-substituted purines (eg 6-fluoropurine), halogen-substiuted pyrimidines, N 6 -ethyladenine, N 4 -(alkyl)- cytosines, 5-ethylcytosine, and the like.
  • an oligonucleotide of the invention will be synthesized in vitro.
  • RNA nucleotides (rX) comprise 2′-OH.
  • DNA nucleotides (dX) comprise 2′-H.
  • Modified nucleotides may include nucleotides having modified base and/or sugar moieties.
  • mX refers to a nucleotide comprising a 2′- and/or 3′- methoxy (2′-OMe and/or 3′-OMe) modification.
  • mX nucleotides at terminal positions of the sequence may be 2′-OMe or 3′-OMe, preferably 2′-OMe.
  • mX nucleotides at non-terminal positions of the sequence may be 2′-OMe.
  • mX is 2′-OMe.
  • “moX” refers to a nucleotide comprising a 2′- 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.
  • moX nucleotides at terminal positions of the sequence may be 2′-OMe or 3′-MOE, preferably 2′-MOE.
  • moX nucleotides at non-terminal positions of the sequence may be 2′-MOE. In a preferred embodiment, moX is 2′-MOE.
  • fX refers to a nucleotide comprising a 2′- and/or 3′-fluoro modification. fX nucleotides at terminal positions of the sequence may be 2′-fluor or 3′- fluor, preferably 2′-fluor. fX nucleotides at non-terminal positions of the sequence may be 2′-fluor. In a preferred embodiment, fX is 2′-fluor.
  • LX refers to a nucleotide comprising a Locked Nucleic Acid (LNA) modification.
  • LNAs are nucleic acids in which the 2′-hydroxyl group is linked to the 3′- or 4′- carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.
  • the linkage is a methylene (-CH2-)n group bridging the 2′-oxygen atom and the 4′-carbon atom, wherein n is 1 or 2, preferably 1.
  • morpholino-X refers to a nucleotide comprising a 1-Oxa-4- azacyclohexane ribose sugar ring (also known as a morpholine ring). Morpholine rings bear methylene groups that are bound to modified phosphates in which the anionic oxygen is replaced by a nonionic dimethylamino group. The substituted phosphate is bound through a phosphorus ⁇ nitrogen bond to the nitrogen atom of another morpholine ring.
  • One standard DNA or RNA nucleobase is bound to each morpholine ring.
  • modified nucleotide which includes “modified dX”, “modified rX”, “modified mx”, “modified moX”, “modified fX”, ““modified LX”, refers to a nucleotide including at least one modification or substitiution.
  • the modification or substitution may be of rX or dX, or may be an additional modification or substitution of an already modified nucleotide such as mX, moX, fX or LX.
  • Modified nucleotides may include modifications or substitutions at positions of the base and/or sugar.
  • modified dX refers to DNA base comprising at least one modification or substitution at one or more positions of the base and/or sugar.
  • Modified rX refers to RNA base comprising at least one modification or substitution at one or more positions of the base and/or sugar.
  • Modified mX refers to a nucleotide including a 2′-OMe and/or 3′-OMe modification, preferably a 2′-OMe modification, and at least one additional modification or substitiution at one or more positions of the base and/or sugar.
  • Modified moX refers to a nucleotide including a 2′-MOE and/or 3′-MOE modification, preferably a 2′-MOE modification, and at least one additional modification or substitiution at one or more positions of the base and/or sugar.
  • Modified fX refers to a nucleotide including a 2′-fluor and/or 3′- fluor modification, preferably a 2′- fluor modification, and at least one additional modification or substitiution at one or more positions of the base and/or sugar.
  • Modified LX refers to a nucleotide including an LNA modification, and at least one additional modification or substitiution at one or more positions of the base and/or sugar.
  • the modification or substitution is selected from the group consisting of: pseudouridine, 3′-deoxy, hydroxyl, des-amino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.
  • the modification or substitution is at one or more positions of the sugar.
  • the modification or substitution of the sugar may be selected from the group consisting of: 3′-deoxy, hydroxyl, amino, thio, halo, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, and combinations thereof.
  • the modification or substitution is at one or more positions of the base.
  • the modification or substitution of the base may be selected from the group consisting of: pseudouridine, hydroxyl, des-amino, 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. [0319] In one embodiment, the modification or substitution is at one or more positions of the base and one or more positions of the sugar.
  • Modified nucleotides include, for example, 2′-OMe-2,6-Diaminopurine (herein referred to as mG1), 2′-OMe-I (2′-O-methylinosine, herein interchangeably referred to as mI or mG2), 2′-OMe-5-Me-U (2′-O-methyl-5-methyluridine, herein referred to as mU1), 2′-OMe-5-Br-U (2′-O-methyl-5-bromouridine herein referred to as mU2), N3-Me-U (3- methyluridine herein referred to as mU3), 2′-OMe-5-Me-C (2′-O-methyl-5- methylcytidine, herein referred to as mC1), N7-methylated guanosine (herein referred to as m7 G), 5-methyl substituted deoxycytidine (herein referred to as 5-Me-dC), 5-bromo
  • 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, wherein 1004921453 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.
  • modified mG includes mG1 and mI.
  • Oligonucleotides of the invention include at least one 2′-OMe or 3′-OMe- modified nucleotide, preferably 2′-OMe-modified nucleotide.
  • reference to an A, T, G, U or C can either mean a naturally occurring base or a modified version thereof.
  • Oligonucleotides of the present disclosure include those having modified backbones or non-natural internucleoside linkages.
  • internucleotide linkage refers to the bond or bonds that connect two nucleotides of an oligonucleotide or nucleic acid and may be a natural phosphodiester linkage or modified linkage. Internucleotide linkages include but are not limited to: biphosphate, triphosphate, phosphorothioate, phosphodiester, thiophosphoramidate, phosphorodiamidate, methylphosphonate, and guanidinopropyl phosphoramidate. In one embodiment, each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester.
  • each internucleotide linkage is independently selected from the group consisting of: triphosphate, phosphorothioate, and phosphodiester. More preferably, each internucleotide linkage is independently selected from phosphorothioate, and phosphodiester. Even more preferably, each internucleotide linkage is phosphorothioate. [0326] Each internucleotide linkage may be the same or different. In a preferred embodiment, each internucleotide linkage is the same, preferably phosphorothioate. [0327] Internucleotide linkages can be introduced at the 2′-, 3′-, or 5′- end of a nucleotide.
  • Each internucleotide linkage may be selected from the group consisting of: 3′-5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, 5′-2′- linkage.
  • each internucletodie linkage is selected from the group consisting of: 3′-5′- and 5′-5′- linkage.
  • a 3′-5′-phosphorothioate internucleotide linkage may be formed by bonding the 3′- phosphate of a first nucleotide and the 5′- hydroxyl group of a second 1004921453 nucleotide, wherein a non-bridging oxygen is substituted with a sulfur.
  • a 3′-5′- phosphodiester internucleotide linkage may be formed by bonding the 3′- hydroxyl group of a first nucleotide and the 5′- phosphate of a second nucleotide.
  • a 5′-5′- ,triphosphate internucloetide linkage may be formed by bonding the 5′- phosphate of a first nucleotide and the 3′- phosphate of a second nucleotide via an additional phosphate group.
  • the sequence includes a 5′-5′- linkage.
  • the 5′-5′- linkage connects nucleotides at a first and second position of the sequence.
  • the sequence includes a 5′-5′- linkage and a 3′-5′- linkage.
  • each internucleotide linkage connects nucleotides at a first and second position of the sequence
  • the 3′-5′- linkage connects nucleotides at a second and third position of the sequence.
  • each internucleotide linkage is a 3′-5′- linkage.
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • Each internucleotide linkage may contain asymmetric or chiral centres and therefore exist in different stereoisomeric forms.
  • Oligonucleotides of the invention may comprise a mixture of different oligonucleotide stereoisomers.
  • a 3-mer oligonucleotide of the invention comprising phosphorothioate internucleotide linkages may comprise a mixture of up to 4 different oligonucleotide phosphorothioate stereoisomers, due to the chirality introduced by the two sulfur atoms in the PS internucleotide linkages.
  • 5′-[mX/modified mX]* y X A * z X B -3′ may comprise up to 4 different stereoisomers resulting from different phosphorothioate stereochemistry configurations at each internucleotide linkage, wherein * y and * z each represent a phosphorothioate internucleotide linkage, wherein * y and * z are each in the R configuration (RR), * y and * z are each in the S configuration (SS), * y is in the R configuration and * z is in the S configuration (RS), and * y is in the S configuration and * z is in the R configuration (SR).
  • the oligonucleotides of the invention may 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 oligonucleotide may comprise additional chiral centres, for example on one or more ribose sugars, and therefore may comprise mixtures of, or single, stereoisomers at the additional chiral centres.
  • Functionalised sequences 1004921453 [0330] In one embodiment, the sequence may be functionalised.
  • the functionalised sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocycyl, arylalkyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the compound is selected from the group consisting of: polyethylene glycol, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, heterocycyl, arylalkyl, branched C1-C20 alkyl, branched C2-C20 alkyenyl, branched C2-C20 alkynyl, substituted C1-C20 alkyl, substituted C2-C20 alkenyl, substituted C2-C20 alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the hydrophobic lipid is selected from cholesterol and tocopherol.
  • the compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol.
  • the compound is conjugated directly to the sequence.
  • the compound is conjugated to the sequence via a linker.
  • the linker may be cleavable or non-cleavable.
  • the linker is a non-cleavable linker.
  • the compound is conjugated to a terminal nucleotide of the sequence, preferably the terminal 3′-nucleotide.
  • the compound is conjugated to the terminal 3′-nucleotide at the 3′-position.
  • Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG, dX-Chol and dX-Toco, wherein dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group, dX-Chol is a DNA base with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol- glyceryl group covalently linked to the 3′-position via a monophosphate group, dX- Toco is a DNA base with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group
  • dX-Chol is a DNA base with an (N-choleste
  • functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dC-TEG, dC-Chol, dC-Toco, wherein dC-TEG is deoxycytidine with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl group covalently linked to the 3′-position via a monophosphate group, dC-Chol, is deoxycytidine with triethylene 1004921453 glycol covalently linked to the 3′-position via a monophosphate group, and dC-Toco is deoxycytidine with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dC-TEG is deoxycytidine with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl group
  • an oligonucleotide comprising or consisting of a sequence consisting of: [mX/modified mX]* y X A * z X B wherein: * y and * z each independently represent an inter-nucleotide linkage, wherein at least one of * y and * z is not phosphorodiamidate;
  • X A and X B 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; 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
  • both * y and * z are not phosphorodiamidate.
  • 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, 1004921453 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.
  • an oligonucleotide comprising or consisting of a sequence consisting of: [mX/modified mX]* y X A * z X B wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • 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, modified moX, modified LX, and modified fX; 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 LNA modified base, fX is a nucleotide comprising
  • any oligonucleotide of the first aspect inhibits TLR7 activity, preferably human TLR7 activity.
  • an oligonucleotide of the first aspect does not potentiate TLR8 activity, preferably human TLR8 activity.
  • an oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity.
  • an 1004921453 oligonucleotide of the first aspect potentiates TLR8 activity, preferably human TLR8 activity.
  • Each internucleotide linkage may be selected from the group consisting of: 3′- 5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, and 5′-2′- linkage.
  • each internucleotide linkage may be selected from: 3′-5′- and 5′-5′- linkage.
  • each internucletodie linkage is a 3′-5′- linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-[mX/modified mX]* y X A * z X B -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage, wherein at least one of * y and * z is not phosphorodiamidate;
  • X A and X B 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; 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 nucleot
  • an oligonucleotide comprising or consisting of a sequence consisting of: 1004921453 5′-[mX/modified mX]* y X A * z X B -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • 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, modified moX, modified LX, and modified fX; 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 LNA modified base, fX is
  • each internucleotide linkage is a 3′-5′ linkage.
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester.
  • Each internucleotide linkage may be the same or different.
  • each internucleotide linkage is independently selected from phosphorothioate and phosphodiester.
  • Most preferably, each internucleotide linkage is phosphorothioate.
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers.
  • the oligonucleotide of the first aspect comprises a single phosphorothioate stereoisomer, preferably wherein * y is in the S configuration.
  • mX is a nucleotide comprising a 2′-OMe modification.
  • moX is a nucleotide comprising a 2′-MOE modification.
  • fX is a nucleotide comprising a 2′-fluor modification.
  • Modified dX, modified rX and modified morpholino comprise at least one modification or substitution at positions of the base and/or sugar.
  • Modified mX, modified moX, modified LX and modified fX comprise at least one additional modification or substitution at additional positions of the base and/or sugar.
  • the modification or substitution is selected from the group consisting of: pseudouridine, 3′-deoxy, hydroxyl, des-amino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl and combinations thereof.
  • Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mU3, mC1, and 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.
  • mG1 is 2′-OMe-2,6-Diaminopurine
  • mI is 2′-OMe-I (2′-O-
  • modified mX is selected from the group consisting of: mG1, mI, mU1, mU2 and mC1. Most preferably, modified mX is mC1.
  • 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, wherein 5-Me-dC is 5-methyl substituted deoxycytidine, 5-Br-dC is 5-bromo substituted deoxycytidine5-CH2OH-dC is 5-hydroxymethyl substituted deoxycytidine, ddC is 2′-deoxy-3′-deoxy cytidine, p
  • Exemplary modified rX includes but is not limited to PSU, 2′-NH2-rX, and ara- rX, wherein 2′-NH2-rX is a 2′-amino modified RNA base, and ara-rX is an arabinose modified RNA base.
  • Exemplary 2′-NH2-rX includes but is not limited to 2′-NH2-U and 2′- NH2-C, wherein 2′-NH2-U is 2′-NH2-uridine, and 2′-NH2-C is 2′-NH2-cytidine.
  • Exemplary ara-rX is ara-C (aracytidine).
  • [mX/modified mX] is selected from the group consisting of: mG, mI, mG1 and mU.
  • [mX/modified mX] is mG or mI.
  • [mX/modified mX] is modified mX.
  • modified mX is mI or mG1, preferably mI.
  • modified mX is not 2′-OMe-N1-Me-G (2′-O-methyl-N1- methylguanosine).
  • [mX/modified mX] is mX.
  • mX is mG or mU, preferably mG.
  • [mX/modified mX] is [mG/modified mG].
  • modified mG is not 2′-OMe-N1-Me-G (2′-O-methyl-N1-methylguanosine).
  • Modified mG includes but is not limited to: mG1 and mI, wherein mG1 is 2′-OMe-2,6- Diaminopurine, and mI is 2′-OMe-I (2′-O-methylinosine).
  • [mG/modified mG] is [mG/mI].
  • [mG/modified mG] is mG. In another embodiment, [mG/modified mG] is mI.
  • 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.
  • X A is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX, and modified rX.
  • X A is selected from the group consisting of: mX, dX, rX, modified mX, modified dX, and modified rX.
  • 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.
  • X A is selected from the group consisting of: mX, dX, rX, and modified mX.
  • 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.
  • X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX, modified dX, modified rX and morpholino-X.
  • 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.
  • X B is selected from the group consisting of: mX, dX, rX, moX, LX, fX, modified mX and modified dX.
  • X B is selected from the group 1004921453 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.
  • X B is selected from the group consisting of: mX, dX, LX, modified mX, modified dX and modified rX.
  • X B is selected from the group consisting of: LC, dC, 5-Me-dC, 5-Br-dC, mC, mC1, ara-C.
  • X B is selected from the group consisting of: LX, modified mX, modified dX and modified rX.
  • X B is selected from the group consisting of: LC, 5-Me-dC, 5-Br-dC, and mC1.
  • X B is LX, preferably LC.
  • at least one of X A and X B is LX.
  • X A and X B are independently LX.
  • one of X A and X B is LX.
  • X B is LX.
  • X B is LX and X A is mX.
  • at least one of X A and X B is dX.
  • X A and X B are independently dX.
  • one of X A and X B is dX.
  • X B is dX.
  • X B is dX and X A is mX. More preferably, X B is dX and X A is mU.
  • at least one of X A and X B is rX.
  • X A and X B are independently rX. In another embodiment, one of X A and X B 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, where at least one of X A and X B is rX, each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-mG*mU*X B -3′ wherein: * each independently represent a 3′-5′- phosphorothioate linkage;
  • 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; 1004921453 wherein 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 LNA modified base, fX is a nucleotide comprising a 2′-fluor and/or 3′-fluor modification, morpholino-X is a nucleotide
  • the sequence may be functionalised.
  • the functionalised sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocycyl, arylalkyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the hydrophobic lipid is selected from cholesterol and tocopherol.
  • the compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol.
  • the compound is conjugated directly to the sequence.
  • the compound is conjugated to the sequence via a linker.
  • the linker may be cleavable or non-cleavable.
  • the linker is a non-cleavable linker.
  • the compound is conjugated to a terminal nucleotide of the sequence, preferably the terminal 3′- nucleotide.
  • the compound is conjugated to the terminal 3′-nucleotide at the 3′- position.
  • Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG, dX-Chol and dX-Toco, wherein dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group, dX-Chol is a DNA base with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol- glyceryl group covalently linked to the 3′-position via a monophosphate group, dX-Toco is a DNA base with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group
  • dX-Chol is a DNA base with an (N-choleste
  • functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dC-TEG, dC-Chol, dC-Toco, wherein dC-TEG is deoxycytidine with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl group covalently linked to the 3′-position via a monophosphate group, dC-Chol, is deoxycytidine with triethylene 1004921453 glycol covalently linked to the 3′-position via a monophosphate group, and dC-Toco is deoxycytidine with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dC-TEG is deoxycytidine with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl group
  • the oligonucleotide of the first aspect is selected from the group of oligonucleotides in Table 1.
  • [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, wherein the sequence is optionally functionalised.
  • X B is selected from the group consisting of: LX, mX, dX, modified mX, modified rX, and modified dX.
  • X B is selected from the group consisting of: LC, mC, ara-C, dC, mC1, and modified dC.
  • modified dC is selected from the group consisting of: 5-Me-dC, 5-Br-dC and 5-I-dC.
  • the sequence is selected from the group consisting of: mG*mU*LC, mI*mU*LC, mG*mU*mC1, 1004921453 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.
  • [mX/modified mX] is [mG/mI];
  • X A is mU;
  • X B is selected from the group consisting of: mX, dX, rX, LX, modified mX modified rX, and modified dX.
  • X B is selected from the group consisting of: LC, mC1, dC, mC, and modified dC.
  • modified dC is selected from the group consisting of: 5-Me-dC, 5-Br-dC and 5-I-dC.
  • 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.
  • [modified mX] is [mG/mI]; and X A and X B are rX.
  • 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.
  • the oligonucleotide of the first aspect further inhibits TLR8 activity, preferably human TLR8 activity.
  • the oligonucleotide that further inhibits TLR8 activity comprises or consists of the sequence mI*mU*mC or mI*mA*dG. [0372] In one embodiment, the oligonucleotide consists of the sequence. [0373] In another embodiment, the oligonucleotide comprises the 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 at the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the oligonucleotide comprises the sequence 5′-mG*mU*X B -3′, wherein X B is dX, preferably X B is dC. Even more preferably, the oligonucleotide comprises the sequence 5′- mG*mU*dC*dC*dC-3′.
  • a method of modifying the TLR7 activity of an oligonucleotide comprising modifying the oligonucleotide by adding the sequence to the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the method 1004921453 reduces the TLR7 potentiating activity of the oligonucleotide. In another embodiment, the method increases the TLR7 inhibitory activity of the oligonucleotide.
  • TLR8 inhibitory oligonucleotides [0375]
  • an oligonucleotide comprising or consisting of a sequence consisting of: X C * y X D * z X E wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X C is selected from the group consisting of: mX, modified mX, dG, and morpholino-X;
  • 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
  • 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;
  • 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.
  • an oligonucleotide comprising or consisting of a sequence consisting of: X C * y X D * z X E wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X C is selected from the group consisting of: mX, modified mX, and dG;
  • X D and X E are 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; 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
  • Any oligonucleotide of the second aspect inhibits TLR8 activity, preferably human TLR8 activity.
  • an oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activtiy.
  • an oligonucleotide of the second aspect does not substantially inhibit TLR7 activity, preferably human TLR7 activtiy.
  • Each internucleotide linkage may be selected from the group consisting of: 3′- 5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, and 5′-2′- linkage.
  • each internucleotide linkage may be selected from: 3′-5′- and 5′-5′- linkage.
  • each internucletodie linkage is a 3′-5′- linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-X C * y X D * z X E -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage; 1004921453 X C is selected from the group consisting of: mX, modified mX, dG, and morpholino-X; 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 morph
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-X C * y X D * z X E -3′ 1004921453 wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X C is selected from the group consisting of: mX, modified mX, and dG;
  • X D and X E are 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; 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/
  • each internucleotide linkage is a 3′-5′ linkage.
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, phosphodiester, phosphoramidate and phosphorodiamidate.
  • Each internucleotide linkage may be the same or different.
  • each internucleotide linkage is 1004921453 independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide linkage is phosphorothioate.
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers. In another embodiment, the oligonucleotide comprises a single stereoisomer.
  • mX is a nucleotide comprising a 2′-OMe modification.
  • moX is a nucleotide comprising a 2′-MOE modification.
  • fX is a nucleotide comprising a 2′-fluor modification.
  • Modified dX, modified rX and modified morpholino-X comprise at least one modification or substitution at positions of the base and/or sugar.
  • Modified mX, modified moX, modified LX and modified fX comprise at least one additional modification or substitution at additional positions of the base and/or sugar.
  • the modification or substitution is selected from the group consisting of: pseudouridine, 3′-deoxy, hydroxyl, des-amino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl and combinations thereof.
  • Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mU3, mC1, and 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 N7-methylated guanosine.
  • mG1 is 2′-OMe-2,6-Diaminopurine
  • mI is 2′-OMe-I (2′-O- methylinosine
  • modified mX is selected from the group consisting of: mG1, mI, mU1, mU2 and mC1.
  • Exemplary modified dX includes but is 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, wherein 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′-deoxy cytidine, pdC is 5-propynyl 1004921453 substituted deoxycyt
  • Exemplary modified rX includes but is not limited to PSU, 2′-NH2-rX, and ara- rX, wherein 2′-NH2-rX is a 2′-amino modified RNA base, and ara-rX is an arabinose modified RNA base.
  • Exemplary 2′-NH2-rX includes but is not limited to 2′-NH2-U and 2′- NH2-C, wherein 2′-NH2-U is 2′-NH2-uridine, and 2′-NH2-C is 2′-NH2-cytidine.
  • Exemplary ara-rX is ara-C (aracytidine).
  • X C is selected from the group consisting of: mG, mU, mC, mI, mG1, and dG.
  • X C is selected from the group consisting of: mX and modified mX.
  • mX is selected from the group consisting of: mG, mC and mU, more preferably mG; and modified mX is mI.
  • X C is selected from the group consisting of: mG and mI.
  • X C is mI.
  • X D is selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX and modified rX.
  • 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.
  • X D is selected from the group consisting of: mX, dX, LX, modified mX, and modified dX.
  • X D is selected from the group consisting of: mX, dX, modified mX, and modified dX.
  • X D is selected from the group consisting of: mA, mU, mC, dA, dT, dG, mU1, mU2, and PSU. More preferably, X D is selected from the group consisting of: mA, mU, dA, dT, and dG.
  • 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.
  • X E is 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.
  • X E is selected from the 1004921453 group consisting of: mX, dX, moX, LX, fX, rX, modified mX and modified dX.
  • X E 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-CH 2 OH-dC, ddC, and pdC.
  • X E 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.
  • X E is selected from the group consisting of: mX, dX, rX and LX.
  • X E is selected from the group consisting of: dA, dC, dG, dT, rG, mC, LA, LC, LG, and LT.
  • X E is selected from the group consisting of: mX and dX.
  • X E is selected from the group consisting of: dC, dG, dT and mC.
  • at least one of X D and X E is LX.
  • X D and X E are independently LX.
  • one of X D and X E is LX.
  • X E is LX.
  • X E is LX and X D is mX.
  • at least one of X D and X E is dX.
  • X D and X E are independently dX.
  • one of X D and X E is dX.
  • X E is dX.
  • X E is dX and X D is mX.
  • at least one of X D and X E is rX.
  • one of X D and X E is rX.
  • X D and X E are each independently rX.
  • X D is selected from rA and rG
  • X E is selected from rA, rG and rC.
  • 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.
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the functionalised sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocycyl, arylalkyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the hydrophobic lipid is selected from cholesterol and tocopherol.
  • the compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol. [0401] In one embodiment, the compound is conjugated directly to the sequence.
  • the compound is conjugated to the sequence via a linker.
  • the 1004921453 linker may be cleavable or non-cleavable.
  • the linker is a non-cleavable linker.
  • the compound is conjugated to a terminal nucleotide of the sequence, preferably the terminal 3′- nucleotide.
  • the compound is conjugated to the terminal 3′-nucleotide at the 3′- position.
  • Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG, dX-Chol and dX-Toco, wherein dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group, dX-Chol is a DNA base with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol- glyceryl group covalently linked to the 3′-position via a monophosphate group, dX-Toco is a DNA base with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group
  • dX-Chol is a DNA base with an (N-choleste
  • the oligonucleotide of the second aspect is selected from the group of oligonucleotides in Table 2.
  • 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*dA*dA, mC*dT*dA, mU*dG*dT, mC*dT*dC, mC*dA*dT, mU*dG*
  • 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. Even more preferably, the sequence is selected from the group consisting of: mI*mA*dG and mI*mU*mC. 1004921453 [0407] In another preferred embodiment, the oligonucleotide of the second aspect further inhibits TLR7 activity, preferably human TLR7 activity.
  • the oligonucleotide that further inhibits TLR7 activity comprises or consists of the sequence of mI*mU*mC or mI*mA*dG. [0408] In one embodiment, the oligonucleotide consists of the sequence. [0409] In another embodiment, the oligonucleotide comprises the 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 at the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • a method of modifying the TLR8 activity of an oligonucleotide comprising modifying the oligonucleotide by adding the sequence to the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the method reduces the TLR8 potentiating activity of the oligonucleotide.
  • the method increases the TLR8 inhibitory activity of the oligonucleotide.
  • an oligonucleotide comprising or consisting of a sequence consisting of: [mX/modified mX]* y X F * z X G wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, LX, rX, modified dX or modified LX; wherein mX is a nucleotide comprising a 2′-OMe and/or 3′-OMe modification, dX is a DNA base, LX is a LNA modified base; when mX is mC, X F is dG, X G is not mG or
  • an oligonucleotide comprising or consisting of a sequence consisting of: mX* y X F * z X G wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, LX, modified dX or modified LX; wherein mX is a nucleotide comprising a 2′-OMe and/or 3′-OMe modification, dX is a DNA base, LX is a LNA modified base; when mX is mC, X F is dG, X G is not mG or dG; or when mX is mG and when: X F is mU, mC or
  • Any oligonucleotide of the third aspect potentiates TLR8 activity, preferably human TLR8 activity.
  • an oligonucleotide of the third aspect does not substantially inhibit TLR7 activity, preferably human TLR7 activity.
  • an oligonucleotide of the third aspect inhibits TLR7 activity, preferably human TLR7 activity.
  • Each internucleotide linkage may be selected from the group consisting of: 3′- 5′-, 5′-5′-, 5′-3′-, 3′-3′-, 3′-2′-, 2′-3′-, 2′-2′-, 2′-5′-, and 5′-2′- linkage.
  • each internucleotide linkage may be selected from: 3′-5′- and 5′-5′- linkage.
  • each internucletodie linkage is a 3′-5′- linkage.
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-[mX/modified mX]* y X F * z X G -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, rX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, rX, LX, modified dX or modified LX; wherein mX is a nucleotide comprising a 2′-OMe and/or 3
  • an oligonucleotide comprising or consisting of a sequence consisting of: 5′-mX* y X F * z X G -3′ wherein: * y and * z each independently represent an inter-nucleotide linkage;
  • X F and X G are each independently selected from the group consisting of: mX, dX, LX, modified mX, modified dX and modified LX; wherein at least one of X F and X G is dX, LX, modified dX or modified LX; wherein mX is a nucleotide comprising a 2′-OMe and/or 3′-OMe modification, dX is a DNA base, LX is a LNA modified base; when mX is mC, X F is dG, X G is not mG or dG; or when mX is mG and when: X F is
  • each internucleotide linkage is a 3′-5′ linkage.
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester.
  • Each internucleotide linkage may be the same or different.
  • each internucleotide linkage is independently selected from phosphorothioate and phosphodiester.
  • each internucleotide linkage is phosphorothioate. 1004921453 [0419]
  • each internucleotide linkage is a 3′-5′- phosphorothioate linkage.
  • the oligonucleotide comprises a mixture of different oligonucleotide stereoisomers, preferably a mixture of different oligonucleotide phosphorothioate stereoisomers.
  • the oligonucleotide of the third aspect comprises a single phosphorothioate stereoisomer, preferably wherein * z is in the R configuration.
  • mX is a nucleotide comprising a 2′-OMe modification.
  • Modified dX and modified rX comprise at least one modification or substitution at positions of the base and/or sugar.
  • Modified mX, modified moX, modified LX and modified fX comprise at least one additional modification or substitution at additional positions of the base and/or sugar.
  • the modification or substitution is selected from the group consisting of: pseudouridine, 3′-deoxy, hydroxyl, des-amino, amino, thio, halo, oxo, aza, deaza, polyethylene glycol, alkyl, alkenyl, alkynyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl and combinations thereof.
  • Exemplary modified mX includes but is not limited to: mG1, mI, mU1, mU2, mC1, m7 G, and N1-Me-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), 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).
  • modified mX is selected from the group consisting of: mG1, mI, mU1, mU2 and mC1.
  • Exemplary modified dX includes but is not limited to: 5-Me-dC, 5-Br-dC, 5- CH2OH-dC, ddC, pdC, and PSU, wherein 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′-deoxy cytidine, pdC is 5- propynyl substituted deoxycytidine, and PSU is pseudo uridine.
  • X F and X G are each independently selected from the group consisting of: mX, dX, and LX; wherein at least one of X F and X G is dX or LX. 1004921453 [0426]
  • mX is selected from the group consisting of: mG, mC, and mU.
  • mX is mG.
  • mX is mC.
  • mX is mU.
  • X F is selected from the group consisting of: mX and dX.
  • X F is selected from the group consisting of: dC, dG, dA, dT, mG, mC, and mU.
  • X F is selected from the group consisting of: dC, dG and mG.
  • X F is dX, preferably dC.
  • X G is selected from the group consisting of: dX and LX.
  • 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.
  • At least one of X F and X G is dX. In one embodiment, one of X F and X G is dX. In a preferred embodiment, X F and X G are independently dX. [0430] 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. [0431] In one embodiment, the sequence may be functionalised.
  • the functionalised sequence comprises a compound selected from the group consisting of: polyethylene glycol, alkyl, alkenyl, alkynyl, heterocycyl, arylalkyl, branched alkyl, branched alkyenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocycyl, substituted arylalkyl, and hydrophobic lipid.
  • the hydrophobic lipid is selected from cholesterol and tocopherol.
  • the compound is selected from the group consisting of: polyethylene glycol, cholesterol and tocopherol. [0432] In one embodiment, the compound is conjugated directly to the sequence.
  • the compound is conjugated to the sequence via a linker.
  • the linker may be cleavable or non-cleavable.
  • the linker is a non-cleavable linker.
  • the compound is conjugated to a terminal nucleotide of the sequence, preferably the terminal 3′- nucleotide.
  • the compound is conjugated to the terminal 3′-nucleotide at the 3′- position.
  • Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG, dX-Chol and dX-Toco, wherein dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group, dX-Chol is a DNA base with an (N-cholesteryl-3-aminopropyl)-triethyleneglycol- glyceryl group covalently linked to the 3′-position via a monophosphate group, dX-Toco is a DNA base with a [(9-DL- ⁇ -tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3′-position via a monophosphate group.
  • dX-TEG is a DNA base with triethylene glycol covalently linked to the 3′-position via a monophosphate group
  • dX-Chol is a DNA base with an (N-choleste
  • the oligonucleotide is selected from the group of oligonucleotides in Table 3.
  • “m” indicates 2′- OMe base, * denotes the phosphorothioate backbone, “d” indicates DNA base.
  • 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.
  • 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.
  • the oligonucleotide is mG*dC*dC.
  • the oligonucleotide consists of the sequence.
  • the oligonucleotide comprises the sequence.
  • 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.
  • the sequence is at the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • 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′.
  • a method of modifying the TLR8 activity of an oligonucleotide comprising modifying the 1004921453 oligonucleotide by adding the sequence to the terminal 5′- and/or 3′- end of the oligonucleotide, preferably the terminal 5′- end.
  • the method increases the TLR8 potentiating activity of the oligonucleotide.
  • the method reduces the TLR8 inhibitory activity of the oligonucleotide.
  • a fusion oligonucleotide comprising: A-[Y-A]n wherein each A independently represents an oligonucleotide described herein, each A may be the same or different; Y represents a cleavable linker, each Y may be the same or different; and n is equal to or greater than 1.
  • each A independently represents an oligonucleotide according to the first aspect.
  • each A independently represents an oligonucleotide according to the second aspect.
  • each A independently represents an oligonucleotide according to the third aspect.
  • At least one A is an oligonucleotide according to the first aspect and at least one further A is an oligonucleotide according to the second aspect.
  • the cleavable linker may be attached to the 5′ and/or 3′ end of the oligonucleotides, 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′.
  • the fusion oligonucleotide comprises 5′-A-3′-Y- 3′-A-5′.
  • Y is cleavable by an enzyme.
  • Y is selected from the group consisting of: a TEG linker, carbon spacers (such as C3, C6, C9, C12), glycerol and PolydT. More preferably Y is a TEG linker.
  • TEG linker refers to Triethylene Glycol linker. 1004921453
  • each A is independently bound to Y by an internucleotide linkage (*).
  • each internucleotide linkage is independently selected from the group consisting of: biphosphate, triphosphate, phosphorothioate, and phosphodiester. Each internucleotide linkage may be the same or different.
  • each internucleotide linkage is independently selected from phosphorothioate and phosphodiester. Most preferably, each internucleotide linkage is phosphorothioate.
  • the fusion oligonucleotide comprises or consists of the sequence 5′-mG*mU*dC-3′-*TEG*-3′-dC*mU*mG-5′.
  • a modified oligonucleotide comprising an agent linked to an oligonucleotide or fusion oligonucleotide described herein by a linker.
  • the agent may be a therapeutic and/or diagnostic agent.
  • Suitable therapeutic agents include but are not limited to: monoclonal antibodies, oligonucleotides, small molecules, cholesterol, radiotherapeutics.
  • Suitable diagnostic agents include but are not limited to: radiolabels, dyes.
  • the agent is a therapeutic agent, more preferably a therapeutic RNA.
  • the therapeutic RNA may be a synthetic oligonucleotide sequence or a naturally occurring oligonucleotide sequence.
  • a synthetic oligonucleotide sequence refers to an oligonucleotide sequence which lacks a corresponding sequence that occurs naturally.
  • the therapeutic RNA may be synthesized in vitro.
  • RNAs include but are not limited to: DNA, RNA, mRNA, siRNA, RNA aptamers, antisense oligonucleotides, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the linker may be cleavable or non-cleavable.
  • the linker is a cleavable linker.
  • Testing for inhibition of TLR7 activity [0449] Some embodiments of the methods of the present invention involve testing for inhibition of TLR7 activity which can be determined using any method known in the art.
  • TLR7 activity in cells may be measured by expression and/or secretion of one or more pro-inflammatory cytokines (e.g. TNF ⁇ ), and/or activation or expression of transcription factors (e.g. NF- ⁇ B).
  • pro-inflammatory cytokines e.g. TNF ⁇
  • transcription factors e.g. NF- ⁇ B
  • an oligonucleotide to inhibit TLR7 activity can, for example, be analysed by incubating cells which express TLR7 with an oligonucleotide, then stimulating said cells with a TLR7 agonist (e.g., R848, guanosine or an immunostimulatory ssRNA such as B-406-AS-1), and analysing the overall TLR7 response in the cell population, or analysing the proportion of cells having TLR7-positive activity after a defined period of time.
  • a TLR7 agonist e.g., R848, guanosine or an immunostimulatory ssRNA such as B-406-AS-1
  • inhibition of TLR7 activity can be identified by observation of an overall decreased TLR7 response of the cell population, or a lower proportion of cells having TLR7-positive activity as compared to positive control condition in which cells are treated with TLR7 agonist in the absence of the oligonucleotide (or in the presence of an appropriate control inhibitory agent).
  • 293XLhTLR7 referred to as HEK-TLR7 cells are transfected with pNF- ⁇ B-Luc4 reporter, incubated with an oligonucleotide, and then stimulated with R848.
  • TLR7 activity can be determined by a luciferase assay, which measures activated NF- ⁇ B by luminescence.
  • TLR7 activity can also be analysed by measuring cytokine levels, for example by ELISA.
  • Testing for inhibition of TLR8 activity [0452] Some embodiments of the methods of the present invention involve testing for inhibition of TLR8 activity which can be determined using any method known in the art.
  • TLR8 activity in cells may be measured by expression and/or secretion of one or more pro-inflammatory cytokines (e.g. IL-6, TNF ⁇ , IP-10), and/or activation or expression of transcription factors (e.g. NF- ⁇ B).
  • pro-inflammatory cytokines e.g. IL-6, TNF ⁇ , IP-10
  • transcription factors e.g. NF- ⁇ B
  • an oligonucleotide to inhibit TLR8 activity can, for example, be analysed by incubating cells which express TLR8 with an oligonucleotide, then stimulating said cells with a TLR8 agonist, and analysing the overall TLR8 response in the cell population, or analysing the proportion of cells having TLR8-positive activity after a defined period of time.
  • inhibition of TLR8 activity can be identified by observation of an overall decreased TLR8 response of the cell population, or a lower proportion of cells having TLR8-positive activity as compared to a positive control condition in which cells are treated with TLR8 agonist in the absence of the oligonucleotide (or in the 1004921453 presence of an appropriate control inhibitory agent).
  • 293XLhTLR8 referred to as HEK-TLR8 cells are transfected with pNF- ⁇ B-Luc4 reporter, incubated with an oligonucleotide, and then stimulated with R848.
  • TLR8 activity can be determined by a luciferase assay, which measures activated NF- ⁇ B by luminescence. TLR8 activity can also be analysed by measuring cytokine levels, for example by ELISA. Testing for potentiating TLR8 activity [0455] Some embodiments of the methods of the present invention involve testing for potentiation of TLR8 activity which can be determined using any method known in the art. In some embodiments TLR8 activity in cells may be measured by expression and/or secretion of one or more pro-inflammatory cytokines (e.g. IL-6, TNF ⁇ , IP-10), and/or activation or expression of transcription factors (e.g. NF- ⁇ B).
  • pro-inflammatory cytokines e.g. IL-6, TNF ⁇ , IP-10
  • transcription factors e.g. NF- ⁇ B
  • the ability of an oligonucleotide to potentiate TLR8 activity can, for example, be analysed by incubating cells which express TLR8 with an oligonucleotide, then stimulating said cells with a TLR8 agonist, and analysing the overall TLR8 response in the cell population, or analysing the proportion of cells having TLR8-positive activity after a defined period of time.
  • potentiation of TLR8 activity can be identified by observation of an overall increased TLR8 response of the cell population, or a higher proportion of cells having TLR8-positive activity as compared to a negative control condition in which cells are treated with TLR8 agonist in the absence of the oligonucleotide (or in the presence of an appropriate control non-potentiating agent).
  • 293XLhTLR8 referred to as HEK-TLR8 cells are transfected with pNF- ⁇ B-Luc4 reporter, incubated with an oligonucleotide, and then stimulated with R848.
  • TLR8 activity can be determined by a luciferase assay, which measures activated NF- ⁇ B by luminescence. TLR8 activity can also be analysed by measuring cytokine levels, for example by ELISA.
  • Oligonucleotides of the invention are designed to be administered to an animal.
  • the oligonucleotide can be administered in combination with another molecule, such as a further nucleic acid (e.g., a mRNA molecule, a short 1004921453 interfering RNA, an antisense oligonucleotide, a CRISPR guide RNA, etc), a peptide, a carrier agent, a therapeutic agent, and the like.
  • a further nucleic acid e.g., a mRNA molecule, a short 1004921453 interfering RNA, an antisense oligonucleotide, a CRISPR guide RNA, etc
  • a peptide e.g., a
  • the oligonucleotide can be conjugated with the other molecule.
  • the oligonucleotide is used to modify a trait of an animal, more typically to treat or prevent a disease or condition.
  • the disease or condition will benefit from the animal not being able to mount a TLR7 and/or TLR8 response following administration of the oligonucleotide.
  • the disease or condition will benefit from the animal being able to mount an increased TLR8 response following administration of the oligonucleotide.
  • a method of inhibiting TLR7 activity in a cell comprising contacting the cell with an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, thereby inhibiting TLR7 activity in the cell.
  • a method of inhibiting TLR7 activity in a subject comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, thereby inhibiting TLR7 activity in the subject.
  • a method of treating or preventing a disease, disorder or condition in a subject is responsive to TLR7 inhibition, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the first aspect, thereby treating or preventing the disease, disorder or condition in the subject.
  • a method of inhibiting TLR8 activity in a cell the method comprising contacting the cell with an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect, thereby inhibiting TLR8 activity in the cell.
  • a method of inhibiting TLR8 activity in a subject comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect, thereby inhibiting TLR8 activity in the subject.
  • a method of treating or preventing a disease, disorder or condition in a subject responsive to TLR8 inhibition comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the second aspect, thereby treating or preventing the disease, disorder or condition in the subject.
  • Diseases, disorders and conditions responsive to TLR7 and/or TLR8 inhibition include immune inflammation-related diseases, allergic diseases, infections, cancers and auto-immune diseases relying on auto-antibodies.
  • Examples of the immune inflammation-related diseases can include diseases of the connective tissue and the musculoskeletal system (such as systemic lupus erythematosus, cutaneous and subcutaneous lupus, rheumatoid arthritis, juvenile idiopathic arthritis, adult-onset Still's disease, ankylosing spondylitis, systemic scleroderma, polymyositis, dermatomyositis, psoriatic arthritis, fibromyalgia, osteoarthritis, mixed connective tissue disease, Guillain-Barre syndrome, and muscular dystrophy), the blood system (such as autoimmune hemolytic anemia, aplastic anemia, and idiopathic thrombocytopenic purpura), the digestive tract system (such as Crohn's disease, ulcerative colitis, and ileitis), the hepatobiliary pancreatic system and the endocrine system (such as autoimmune hepatitis, viral hepatitis, alcoholic
  • the immune inflammation-related disease is selected from: systemic lupus erythematosus, cutaneous lupus, and psoriasis.
  • the allergic diseases can include atopic dermatitis, hay fever, asthma, anaphylaxis, anaphylactoid reactions, food allergy, rhinitis, otitis media, drug reactions, insect sting reactions, plant reactions, latex allergy, conjunctivitis, and urticaria.
  • infections can include diseases caused by infections by viruses (such as a single-stranded RNA virus, a double-stranded RNA virus, a single- stranded DNA virus, and a double-stranded DNA virus), bacteria (such as gram- negative bacteria, gram-positive bacteria, acid-fast bacteria, actinomycetes, spirochetes, spiral bacteria, 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, Ascaridida, and Oxyuridae).
  • viruses such as a single-stranded RNA virus, a double-stranded RNA virus, a single- strand
  • Examples of the cancer treatment can include treatments for blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, skin cancers (including melanoma), leukemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SGLG), non-small cell lung cancer (NSGLG), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including blastom
  • the present invention also provides a method of reducing or minimising a symptom 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 aches, fatigue.
  • the condition responsive to TLR7 and/or TLR8 inhibition is mRNA administration.
  • a method of potentiating TLR8 activity in a cell comprising contacting the cell with an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect, thereby potentiating TLR8 activity in the subject.
  • a method of potentiating TLR8 activity in a subject comprising administering to the subject a therapeutically effective amount of an oligonucleotide, a fusion oligonucleotide, or a composition according to the third aspect, thereby potentiating TLR8 activity in the subject.
  • a method of treating or preventing a disease, disorder or condition in a subject responsive to increased TLR8 signalling 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.
  • Diseases, disorders and conditions associated with decreased TLR8 signalling include cancer, viral and bacterial infections.
  • cancers include blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, skin cancers (including basal cell carcinoma, melanoma), leukemia or lymphoid malignancies, lung cancer including small-cell lung cancer (SGLG), non-small cell lung cancer (NSGLG), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer 1004921453 including gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, cervical cancer, liver cancer, bladder cancer, he
  • the oligonucleotides of the first and second aspect may be used in methods of preventing or inhibiting inflammation associated with administration of a therapeutic RNA, such as those known in the art, to a subject.
  • the oligonucleotides described herein may be used in the prevention or inhibition of inflammation mediated by one or more nucleic acid sensors (e.g., TLR7, TLR8) during or following administration of the therapeutic RNA.
  • the inflammation may involve or include any cells, tissues or organs of the body.
  • the inflammation is or comprises hepatic inflammation.
  • the therapeutic RNA may be conjugated to N-acetylgalactosamine (GalNAc), which enhances asialoglycoprotein receptor (ASGR)-mediated uptake into liver hepatocytes (Nair et al., 2014), and thereby enabling their specific targeting to the liver.
  • GalNAc N-acetylgalactosamine
  • ASGR asialoglycoprotein receptor
  • the oligonucleotides of the first aspect may be utilised to prevent or inhibit a TLR7-dependent inflammatory response associated with the administration of a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in vitro or in vivo.
  • a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in vitro or in vivo.
  • the therapeutic RNA may be part of RNA-based therapeutic agent, such as an mRNA vaccine.
  • the oligonucleotide can at least partly inhibit the engagement or sensing of these therapeutic RNA molecules by TLR7.
  • the oligonucleotides of the first aspect may therefore minimise the need for the use of modified bases, such as pseudo-uridines, and/or other modifications that reduce the immunogenicity of mRNA molecules for their inclusion in mRNA vaccine compositions.
  • the oligonucleotides of the second aspect and more particularly those described herein that exhibit TLR8-inhibitory activity, may be utilised to prevent or inhibit a TLR8-dependent inflammatory response associated with the administration of a therapeutic RNA selected from the group consisting of: RNA, mRNA, 1004921453 siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof in vitro or in vivo.
  • the therapeutic RNA may be part of RNA-based therapeutic agent, such as an mRNA vaccine.
  • the oligonucleotide can at least partly inhibit the engagement or sensing of these therapeutic RNA molecules by TLR8.
  • the oligonucleotides of the second aspect may therefore minimise the need for the use of modified bases, such as pseudo-uridines, and/or other modifications that reduce the immunogenicity of mRNA molecules for their inclusion in mRNA vaccine compositions.
  • the oligonucleotides of the first and second aspects may be a component or included within an immunogenic composition, such as an RNA or mRNA vaccine composition, as are known in the art.
  • RNA vaccine refers to vaccines comprising RNA that encodes one or more nucleotide sequences encoding antigens capable of inducing an immune response in a mammal.
  • mRNA vaccines are described, for example, in International Patent Application Nos. PCT/US2015/027400 and PCT/US2016/044918, herein incorporated by reference in their entirety.
  • the present invention provides an immunogenic composition, such as a vaccine composition, comprising a therapeutic RNA and an oligonucleotide or fusion oligonucleotide provided herein.
  • the therapeutic RNA is selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self-amplifying RNAs, circular RNAs and combinations thereof.
  • the modified oligonucleotide comprises a therapeutic RNA selected from the group consisting of: RNA, mRNA, siRNA, RNA aptamers, single guide RNA, self- amplifying RNAs, circular RNAs and combinations thereof.
  • 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.
  • the oligonucleotide of the immunogenic composition exhibits TLR8 inhibitory activity. In some embodiments, the oligonucleotide of the immunogenic composition exhibits TLR7 and TLR8 inhibitory activity.
  • the immunogenic composition is suitably for use in a method of: (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.
  • the mRNA vaccine When the mRNA vaccine is delivered 1004921453 to a cell, the mRNA will be processed into a polypeptide or peptide by the intracellular machinery which can then process the polypeptide or peptide into immunogenic fragments capable of stimulating an immune response.
  • the oligonucleotide may be included as a separate or discrete component and/or conjugated with a therapeutic RNA of the vaccine composition.
  • the therapeutic RNA is selected from RNA or mRNA.
  • the therapeutic RNA of the RNA vaccine may be unmodified or substantially unmodified (e.g., does not include any modified bases).
  • the therapeutic RNA may contain one or more modifications that typically enhance stability, such as modified nucleotides, modified sugar phosphate backbones, and 5′ and/or 3′ untranslated regions (UTR).
  • the therapeutic RNA may be included or incorporated within a delivery, transfer or carrier system of the immunogenic composition, as are known in the art.
  • the therapeutic RNA of the immunogenic composition may be encapsulated or complexed in nanoparticles, and more particularly lipid nanoparticles.
  • suitable nanoparticles include, but are not limited to polymer based carriers, such as polyethylenimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocry stalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi- domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic poly conjugates) and dry powder formulations.
  • PEI polyethylenimine
  • lipid nanoparticles and liposomes such as polyethylenimine (PEI),
  • the oligonucleotide is included in the immunogenic composition separate from the carrier system. In other embodiments, the oligonucleotide is included or incorporated within the carrier system of the immunogenic composition, such as incorporated into a lipid nanoparticle together with the therapeutic RNA of the RNA vaccine. [0485] In some embodiment, the oligonucleotide may be applied to the surface of an implantable biomaterial, such as a prosthetic. 1004921453 [0486] In particular examples, therapeutically effective amounts of the therapeutic RNA and the oligonucleotide of the invention may be administered simultaneously, concurrently, sequentially, successively, alternately or separately in any particular combination and/or order.
  • Oligonucleotides of the disclosure may be admixed, encapsulated, conjugated (such as fused) or otherwise associated with other molecules, molecule structures or mixtures of compounds, resulting in, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Oligonucleotides of the disclosure may be administered in a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be solid or liquid.
  • compositions include, but are not limited to, diluents, solvents, surfactants, excipients, suspending agents, buffering agents, lubricating agents, adjuvants, vehicles, emulsifiers, absorbants, dispersion media, coatings, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetration agents, sequestering agents, isotonic and absorption delaying agents that do not affect the activity of the active agents of the disclosure.
  • the pharmaceutical carrier is water for injection (WFI) and the pharmaceutical composition is adjusted to pH 7.4, 7.2-7.6.
  • the salt is a sodium or potassium salt.
  • the oligonucleotides may contain chiral (asymmetric) centres or the molecule as a whole may be chiral. Preferably, the oligonucleotides contain chiral centres at the phosphorothioate linkages. The individual stereoisomers and mixtures of these are within the scope of the present disclosure.
  • Oligonucleotides of the disclosure may be pharmaceutically acceptable salts, esters, or salts of the esters, or any other compounds which, upon administration are capable of providing (directly or indirectly) the biologically active metabolite.
  • oligonucleotide refers to physiologically and pharmaceutically acceptable salts of the oligonucleotide that retain the desired biological activities of the parent compounds and do not impart undesired toxicological 1004921453 effects upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860.
  • Oligonucleotides of the disclosure may be prodrugs or pharmaceutically acceptable salts of the prodrugs, or other bioequivalents.
  • prodrugs refers to therapeutic agents that are prepared in an inactive form that is converted to an active form (i.e., drug) upon administration by the action of endogenous enzymes or other chemicals and/or conditions.
  • prodrug forms of the oligonucleotide of the 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.
  • a prodrug may, for example, be converted within the body, e. g. by hydrolysis in the blood, into its active form that has medical effects.
  • Pharmaceutical 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” ed. H. Bundgaard, Elsevier, 1985; and in Edward B.
  • oligonucleotides of the invention can be complexed with a complexing agent to increase cellular uptake of oligonucleotides.
  • a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells.
  • cationic lipid includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells.
  • cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof.
  • Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms.
  • Preferred straight chain or branched alkyl or alkene groups have six or more carbon atoms.
  • Alicyclic groups include 1004921453 cholesterol and other steroid groups.
  • Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
  • counterions e.g., Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
  • cationic lipids examples include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE TM (e.g., LIPOFECTAMINE TM 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).
  • PAMAM polyamidoamine
  • DOPE Lipofectase
  • LIPOFECTAMINE TM e.g., LIPOFECTAMINE TM 2000
  • DOPE Cytofectin
  • Cytofectin Gilead Sciences, Foster City, Calif.
  • Eufectins JBL, San Luis Obispo, Calif.
  • Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N- trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N- trimethylammonium methylsulfate (DOTAP), 3.beta.-[N-(N′,N′- dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB).
  • DOTMA N-[1
  • Oligonucleotides can also be complexed with, e.g., poly (L-lysine) or avidin and lipids may, or may not, be included in this mixture, e.g., steryl-poly (L-lysine).
  • Cationic lipids have been used in the art to deliver oligonucleotides (as well as mRNA vaccines) to cells.
  • Other lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the methods of the invention.
  • lipid compositions are also known in the art and include, e.g., those taught in US 4,235,871; US 4,501,728; 4,837,028; 4,737,323.
  • lipid compositions can further comprise agents, e.g., viral proteins to enhance lipid-mediated transfections of oligonucleotides.
  • agents e.g., viral proteins to enhance lipid-mediated transfections of oligonucleotides.
  • N-substituted glycine oligonucleotides peptoids
  • a composition for delivering oligonucleotides of the invention comprises a peptide having from between about one to about four basic residues.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic 1004921453 acid
  • uncharged polar side chains e.g., glycine (can also be considered non-polar
  • asparagine, glutamine, serine, threonine, tyrosine, cysteine nonpolar side chains
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine,
  • oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide”.
  • the composition includes an oligonucleotide which is complementary to a target nucleic acid molecule encoding the protein, and a covalently attached transporting peptide.
  • the oligonucleotide is attached to a targeting moiety such as N-acetylgalactosamine (GalNAc), an antibody, antibody-like molecule or aptamer (see, for example, Toloue and Ford (2011) and Esposito et al. (2016)).
  • GalNAc N-acetylgalactosamine
  • the oligonucleotide of the disclosure is administered systemically. In another embodiment, the oligonucleotide of the disclosure is administered topically.
  • systemic administration is a route of administration that is either enteral or parenteral.
  • enteral refers to a form of administration that involves any part of the gastrointestinal tract and includes oral administration of, for example, the oligonucleotide in tablet, capsule or drop form; gastric feeding tube, duodenal feeding tube, or gastrostomy; and rectal administration of, for example, the oligonucleotide in suppository or enema form.
  • parenteral includes administration by injection or infusion.
  • Examples include, intravenous (into a vein), intra-arterial (into an artery), intramuscular (into a muscle), intra-cardiac (into the heart), subcutaneous (under the skin), 1004921453 intraosseous infusion (into the bone marrow), intradermal, (into the skin itself), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intra-vesical (infusion into the urinary bladder). transdermal (diffusion through the intact skin), transmucosal (diffusion through a mucous membrane), inhalational. [0506] In one embodiment, administration of the pharmaceutical composition is subcutaneous.
  • administration of the pharmaceutical composition is intravenously or topically, preferably intravenously.
  • the oligonucleotide may be administered as single dose or as repeated doses on a period basis, for example, daily, once every two days, three, four, five, six seven, eight, nine, ten, eleven, twelve, thirteen or fourteen days, once weekly, twice weekly, three times weekly, every two weeks, every three weeks, every month, every two months, every three months to six months or every 12 months.
  • administration is 1 to 3 times per week, or once every week, two weeks, three weeks, four weeks, or once every two months.
  • administration is once weekly.
  • a low dose administered for 3 to 6 months such as about 25-50mg/week for at least three to six months and then up to 12 months and chronically.
  • Illustrative doses are between about 10 to 5,000mg.
  • Illustrative doses include 25, 50, 100, 150, 200, 1,000, 2,000mg.
  • Illustrative doses include 1.5 mg/kg (about 50 to 100mg) and 3 mg/kg (100-200mg), 4.5 mg/kg (150-300mg), 10 mg/kg, 20 mg/kg or 30mg/kg.
  • doses are administered once per week.
  • a low dose of approximately 10 to 30, or 20 to 40, or 20 to 28 mg may be administered to subjects typically weighing between about 25 and 65kg.
  • the oligonucleotide is administered at a dose of less than 50 mg, or less than 30 mg, or about 25 mg per dose to produce a therapeutic effect.
  • Example 1 Materials and Methods Cell Culture and Stimulation
  • 293XL-hTLR7-HA, 293XL-hTLR8-HA stably expressing human TLR7 or TLR8 were purchased from Invivogen, and were maintained in Dulbecco’s modified Eagle’s medium plus L-glutamine supplemented with 1 ⁇ antibiotic/antimycotic (Thermo Fisher Scientific) and 10% heat-inactivated foetal bovine serum (referred to as complete DMEM), with 10 ⁇ g/ml Blasticidin (Invivogen).
  • Dulbecco’s modified Eagle’s medium plus L-glutamine supplemented with 1 ⁇ antibiotic/antimycotic (Thermo Fisher Scientific) and 10% heat-inactivated foetal bovine serum (referred to as complete DMEM), with 10 ⁇ g/ml Blasticidin (Invivogen).
  • THP-1 cells Human acute myeloid leukemia THP-1 cells were grown in RPMI 1640 plus L-glutamine medium (Life Technologies) complemented with 1x antibiotic/antimycotic and 10% heat inactivated foetal bovine serum (referred to as complete RPMI). THP-1 cells were not differentiated with PMA in any experiments unless otherwise noted, and rather used in suspension. RAW264.7- ELAM macrophages , TLR7-deficient RAW264.7 cells and immortalized wild-type BMDMs (Ferrand et al., Frontiers in Cellular and Infection Microbiology, 2018; 8: 87) , along with TLR7/8 double-deficient immortalized BMDMs were grown in complete DMEM.
  • the oligonucleotides were HPLC-purified and confirmed to be endotoxin free by Lonza PYROGENT Ultra 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 inter-nucleotide linkages are denoted with a *.
  • HEK293 cells stably expressing TLR8 or TLR7 were reverse-transfected with pNF- ⁇ B-Luc4 reporter (Clontech), with Lipofectamine 2000 (Thermo Fisher Scientific), 1004921453 according to the manufacturer’s protocol. Briefly, 500,000-700,000 cells were reverse- transfected with 200-400 ng of reporter with 1.2 ⁇ l of Lipofectamine 2000 per well of a 6-well plate, and incubated for 3-24 h at 37 °C with 5% CO 2 . Following transfection, the cells were collected from the 6-wells and aliquoted into 96-wells, just before trimer and overnight TLR stimulation (as above described).
  • RNA Reverse Transcription Quantitative Real-Time PCR [0516] Total RNA was purified from cells using the ISOLATE II RNA Mini Kit (Bioline).
  • Random hexamer cDNA was synthesized from isolated RNA using the High-Capacity cDNA Archive kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
  • RT-qPCR was carried out with the Power SYBR Green Master Mix (Thermo Fisher Scientific) on the QuantStudio 6 RT-PCR system (Thermo Fisher Scientific). Each PCR was carried out in technical duplicate and human 18S was used as reference gene. Each amplicon was gel-purified and used to generate a standard curve for the quantification of gene expression (used in each run). Melting curves were used in each run to confirm specificity of amplification.
  • TLR8 Potentiation of TLR8 in humanised TLR8/TLR7 mice
  • Spleens were harvested from 3 C57/BL6 and 3 humanised C57BL/6- Tlr8 tm1(TLR8) Tlr7 tm1(TLR7) /Bcgen (B-hTLR8/hTLR7) mice, and splenocytes isolated by passing the spleens through a 70 ⁇ m cell strainer.
  • cells 1004921453 were washed and resuspended in RPMI 1640 (Gibco) supplemented with 10 % heat- inactivated foetal bovine serum (FBS) (ExCell) before seeding in technical triplicates at 3 ⁇ 10 5 cells/well in a 96-well flat-bottom polystyrene TC-treated microplate.
  • Cells were pre-treated with 5 ⁇ M of TLR8 potentiating oligo 38-2, or a vehicle control (TE buffer) for 1 h before addition of 1 ⁇ g/mL Resiquimod (R848) (MedChemExpress; HY-13740) overnight.
  • biopsies were inserted into Transwell filters (Corning) in 12-well culture plates, with the epidermis facing upwards at the air- liquid interface and the dermis suspended in 1 mL culture medium, and incubated at 37 ⁇ C + 5 % CO2.
  • the biopsies were then pre-treated with 5 ⁇ M TLR8 potentiating oligo, 38-2, or a vehicle control (TE buffer), for 30 min before addition of 600 nM Motolimod (Cambridge Bioscience; CAY22952) or a vehicle control (DMSO) for 24 h. Media were harvested for measurement of IL-8 levels by flow cytometry beads.
  • LNP particle size, polydispersity index (PDI), zeta potential, and mRNA encapsulation rate were assessed.
  • LC-MS/MS and RT-qPCR were used to quantitate GGC-v1 and mRNA, respectively, in the LNPs.
  • Co-delivery of GGC-v1 with mRNA in vivo Female 8-week-old 129X1/SvJ mice (Jackson Laboratories) ( ⁇ 25 g) were injected intravenously (i.v.) with ⁇ 20 ⁇ g FLuc mRNA (actual 20.349 ⁇ g and 20.484 ⁇ g) encapsulated in LNPs with, or without, GGC-v1 (see details above).
  • Bioluminescence 1004921453 imaging was performed at 6 and 24 h post-injection using an IVIS Spectrum®. Briefly, mice were anaesthetised with 4 % isoflurane, and 3 mg/mouse d-luciferin potassium salt was administered i.v. in order to quantify luminescence expression using images recorded for 3 minutes starting 5 minutes post-luciferin injection. For bioluminescence image analysis, regions of interest encompassing the area of signal were defined using the IVIS Spectrum, and the total number of photons per second [counts/second (cps)] was recorded.
  • Luciferase Cell Culture Lysis Reagent Promega
  • Example 2 Structure activity relationship of GUC inhibition on human TLR7 [0523]
  • the 2′-OMe/DNA-containing C2Mut1-dC oligonucleotide (mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC where mX is a 2′-OMe modified base, dX a DNA base and * a phosphorothioate inter-nucleotide linkage) has been demonstrated to inhibit human TLR7 sensing.
  • C2Mut1-dC To determine the motif exhibiting the inhibitory effects of C2Mut1-dC the inventors investigated mutants of the 5′-end region of C2Mut1-dC, generating 3′-end variants C2Mut1-dC2 and C2Mut1-dC3 which harbor a mG*mG*mU*dA or a mG*mG*mU*dC motif. Both C2Mut1-dC2 and C2Mut1-dC3 inhibited TLR7 sensing.
  • substitution of mG with 2′-OMe-2,6-diaminopurine or 2′- OMe-I, or mU with 2′-OMe-5-Me-U or 2′-OMe-5-Br-U retained strong inhibitory activity at 5 ⁇ M, however, substitution of mG with 2′-OMe-2,6-diaminopurine or mU with 2′- OMe-5-Me-U or 2′-OMe-5-Br-U significantly decreased the inhibitory activity of GUC at 200 nM (while substitution of mG 2′-OMe-I inhibited as well as GUC at this dose).
  • trimers were clearly very potent inhibitors with GUC-v19 being the most potent with an IC50 projected below 100 nM.
  • screen analyses of 2′-OMe trimers suggested the strongest potency for GUX motifs, where X could be mC, mG, mU or mA.
  • mG*mU*dA and mG*mU*dC both retained inhibitory function suggested that mG*mU*dX (where dX is a DNA base) would also be inhibiting TLR7.
  • the inventors next tested whether the di-nucleotide mG*mU could also inhibit TLR7.
  • the inventors compared the inhibitory activity of mG*mU*mA, mG*mG*mU*mA and mG*mG*mU*mA*mU on TLR7 inhibition.
  • the inventors also compared mG*mU*dC to the 5 mer mG*mG*mU*dC*dT, along with the activity of two mG*mU*dC trimers linked in 5′-3′-3′-5′ orientation with a Triethylene glycol (TEG) linker (Figure 10).
  • TAG Triethylene glycol
  • the GUC-v1 trimer was the optimal length for maximum inhibition at lower dose (500 nM).
  • fusion of two GUC-v1 trimers with a TEG linker retained strong inhibitory activity even at 50 nM - concentration at which a single GUC-v1 trimer is not inhibitory. This suggests that the TEG linker is cleaved to release two molecules of GUC-v1 for each molecule of linked oligo, underlying their stronger activity.
  • trimers containing as few as a single 2′-OMe moiety combined with two DNA moieties retained inhibitory activity on TLR7 (as seen with GUC-v4)
  • the inventors next tested all possible 64 combinations of DNA trimers on TLR7 inhibition. Comparison of inhibition at 2 ⁇ M showed that only 3 DNA trimers inhibited TLR7 by more than 20% (TCT, TTT and TTG), versus 26 for 2′-OMe ( Figure 11).
  • the inventors therefore tested a panel of 642′-OMe trimers on mTLR7 sensing in RAW 264.7 cells stably expressing an ELAM-luciferase reporter, which is activated by R848 sensing (Zamanian-Daryoush et al., J Interferon Cytokine Res, 2008; 28(4): 221-33).
  • This screen was carried out at two doses of R848 (0.5ug/ml and 0.125ug/ml), and identified GGC, GAC and GAG as the most potent inhibitors of mouse TLR7 sensing (Figure 13).
  • the inventors also tested the panel of 64 DNA trimers on mouse TLR7 sensing and observed a mild inhibition with 8 trimers inhibiting more than 20% at 5 ⁇ M (the maximum being 27% inhibition with ACG). At this dose however, 162′-OMe trimers inhibited mTLR7 by more than 20%, showing a clear superiority for 2′-OMe trimers (Figure 13). [0537] To define whether, like human TLR7, mouse TLR7 inhibition could be increased upon DNA substitutions of selected bases of the trimers, the inventors next tested a panel of 6 variants for 2′-OMe GGC and GAG ( Figure 14).
  • GGC-v1 was slightly more potent than GGC, and critically GGC-v3 was devoid of any inhibitory activity (Figure 15). 1004921453 [0539] The inventors next tested whether the trimers could also inhibit TLR7 sensing driven by an immunostimulatory ssRNA, referred to as B-406-AS-1 (Sarvestani et al., Nucleic Acids Res, 2015; 43(2): 1177-88). As shown in Figure 16, GGC significantly inhibited ELAM-luc driven by transfection of B-406-AS-1 in RAW-ELAM cells. [0540] Having shown that trimers harbouring 2 DNA bases (e.g.
  • GAG-v4 – Figure 14 were still very potent inhibitors of mouse TLR7, the inventors next assessed the inhibitory effect of the 64 DNA trimers. As shown in Figure 17, DNA trimers were only modestly inhibitory, with only 8 oligos inhibiting more than 20% (with maximum inhibition at 28% at 5 ⁇ M for ACG). There was little overlap of inhibition between the chemistries (trimers inhibiting TLR7 with 2′-OMe were not inhibitory when composed of DNA bases) and ACG was the only trimer displaying >20% inhibition for both DNA and 2′-OMe chemistries. [0541] These observations demonstrate that at least one 2′-OMe base is critical for the activity of the trimers on mouse TLR7.
  • Example 4 Modulation of TLR8 sensing by trimers
  • the inventors screened single doses of various trimers in HEK-TLR8 and THP-1 cells. As shown in Figure 19, there was a significant correlation of potentiation and inhibition in response to the two doses in each cell type (slightly better in HEK-TLR8). In both cell types, 2′-OMe CGG was the strongest potentiator of R848 or Motolimod sensing. UCG was also a top potentiator in both cells.
  • AGG was very potent in HEK-TLR8 but not in THP-1 cells, and UCA/CGC being strong in THP-1 but not in HEK-TLR8.
  • GAX were strong inhibitors of TLR8 in both cell types, with GAG the most potent in THP-1 cells at 1uM.
  • mG*dC*dC was a stronger potentiator of TLR8 sensing than mC*mG*mG at this low dose (500nM).
  • Example 5 Alternative modifications for GUC trimers and inhibition on human TLR7
  • modification of the 2′-position of the sugar for instance with use of 2′-deoxycytidine to replace the 2′-OMe-cytidine in GUC- v1, or modification of the cytosine portion in GUC-v19 ( Figures 5 and 6), could both be used to improve inhibitory activity of GUC trimers.
  • trimers containing GU[X] and GA[X] are the most potent inhibitors.
  • 3 rd base LNA modification increased the potency of inhibition in a sequence specific manner, with 17/32 trimers inhibiting more than 20% at 400 nM, versus 5/32 for full 2′-OMe trimers (GU[X] and UUC). This was particularly evident with the mG*mA*LX trimers which were much stronger inhibitors with the LNA moiety.
  • the majority of the strongest inhibitors contained a 5′-mG (with 14/16 mG*mX*LX trimers inhibiting 20% or more), underlining further the importance of this base in human TLR7 inhibition.
  • the inventors next investigated whether the TEG group alone, appended to GUC-v1 could increase TLR7 inhibition. The inventors also tested the activity of Cholesterol and Tocopherol 3′-end conjugation to GUC-v1 to define whether these groups could be later used for delivery of naked trimers (Figure 27).
  • mUdTdC, mUdTdA, mUdAdA, mUdGdC, mUdTdT, mUdGdT were the most potent inhibitors but only reaching ⁇ 50-75% inhibition which was rather weak at this very high dose.
  • trimers exhibiting inhibitory activity here started with mU and 1004921453 mUdTd[X] was over-represented. None of the mCd[X]d[X] trimers inhibited TLR7 sensing.
  • Example 6 Alternate trimers for inhibiting mouse TLR7
  • mice TLR7 inhibition could be modulated by DNA modification of the 3′-end nucleotide, and 5′ inosine modification (as seen with GUC-v16). Having shown that mG*mA*mG, mG*mA*dG and mG*mA*LG were potent inhibitors of mouse TLR7, the inventors next tested the inhibitory effect of GAG-v7, containing a 5′ inosine modification (the inventors also tested GAG-v8 with an 3 rd LNA base).
  • GUC-v16 was also a potent inhibitor at lower doses
  • the inventors next tested its potency at 500 nM and compared it to GAG-v4 (mGdAdG) and mGmAdT which were also potent TLR8 inhibitors at 500 nM (see Figure 22).
  • GUC-v16 was significantly more inhibitory than GAG-v4 at 500 nM ( Figure 44).
  • the IC50 for GUC-v16 was 190 nM – suggesting that much lower IC50 could be attained in the optimal mGmA(dG/dT) sequence context ( Figure 44).
  • GAG-v7 led to stronger inhibition of TLR8 sensing (Figure 45).
  • GAG-v7 had an IC50 of 260 nM in these experiments – suggesting a similar activity to that of GUC-v16.
  • combination of a 5′-inosine with a 3′-LNA cytosine as in GUC-v30 rather decreased the inhibition of TLR8 (Figure 45A&B).
  • Other inosine variants in GUC-v35-36 were only poorly inhibiting TLR8 (suggesting that these modifications rather reduced the inhibitory activity of inosine in this sequence context).
  • trimers inhibiting TLR8 by >40% at 5 ⁇ M in this screen were exclusively starting with 2′-OMe uridine with preferential mUdA(dT/C/G) or mUdT(dG/A/C) sequences.
  • mC*dC*dC was the strongest potentiator of TLR8 sensing, with a clear enrichment for mC*dC*d[X] variants among the top potentiators (which also included mU*dC*dA, mU*dC*dC).
  • mG*dC*dC and mG*dC*dT were significantly potentiating uridine sensing in HEK TLR8 cells.
  • This inventors next tested whether mG*dC*dC could be used to sensitize TLR8 to sense otherwise non-immunostimulatory cellular RNA. To test this, total mouse RNA was transfected into HEK TLR8 cells in the presence or absence of the mG*dC*dC trimer. [0600] Analysis of HEK TLR8 cells transfected with high dose of total RNA only marginally activated TLR8-driven NF- ⁇ B luciferase.
  • mGmAdG is one of the best trimers inhibiting TLR8 in the experiments described herein. The inventors have previously found that similar to TLR7 inhibition, modification of the 3 rd base of mG*mA*mG with a DNA base was tolerated.
  • ssRNA40 strongly activated TNF production by IFN ⁇ primed THP-1 cells, which depends on TLR7/8 activation (Gantier et al., Journal of Immunology, 180 (4) , pp.2117-2124).
  • the inhibitory effect of the TriLink GAG trimer was comparable to that seen with PS-modified mG*mA*mG in HEK TLR8 cells, which was surprising given that the TriLink trimer should be much more prone to degradation prior to reaching the TLR8 containing endosome due to the PPP group and lack of PS modification. This specific observation suggests that the N7-methyl-Guanosine modification may play an important role in TLR8 inhibition. [0605] Critically, as shown in Figure 55, the TriLink GAG did not have any inhibitory activity on human or mouse TLR7 sensing of R848 when used with the same conditions as in Figure 54, while mGmAmG was modestly inhibitory in both system at this high dose of trimer.
  • Example 8 Structure activity relationship of alternate GUC inhibition on human TLR7 [0609]
  • Example 5 demonstrated that changing the last base of a fully 2′OMe modified mG*mU*mC (GUC) 3-mer into a DNA base (mG*mU*dC; GUC-v1) or LNA base (mG*mU*lC; GUC-v13) significantly improved hTLR7 inhibition ( Figures 23 and 24).
  • GUC 2′OMe modified mG*mU*mC
  • GAG-v10 mG*mA*dG*dC*dC*dC
  • GAG-v8 mGmAlG
  • GAG-v14, v16, v17, v20, and v21 were only poorly inhibiting TLR7 at 5 ⁇ M (i.e. less than ⁇ 50%), indicating that these modifications are deleterious to TLR7 inhibition.
  • Other variants including GAG-v12, GAG-v13, GAG-v19 were inhibiting hTLR7 as potently as GAG-v7 ( Figure 58).
  • GAG-v11, GAG-v15, GAG-v16 and GAG-v18 were also significantly inhibiting hTLR7 at the high dose of 5 ⁇ M, although not as much as GAG-v12, v13, v19 and GAG-v7.
  • all the GAG variants tested showed only weak inhibitory activity on hTLR7 ( ⁇ 20-30%), with GAG-v7 remaining arguably the most potent inhibitor (Figure 58).
  • the inventors also tested a panel of 16 mG*rX*rX 3-mers where the first base is 2′OMe and the 2 nd and 3 rd base are RNA bases – to extend the data the inventors previously obtained with the mG*dX*dX series shown in Figure 12. These sequences were tested in HEK-TLR7 cells at the dose of 2 ⁇ M ( Figure 59). The inventors observed that mG*rA*rA and mG*rU*rC were equally effective as tested mG*mU*dC (GUC-v1).
  • Example 9 Trimer inhibition of mouse TLR7
  • the inventors tested the length variants of GUC including GUC-v38 and GUC-v39 and compared their activity to 1004921453 parental 2′OMe GUC, GUC-v13 and GUC-v1 Linked 3′-5′ on mTLR7 inhibition – noting that mGmUmC was a poor inhibitor of mouse TLR7 (see Figure 13).
  • the inventors also tested the GAG variants (v11-21) in these experiments since mGmAmG is a potent inhibitor of mouse TLR7.
  • the inventors also tested the 9 GUC (GUC-v41 to GUC-v49) and 11 GAG (GAG-v11 to GAG-v21) variants at both the high (5 ⁇ M) and intermediate (1 ⁇ M) doses on mTLR7 inhibition.
  • GUC-v42 and GUC-v44 were the strongest inhibitors of mTLR7.
  • GUC-v13 also inhibited mTLR7 by ⁇ 40%.
  • Additional GUC variants including GUC-v43, GUC-v45 and GUC-v47 were also showing significant inhibition of mTLR7 ( Figure 61).
  • GAG-v1 (mG*mA*dG) had an improved hTLR8 inhibiting ability compared to the parental GAG (mG*mA*mG) sequence ( Figure 20).
  • the inventors observed that the inosine variant of the first base in GAG-v7 was more potent than GAG-v1 in inhibiting hTLR8 ( Figure 45 and 62) at 1 ⁇ M and 200 nM.
  • LNA modification of the 3 rd base in GAG-v8 had a similar activity to that of GAG-v1.
  • the inventors also tested a panel of 16 mG*rX*rX 3-mers where the first base is 2′OMe and the 2 nd and 3 rd base are RNA bases – to extend the data the inventors obtained with the mG*dX*dX series shown in Figure 22.
  • mG*rA*rA and mG*rG*rA completely abolished hTLR8 sensing at 5 ⁇ M (Figure 64).
  • mG*rA*rA was also inhibitory on hTLR7 ( Figure 64), suggesting a potential functional activity in homeostasis since mGrArA is the most prevalent 2′OMe guanosine motif observed in human ribosomal RNA (18/28S).
  • Some additional sequences including mG*rA*rG and mG*rA*rU also showed more than 50% inhibition of hTLR8, aligning with the inventors observations that GAX like 3-mers starting with a 2′OMe guanosine were inhibitory with DNA and 2′OMe bases.
  • Pre-treatment with the oligo 38-2 resulted in potentiation of TLR8 activity – measured by TNF production – in R848-treated splenocytes derived from B- hTLR8/hTLR7 mice, but not in R848-treated splenocytes from WT mice that express only the non-functional murine TLR8 protein (Figure 65).
  • Example 12 Potentiation of TLR8 in human skin explants [0628] The ability of 38-2 (mG*dC*dC) to potentiate TLR8 signalling was then assessed in human skin tissue using full thickness healthy skin punch biopsies in the culture medium.
  • the mRNA and GGC-v1 oligo were pre-mixed by pipetting at a ratio of 5 mRNA:1 GGC-v1 (by mass) before encapsulation. Concentrations of mRNA and GGC-v1 were then quantified by RT-qPCR and LC-MS/MS, respectively (Table 4). As expected, GGC-v1 was not detected in the control LNPs containing mRNA alone. In the LNPs containing both mRNA and GGC-v1, the measured ratio of mRNA:GGC-v1 was 8.5:1, indicating 12 % of the LNP contents were GGC-v1 oligo by weight – down from the intended 20 % at pre-mixing (Table 4).
  • the LNPs fell within acceptable parameters in all measured criteria, and there were no significant differences between the LNPs containing mRNA alone and the LNPs containing both mRNA and the GGC-v1 oligo (Table 5). Table 5. Physico-chemical properties of the LNPs formulated with mRNA alone, or mRNA and GGC-v1. PDI, poly-dispersity index. Data are presented as Mean ⁇ SD. 1004921453 Example 14 – Inhibition of mRNA vaccine-induced reactogenicity [0631] Finally, to test the ability of the GGC-v1 oligo to block mRNA vaccine-induced reactogenicity in vivo, WT 129X1/SvJ mice were injected i.v.
  • RR the PS backbone
  • RS the RS
  • SR the SR
  • SS the SS

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Abstract

L'invention concerne des oligonucléotides qui inhibent le récepteur de type Toll 7 (TLR7) et/ou le récepteur de type Toll 8 (TLR8), ou potentialisent TLR8, et leurs utilisations.
PCT/AU2023/051007 2022-10-12 2023-10-12 Oligonucléotides modifiés WO2024077351A1 (fr)

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WO2008019486A1 (fr) * 2006-08-16 2008-02-21 Protiva Biotherapeutics, Inc. Modulation par acide nucléique de stimulation immune facilitée par un récepteur similaire au récepteur toll
WO2010105819A1 (fr) * 2009-03-17 2010-09-23 Gunther Hartmann Ligand du tlr7 et ses utilisations
EP3398955A1 (fr) * 2015-12-16 2018-11-07 Ajinomoto Co., Inc. Procédé de production d'oligonucléotides et nucléoside, nucléotide ou oligonucléotide
WO2021198883A1 (fr) * 2020-03-31 2021-10-07 Janssen Biopharma, Inc. Synthèse d'oligonucléotides et de composés associés
WO2022036858A1 (fr) * 2020-08-20 2022-02-24 深圳市瑞吉生物科技有限公司 Nouvel analogue de coiffe 5' ayant une structure cap2 et son procédé de préparation
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WO2001002419A1 (fr) * 1999-07-07 2001-01-11 Isis Pharmaceuticals, Inc. Oligomeres contenant c3'-methylene hydrogene phosphonate et composes associes
WO2008019486A1 (fr) * 2006-08-16 2008-02-21 Protiva Biotherapeutics, Inc. Modulation par acide nucléique de stimulation immune facilitée par un récepteur similaire au récepteur toll
WO2010105819A1 (fr) * 2009-03-17 2010-09-23 Gunther Hartmann Ligand du tlr7 et ses utilisations
EP3398955A1 (fr) * 2015-12-16 2018-11-07 Ajinomoto Co., Inc. Procédé de production d'oligonucléotides et nucléoside, nucléotide ou oligonucléotide
WO2021198883A1 (fr) * 2020-03-31 2021-10-07 Janssen Biopharma, Inc. Synthèse d'oligonucléotides et de composés associés
WO2022036858A1 (fr) * 2020-08-20 2022-02-24 深圳市瑞吉生物科技有限公司 Nouvel analogue de coiffe 5' ayant une structure cap2 et son procédé de préparation
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