RIG-i inhibitors

Abstract

The present invention relates to oligonucleotides that inhibit retinoic acid-inducible gene-I (RIG-I), and uses thereof.

Description

RIG-1 inhibitorsField of the invention

[0001] The present invention relates to oligonucleotides that inhibit retinoic acidinducible gene-l (RIG-I), and uses thereof.Related application

[0002] This application claims the benefit of priority to Australian provisional application no 2024903630 filed on 6 November 2024, the disclosure of which is incorporated herein by reference in its entirety.Background of the invention

[0003] RNA therapeutics based on synthetic oligonucleotides have been gaining a lot of interest, with several regulatory approvals in the US and European Union, and multibillion license deals in recent years.

[0004] In mammals, recognition of exogenous nucleic acids is a critical component of immune responses to pathogens and is achieved by a variety of innate immune sensors, such as Toll like receptors (TLRs) 3, 7, 8 and 9, and retinoic acid-inducible gene-l (RIG-I). However, these innate immune sensors can be activated by synthetic oligonucleotides leading to unwanted pro-inflammatory responses. Therefore, there has been a focus on designing oligonucleotides that evade activation of innate immune sensors and avoid strong off-target pro-inflammatory immune responses in patients. In addition to evading nucleic acid sensing, there is growing evidence that select sequences of synthetic oligonucleotides can actively antagonise innate immune sensors (e.g. for TLR7, TLR9, cGAS etc). Novel insights into the activities of TLR7 antagonism by selected oligonucleotides revealed that motifs as short as 2 to 3 nucleotides were directly interacting with a novel site in TLR7, promoting an inactive conformation blocking TLR7 function. Critically, the site underlying this interaction with the antagonistic oligonucleotides was found to be mutated in rare patients with systemic lupus - indicating that the oligonucleotides harness a naturally existing antagonism of TLR7. These discoveries suggest that the antagonistic activities of selected oligonucleotide sequences is rather dependent on a mimicry of natural antagonists.

[0005] Nonetheless, whether modified oligonucleotides can exhibit immunosuppressive sequence effects on RIG-1, and the sequence determinants of such activities, are currently lacking. Further, our understanding of the immunosuppressive effects of oligonucleotides combining base, sugar and / or backbone modifications, as is seen in most oligonucleotide therapeutics approved and in development, is nearly nonexistent.

[0006] There is a need for inhibitors of RIG-1 activation, including those that are suitable for use with RNA technologies, such as mRNA therapeutics.

[0007] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0008] In a first aspect there is provided an oligonucleotide comprising a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-1 activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 1.

[0009] In one embodiment of the first aspect, there is provided an oligonucleotide about 3 to about 20 nucleotides in length comprising the following sequence:mG*mC*XA;whereinXAis mG, dG, +G or mU;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mil is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group;* is a 3’-5’ phosphorothioate internucleotide linkage;the oligonucleotide inhibits RIG-I activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 2.

[0010] In another embodiment of the first aspect, there is provided an oligonucleotide comprising the following sequence:mG*mC*XA*Xc*XD*XE;whereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mU or dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-0Me guanosine, mC is 2'-OMe cytidine, mil is 2'-OMe uridine, mA is 2'-OMe adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-1 activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 2.

[0011] Preferably, when the sequence is mG*mC*XA*Xc*XD*XE, XAis mG, dG or +G.

[0012] In a second aspect there is provided an oligonucleotide comprising a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG or +G; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mil is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is LNA guanosine, and +T is LNA thymidine; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 1.

[0013] In a preferred embodiment of the second aspect, there is provided an oligonucleotide about 3 to about 20 nucleotides in length comprising the following sequence:mG*mC*XA;whereinXAis mG, dG or +G;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, dG is 2’-deoxyribose guanosine, and +G is LNA guanosine;* is a 3’-5’ phosphorothioate internucleotide linkage;the oligonucleotide inhibits RIG-1 activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 2.

[0014] In one embodiment of the first or second aspect, there is provided an oligonucleotide comprising or consisting of a sequence selected from the group of oligonucleotides in Table 3.

[0015] In a preferred embodiment of the first or second aspect, the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 4.

[0016] In another preferred embodiment of the first or second aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dG*dG*dG*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 70), mG*mC*mG*mG*mU*dA*dG*dG*dG*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 71), mG*mC*mG*mG*mU*dG*dG*dG*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 72) mG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC (SEQ ID NO: 46), or mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0017] In another preferred embodiment of the first or second aspect, the oligonucleotide comprises or consists of the sequencemG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC (SEQ ID NO: 46) or mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0018] In another preferred embodiment of the first or second aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0019] There is also provided use of an oligonucleotide or composition according to the first or second aspect for inhibiting RIG-I activity.

[0020] In a third aspect, there is provided use of an oligonucleotide for inhibiting RIG-I, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

[0021] In one embodiment of the third aspect, there is provided an oligonucleotide comprising a sequence defined by:mG*mC*XA*Xc*XD*XE,whereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mU, dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, mA is 2'-0Me adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

[0022] In a fourth aspect there is provided use of an oligonucleotide for inhibiting RIG-I, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XB;whereinXAis mG, dG or +G; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is LNA guanosine, and +T is LNA thymidine; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

[0023] In one embodiment of the third or fourth aspect, there is provided use of an oligonucleotide for inhibiting RIG-1, wherein the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 5.

[0024] In one embodiment of the third or fourth aspect, there is provided use of an oligonucleotide for inhibiting RIG-1, wherein the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 6.

[0025] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0026] Figure 1. P125 HEK293 cells stably expressing an IFN-p-luciferase reporter were pre-treated ~60 min with 50 nM of 20-mer phosphorothioate (PS) oligos, prior to 3phpRNA transfection (50 ng / ml) overnight. Data shown are from two independent biological replicate plates analysed on the same day (Plate A and Plate B). The IFN-p-luciferase values are reported to the 3phpRNA condition after background correction to non-treated control.

[0027] Figure 2. P125 HEK293 cells stably expressing an IFNb-luciferase reporter were pre-treated ~60 min with 20 nM of indicated 20-mer phosphorothioate (PS) oligos, prior to 3phpRNA transfection (50 ng / ml) overnight. Data shown are averages from three independent experiments. The IFN-p-luciferase values are reported to the 3phpRNA condition after background correction to NT control. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests are shown. NT is non-treated.

[0028] Figure 3. MEME analyses showing one enriched motif in H2 and F10 ASOs.

[0029] Figure 4. P125 HEK293 cells stably expressing an IFN-p-luciferase reporter were pre-treated ~60 min with 20 nM of indicated phosphorothioate (PS) oligos, prior to 3phpRNA transfection (50 ng / ml) overnight. Data shown are averages from three independent experiments. The IFN-p-luciferase values are reported to the 3phpRNA condition after background correction to NT control. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “C2P2-Mut1” condition with P<0.05 areshown. NT is non-treated. mX is 2'-0Me and dX is DNA. * denotes a phosphorothioate internucleotide linkage.

[0030] Figure 5. Undifferentiated THP-1 were pre-treated ~60 min with 50 nM of indicated phosphorothioate (PS) oligos, prior to 3phpRNA transfection (50 ng / ml) for 6 h, and analysis of secreted IP10 levels by specific ELISA. Data shown are averages from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “C2P2-Mut1” condition with P<0.05 are shown. NT is non-treated.

[0031] Figure 6. Undifferentiated THP-1 were pre-treated ~60 min with 50 nM of indicated phosphorothioate (PS) oligos, prior to 3phpRNA transfection (50 ng / ml) for 6h, and analysis of secreted I FN-p levels by specific ELISA. Data shown are averages from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “C2P2-Mut1” condition with P<0.05 are shown. NT is nontreated.

[0032] Figure 7. Undifferentiated THP-1 were pre-treated ~60 min with 50 nM of indicated phosphorothioate (PS) oligos, prior to 3phpRNA transfection (50 ng / ml) for 6h, and analysis of secreted IP-10 and IFN-p levels by specific ELISAs. Data shown are shown relative to agonist only condition from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “C2-Mut1dC-5GC” condition with P<0.05 are shown. NT is non-treated. mX is 2'-OMe and dX is DNA. * denotes a phosphorothioate internucleotide linkage.

[0033] Figure 8. Undifferentiated THP-1 were pre-treated ~60 min with 50 nM of indicated phosphorothioate (PS) oligos, prior to poly(IC) transfection (1 mg / ml) (A), or transfection of 5 mg / ml of 5'-capped EGFP mRNA (B) for 6h and analysis of secreted IP-10 levels by specific ELISA. Data shown are averages from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “mRNA only” condition with P<0.05 are shown (B). NT is non-treated.

[0034] Figure 9. Undifferentiated THP-1 were pre-treated ~60 min with 5μM of indicated phosphorothioate (PS) 3-mer oligos, prior to 3phpRNA transfection (50 ng / ml) for 6 h, and analysis of secreted IP-10 levels by specific ELISA. Data shown are fromtwo independent biological replicate plates analysed on the same day (Plate A and Plate B). NT is non-treated.

[0035] Figure 10. Undifferentiated THP-1 were pre-treated ~60 min with 5pM of indicated phosphorothioate (PS) 2'-OMe 3-mer oligos, prior to 3phpRNA transfection (50 ng / ml) for 6 h, and analysis of secreted IP-10 levels by specific ELISA. Data shown are averages from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “3phpRNA only” condition with P<0.05 are shown. NT is non-treated.

[0036] Figure 11. hTERT immortalised fibroblasts from two different sources were pretreated ~60 min with 5μM of GCG 2'-OMe 3-mer oligo, prior to 3phpRNA transfection (50 ng / ml) for 16h, and analysis of secreted IP-10 levels by specific ELISA. Data shown are averages from 3 biological replicate from a single experiment. SEM are shown. NT is non-treated.

[0037] Figure 12. hTERT immortalised fibroblasts were pre-treated ~60 min with 5pM of mGmCmG 3-mer oligos or its 3’end variants (mGmCdG and mGmCLG, with DNA and LNA 3’end, respectively, where LG is LNA guanosine), prior to 3phpRNA transfection (50 ng / ml) for 16h, and analysis of secreted IP-10 levels by specific ELISA. Data shown are average from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “3phpRNA only” condition with P<0.05 are shown. NT is non-treated.

[0038] Figure 13. Undifferentiated THP-1 were pre-treated ~60 min with 5pM of indicated phosphorothioate (PS) 3-mer oligos, prior to 3phpRNA transfection (50 ng / ml) for 6 h, and analysis of secreted IP-10 levels by specific ELISA. Data shown are averages from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “3phpRNA only” condition with P<0.05 are shown. NT is non-treated.

[0039] Figure 14. hTERT immortalised fibroblasts were pre-treated ~60 min with 200 nM of indicated oligos prior to 3phpRNA transfection (50 ng / ml) for 16h, and analysis of secreted IP-10 and IL-8 levels by specific ELISAs. Data shown are average from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSDtests compared to the “3phpRNA only” condition with P<0.05 are shown. NT is nontreated.

[0040] Figure 15. Effect of C2Mut1-3Mod1 on RIG-I Like Receptor (RLR) sensing in PBMCs. PBMCs from healthy donors were purified and pre-treated with 50 nM of C2Mut1-3Mod1 for 60 min prior to overnight transfection of 50 ng / ml polylC and analysis of secreted IP10 levels by specific ELISA. Data are shown relative to pIC condition, and are averaged from 3 independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests are shown. NT is non-treated.

[0041] Figure 16. Inhibition of saRNA sensing. Undifferentiated THP-1 cells were pretreated ~60 min with 50 nM of indicated PS oligos, prior to transfection of 5 pg / ml of 5'-capped EGFP saRNA for 16h and analysis of secreted IP-10 levels by specific ELISA. Data shown are averaged from two independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “saRNA only” condition are shown. NT is non-treated.

[0042] Figure 17. Undifferentiated THP-1 were pre-treated ~60 min with 50 nM of indicated PS oligos, prior to 3phpRNA transfection (50 ng / ml) for 6 h, and analysis of secreted IP10 levels by specific ELISA. Data shown are averaged from three independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “C2P2-Mut1-3mod1” condition with P<0.05 are shown. NT is nontreated. Bold underlined residues indicate modifications of 3mod1 variants compared to the parental oligonucleotide.

[0043] Figure 18. Undifferentiated THP-1 were pre-treated ~60 min with 50 nM of indicated PS oligos, prior to 3phpRNA transfection (50 ng / ml) for 6 h (top) or 16 h (bottom), and analysis of secreted IP10 levels by specific ELISA. Data shown are averaged from three independent experiments. SEM and One-way ANOVA with uncorrected Fisher’s LSD tests compared to the “C2P2-Mut1-3mod1” condition with P<0.05 are shown. NT is non-treated. Bold underlined residues indicate modifications of 3mod1 variants compared to the parental oligonucleotide.Detailed description of the embodiments

[0044] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individualfeatures mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.Definitions

[0045] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0046] As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.

[0047] “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% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0048] 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. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, 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.

[0049] As used herein, 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 condition, or the risk of (or susceptibility to) the disease or condition. The term “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.

[0050] In particularly preferred embodiments, 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.

[0051] As used herein, the terms “preventing”, “prevent” or “prevention” include administering a therapeutically effective amount of an oligonucleotide(s) described herein sufficient to stop or hinder the development of at least one symptom of a disease, disorder or condition. As used herein, “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.

[0052] Herein, 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. In one example, the animal is a vertebrate. For example, the animal can be a mammal, avian, chordate, amphibian or reptile. Exemplary subjects include but are not limited to 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). In one example, the mammal is a human.

[0053] 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 cellularresponses (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.

[0054] As used herein, the phrase “inhibits RIG-I 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 RIG-I based immune response or is only able to elicit a reduced or partial RIG-I based immune response, such as to a pathogen or a damaged endogenous nucleic acid. In an embodiment, the RIG-I based immune response is less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the response in the absence of the oligonucleotide. In an embodiment, an oligonucleotide of the invention inhibits or reduces RIG-I activity by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0055] As used herein 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. For example, the method may include obtaining data from multiple individuals to develop an appropriate reference data set. Alternatively, 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.

[0056] As used herein the terms “disease”, “disorder” or “condition” relate to any unhealthy or abnormal state.

[0057] The term “disease, disorder or condition in a subject responsive to RIG-1 inhibition” includes diseases, conditions, and disorders in which the inhibition of RIG-1 provides a therapeutic benefit. This includes diseases, disorders and conditions associated with increased RIG-1 signalling. This also includes diseases, disorders and conditions wherein RIG-1 signalling exacerbates an aberrant autoimmune response. Diseases, disorders and conditions responsive to RIG-1 inhibition include inflammation-related diseases, allergic diseases, infections, cancers and auto-immune diseases.

[0058] The phrase “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.

[0059] The terms “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. For example, this can be the amount of a composition comprising one or more agents that inhibit the activity of RIG-1, necessary to reduce, alleviate and / or prevent a disease, disorder or condition. In some embodiments, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of a disease, disorder or condition. In other embodiments, 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.

[0060] Ideally, 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.

[0061] 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. Preferably, a sub-therapeutic dose is one which does not cause significant adverse side effects in the subject.Oligonucleotides

[0062] In the context of this invention, the term “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 comprising bases (also referred to herein as “nucleobases”), modified bases, sugars, modified sugars, internucleotide linkages or modified internucleotide linkages.

[0063] "Gapmer" refers to an oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings."

[0064] As used herein, 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. For the present invention, the target is at least RIG-I.

[0065] Typically, an oligonucleotide of the invention will be synthesized in vitro.

[0066] As used herein, the term “nucleotide” includes all naturally occurring nucleotides, including all forms of nucleotide bases found in nature.

[0067] As used herein, the term “modification group” or “modified” refers to any chemical moiety that may be attached to the oligonucleotide at locations, which include, but are not limited to, the sugar, base, and / or internucleotide linkage.

[0068] As used herein, the terms “nucleotide analogs”, “modified nucleotides”, or “nucleotide derivatives” include synthetic nucleotides as described herein.Bases

[0069] Oligonucleotides of the invention comprise nucleobases ("bases"). The bases may be modified or unmodified.

[0070] Nitrogenous bases most commonly found in naturally occurring nucleotides are purine and pyrimidine bases. Naturally occurring purine bases include, for example, adenine, guanine, and N6-methyladenine. Naturally occurring pyrimidine bases include, for example, uracil, cytosine, thymine, 5-methylcytosine, pseudouracil.

[0071] Unmodified bases refer to the canonical naturally occurring bases, which include: guanine (G), cytosine (C), thymine (T), adenine (A), uracil (U) and hypoxanthine (I).

[0072] Modified bases include but are not limited to: N6-methyladenine, N7-methylguanine, 5-methylcytosine, pseudouracil and N1-methylpseudouracil.

[0073] Unless stated to the contrary, reference to an A, T, G, U, I or C can either mean an unmodified base or a modified version thereof.

[0074] Oligonucleotides of the invention may comprise modified bases, unmodified bases, or a combination thereof. In particular embodiments, one or more bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases inclusive of any range therein) of the oligonucleotide described herein are modified. In some embodiments, all bases of the oligonucleotide described herein are modified. In alternative embodiments, no bases of the oligonucleotide described herein are modified.

[0075] In some embodiments, the oligonucleotide may comprise a modified guanine base at the 5’-terminal end comprising a N7-methylation. Preferably, the oligonucleotide does not comprise a modified guanine base at the 5’-terminal end comprising a N7-methylation.

[0076] In a preferred embodiment, oligonucleotides of the invention comprise unmodified bases selected from: A, T, G, U, I and C, more preferably selected from: A, T, G, U, and C. In other words, preferably, no bases of the oligonucleotide describedherein are modified such that in preferred embodiments, A, T, G, II, I or C refer to naturally occurring, unmodified bases.Sugars

[0077] Oligonucleotides of the invention comprise sugars (also referred to herein interchangeably as “ribose sugar” or “ribofuranose sugar”). The sugars may be modified or unmodified.

[0078] Sugars include but are not limited to: ribose, 2'-deoxyribose, 2'-O-(methyl) ribose (also known as 2-O-CH3, also known as 2'-OMe), and conformationally constrained sugars comprising a 2’-O,4’-C bridging group, such as Locked Nucleic Acid ribose (LNA).

[0079] Examples of 2’-O,4’-C bridging groups include but are not limited to 2-0, 4-C-methylene bridge (LNA modified sugar), 2'-O,4'-C-ethylene bridge (ENA modified sugar), 2',4'-constrained-2'-O-ethyl (cEt modified sugar) or 2',4'-constrained-2'-O-methoxyethyl (cMOE modified sugar).

[0080] Locked Nucleic Acids (LNAs) refer to ribose sugars 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. In one embodiment, the linkage is a methylene (-CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98 / 39352 and WO 99 / 14226. In some embodiments, however, the modification does not comprise LNA.

[0081] As used herein, unmodified sugars refer to: ribose and 2'-deoxyribose. As used herein, modified sugars refer to: 2'-OMe, LNA, ENA, cEt and cMOE.

[0082] As used herein rX denotes a base comprising a ribose sugar, dX denotes a base comprising a 2'-deoxyribose sugar, mX denotes a base comprising a 2'-OMe ribose sugar, +X denotes a nucleoside analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group.

[0083] In one embodiment, +G is locked nucleic acid (LNA) guanosine, ethylene-bridged nucleic acid (ENA) guanosine, constrained ethyl (cEt) guanosine or constrained 2’-MOE (cMOE) guanosine. Preferably, +G is LNA guanosine.

[0084] In one embodiment, +T is locked nucleic acid (LNA) thymidine, ethylene-bridged nucleic acid (ENA) thymine, constrained ethyl (cEt) thymidine or constrained 2’-MOE (cMOE) thymidine. Preferably, +T is LNA thymidine.

[0085] In one embodiment, +C is locked nucleic acid (LNA) cytidine, ethylene-bridged nucleic acid (ENA) cytidine, constrained ethyl (cEt) cytidine or constrained 2’-MOE (cMOE) cytidine. Preferably, +C is LNA cytidine. More preferably, +C comprises a 5-methylcytosine nucleobase.

[0086] Oligonucleotides of the invention may comprise modified sugars, unmodified sugars, or a combination thereof. In particular embodiments, one or more sugars (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 sugars inclusive of any range therein) of the oligonucleotide described herein are modified. In some embodiments, all sugars of the oligonucleotide described herein are modified.

[0087] Preferably, one or more sugars of the oligonucleotide described herein are modified, wherein the sugar modification is selected from: 2'-OMe, LNA, ENA, cEt, cMOE, and combinations thereof. More preferably, the sugar modification is selected from: 2'-OMe, LNA, and combinations thereof.

[0088] Preferably at least one, more preferably at least two or more sugars of the oligonucleotide described herein include a 2'-OMe modification.Internucleotide linkages

[0089] Oligonucleotides of the present disclosure include internucleotide linkages. The internucleotide linkages may be modified or unmodified.

[0090] As used herein, the term “internucleotide linkage” refers to the bond or bonds that connect two nucleotides of an oligonucleotide or nucleic acid.

[0091] Internucleotide linkages most commonly found in naturally occurring nucleotides include phosphodiester linkages. As used herein, unmodified internucleotide linkage refers to a phosphodiester linkage.

[0092] Modified oligonucleotide internucleotide linkages containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, and phosphoramidates, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage, that is, a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Preferably, the modified internucleotide linkage is phosphorothioate.

[0093] In one embodiment, each internucleotide linkage is independently selected from phosphorothioate and phosphodiester. Each internucleotide linkage may be the same or different. In a preferred embodiment, each internucleotide linkage is the same, preferably phosphorothioate.

[0094] 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. Preferably each internucleotide linkage is selected from the group consisting of: 3'-5'- and 5'-5'- linkage. For example, 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 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. In one embodiment, the sequence includes a 5'-5'- linkage.Preferably, the 5'-5'- linkage connects nucleotides at a first and second position of the sequence. In one embodiment, the sequence includes a 5'-5'- linkage and a 3'-5'-linkage. Preferably, the 5'-5'- linkage connects nucleotides at a first and second position of the sequence, and the 3'-5'- linkage connects nucleotides at a second and third position of the sequence. In a preferred embodiment, each internucleotide linkage is a 3'-5'- linkage.

[0095] In a particularly preferred embodiment, each internucleotide linkage is a 3'-5'-phosphorothioate linkage.Oligonucleotides that inhibit RIG-I activity

[0096] The inventors have surprisingly identified a number of oligonucleotide sequences that inhibit RIG-I activity, and have identified core structural sequencefeatures that confer RIG-I inhibitory activity.

[0097] The inventors have identified a minimum structural motif required for RIG-I inhibitory activity selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage.

[0098] In a first aspect, the inventors have identified novel oligonucleotides consisting of or comprising a minimum structural motif required for RIG-1 inhibition, and uses thereof.

[0099] In another aspect, the inventors have identified a series of previously disclosed oligonucleotides that surprisingly inhibit RIG-1 and have therefore discovered new uses for these oligonucleotides as RIG-1 inhibitors.

[0100] Accordingly in a first aspect, there is provided an oligonucleotide comprising a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-1 activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 1.Table 1. “m” indicates 2'-0Me base, * denotes a 3’-5’ phosphorothioate linkage, “d” indicates DNA base, “+” indicates LNA base. Sequence is from 5’3’.

[0101] In one embodiment of the first aspect, there is provided an oligonucleotide about 3 to about 20 nucleotides in length comprising the following sequence:mG*mC*XA;whereinXAis mG, dG, +G or mU;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mil is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group;* is a 3’-5’ phosphorothioate internucleotide linkage;the oligonucleotide inhibits RIG-I activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 2.

[0102] In another embodiment of the first aspect, there is provided an oligonucleotide comprising the following sequence:mG*mC*XA*Xc*XD*XE;whereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mil or dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, mA is 2'-0Me adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-1 activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 2.Table 2. “m” indicates 2'-0Me base, * denotes a 3’-5’ phosphorothioate linkage, “d” indicates DNA base, “+” indicates LNA base. Sequence is from 5’3’.

[0103] Preferably, when the sequence is mG*mC*XA*Xc*XD*XE, XAis mG, dG or +G.

[0104] Preferably, the sequence is mG*mC*mG*Xc*XD*XE. More preferably, the sequence is mG*mC*mG*[mG / mU]*XD*XE. Even more preferably, the sequence is mG*mC*mG*mG*XD*XE. Even more preferably, the sequence is mG*mC*mG*mG*[mG / mU]*XE. Even more preferably, the sequence is mG*mC*mG*mG*mU*XE. Most preferably, the sequence is mG*mC*mG*mG*mU*[dA / dG].

[0105] In one embodiment of the first aspect, the oligonucleotide comprises the sequence mG*mC*XA*Xc*XD*XE, wherein the sequence is located at the 5’ terminal end, middle or 3’ terminal end of the oligonucleotide. Preferably the sequence is located at the 5’ terminal end.

[0106] In a second aspect there is provided an oligonucleotide comprising a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG or +G; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mil is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is LNA guanosine, and +T is LNA thymidine; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-1 activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 1.

[0107] each internucleotide linkage is a phosphorothioate linkage, more preferably a 3’-5’ phosphorothioate linkage.

[0108] In a preferred embodiment of the first or second aspect, the oligonucleotide is between about 3 and about 50 nucleotides, between about 3 and 20 nucleotides, including any value therein, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0109] In one embodiment of the first or second aspect, the oligonucleotide consists of the sequence. For example, in an embodiment of the second aspect the oligonucleotide may consist of the sequence, such that the oligonucleotide is 3 nucleotides in length. In such embodiments the oligonucleotide consists of the sequence mG*mC*+G.

[0110] In another embodiment of the first or second aspect, the oligonucleotide is about 10 to about 20 nucleotides in length, including any value therein, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, most preferably about 15 to about 20 nucleotides in length, even more preferably about 15 nucleotides in length.

[0111] The sequence may be located at any position within the oligonucleotide, including at the 5’ terminal end, the middle region, or the 3’ terminal end.

[0112] In a preferred embodiment of the first or second aspect, the oligonucleotide comprises a sequence defined by mG*mC*XA.

[0113] In one embodiment of the first or second aspect, the oligonucleotide comprises the sequence mG*mC*mG. In such embodiments, the sequence mG*mC*mG is preferably located at the 5’ terminal end of the oligonucleotide.

[0114] In one embodiment of the first or second aspect, the oligonucleotide comprises the sequence mG*mC*mG*mG. Preferably, the sequence mG*mC*mG*mG is located at the 5’ terminal end of the oligonucleotide.

[0115] In one embodiment of the first or second aspect, the oligonucleotide comprises the sequence mG*mC*mG*mG*mU. Preferably, the sequence mG*mC*mG*mG*mU is located at the 5’ terminal end of the oligonucleotide.

[0116] In one embodiment of the first or second aspect, the oligonucleotide comprises the sequence mG*mC*mG*mG*mU*dA or mG*mC*mG*mG*mU*dG.Preferably, the sequence mG*mC*mG*mG*mU*dA or mG*mC*mG*mG*mU*dG is located at the 5’ terminal end of the oligonucleotide.

[0117] In a particularly preferred embodiment of the first or second aspect, there is provided an oligonucleotide about 3 to about 20 nucleotides in length comprising the following sequence:mG*mC*XA;whereinXAis mG, dG or +G;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, dG is 2’-deoxyribose guanosine, and +G is LNA guanosine;* is a 3’-5’ phosphorothioate internucleotide linkage;the oligonucleotide inhibits RIG-I activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 2.

[0118] In one embodiment of the first or second aspect, there is provided an oligonucleotide comprising or consisting of a sequence selected from the group of oligonucleotides in Figures 4, 7, 17 and 18.

[0119] In one embodiment of the first or second aspect, there is provided an oligonucleotide comprising or consisting of a sequence selected from the group of oligonucleotides in Table 3.Table 3. Oligonucleotides that are inhibitors of RIG-1 sensing, “m” indicates 2'-0Me base, * denotes a 3’-5’ phosphorothioate linkage, “d” indicates DNA base, “+” indicates LNA base. Sequence is from

[0120] In a preferred embodiment of the first or second aspect, the sequence mG*mC*XAis located at the 5’ terminal end of the oligonucleotide.

[0121] In another preferred embodiment of the first or second aspect, the oligonucleotide is about 10 to about 20 nucleotides in length and the sequence mG*mC*XAis located at the 5’ terminal end.

[0122] In another preferred embodiment of the first or second aspect, the sequence mG*mC*XAis mG*mC*mG.

[0123] In one embodiment of the first or second aspect, the 3’ terminal end of the oligonucleotide comprises of the following sequence:mX*[mX / dX]*[dX / mX]*[dX / mX]*[dX / mX / +X];wherein mX is a nucleotide comprising a 2’-OMe modification, dX is a deoxyribonucleotide and +X is an LNA nucleotide, and* is independently an internucleotide linkage.

[0124] Preferably, the 3’ terminal end of the oligonucleotide comprises the sequence mC*[mA / dA]*[dG / mG]*[dC / mC]*[dC / mC / +C].

[0125] Preferably, the last two nucleotides at the 3’ terminal end of the oligonucleotide are not mC*+C.

[0126] In another preferred embodiment, the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 4.Table 4. Oligonucleotides that are inhibitors of RIG-I sensing, “m” indicates 2'-OMe base, * denotes a 3’-5’ phosphorothioate linkage, “d” indicates DNA base, “+” indicates LNA base. Sequence is from

[0127] In another preferred embodiment of the first or second aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dG*dG*dG*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 70), mG*mC*mG*mG*mU*dA*dG*dG*dG*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 71), mG*mC*mG*mG*mU*dG*dG*dG*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 72) mG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC (SEQ ID NO: 46), or mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0128] In another preferred embodiment of the first or second aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC (SEQ ID NO: 46) ormG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0129] In another preferred embodiment of the first or second aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0130] The oligonucleotide may further include a sequence comprising mG*mU*XB. Preferably the further sequence is located in a middle region of the oligonucleotide.

[0131] Optionally the oligonucleotide according to the first or second aspect may inhibit TLR7 and / or TLR8.Testing for inhibition of RIG-I activity

[0132] Some embodiments of the methods of the present invention involve testing for inhibition of RIG-I activity which can be determined using any method known in the art. In some embodiments, RIG-I activity in cells may be measured by expression and / or secretion of one or more pro-inflammatory cytokines (e.g. IFN-p or IP-10), and / or activation or expression of transcription factors (e.g. IRF3).

[0133] The ability of an oligonucleotide to inhibit RIG-I activity can, for example, be analysed by incubating cells which express RIG-I with an oligonucleotide, then stimulating said cells with a RIG-I agonist (e.g., the commercially available RIG-I agonist 3phpRNA (Invivogen)), and analysing the overall RIG-I response in the cell population, or analysing the proportion of cells having RIG-l-positive activity after a defined period of time.

[0134] In such examples, inhibition of RIG-I activity can be identified by observation of an overall decreased RIG-I response of the cell population, or a lower proportion of cells having RIG-I -positive activity as compared to positive control condition in which cells are treated with RIG-I agonist in the absence of the oligonucleotide (or in the presence of an appropriate control inhibitory agent). In one example, HEK293 cells constitutively expressing an pIFN-p-Luc reporter (referred to as p125 HEK 293 cells), incubated with an oligonucleotide, and then stimulated with 3phpRNA. RIG-I activity canbe determined by a luciferase assay, which measures activated IFN-p by luminescence. RIG-1 activity can also be analysed by measuring cytokine levels, for example by ELISA.Compositions

[0135] Oligonucleotides of the first or second aspect 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.

[0136] Oligonucleotides of the first or second aspect may be administered in a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be solid or liquid. Useful examples of pharmaceutically acceptable carriers 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.

[0137] In one embodiment, the pharmaceutical carrier is water for injection (WFI) and the pharmaceutical composition is adjusted to pH 7.4, 7.2-7.6. In one embodiment, the salt is a sodium or potassium salt.

[0138] 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.

[0139] Oligonucleotides of the first or second aspect 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. The term "pharmaceutically acceptable salts" as used herein 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 effects upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860.

[0140] Oligonucleotides of the first or second aspect may be prodrugs or pharmaceutically acceptable salts of the prodrugs, or other bioequivalents. The term "prodrugs" as used herein 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. In particular, 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.

[0141] 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. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987. Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate".

[0142] In one embodiment, oligonucleotides of the first or second aspect can be complexed with a complexing agent to increase cellular uptake of oligonucleotides. An example of a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells.

[0143] The term "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. In general, 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 cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., CI-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

[0144] Examples of cationic lipids include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-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). 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).

[0145] 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. In addition to those listed above, other 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.

[0146] In one embodiment, lipid compositions can further comprise agents, e.g., viral proteins to enhance lipid-mediated transfections of oligonucleotides. In another embodiment, N-substituted glycine oligonucleotides (peptoids) can be used to optimize uptake of oligonucleotides.

[0147] In another embodiment, a composition for delivering oligonucleotides of the first or second aspect comprises a peptide having from between about one to about four basic residues. These basic residues can be located, e.g., on the amino terminal, C-terminal, or internal region of the peptide. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (can also be considered nonpolar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromaticside chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Apart from the basic amino acids, a majority or all of the other residues of the peptide can be selected from the non-basic amino acids, e.g., amino acids other than lysine, arginine, or histidine. Preferably a preponderance of neutral amino acids with long neutral side chains are used.

[0148] In one embodiment, oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide”. In one embodiment, the composition includes an oligonucleotide which is complementary to a target nucleic acid molecule encoding the protein, and a covalently attached transporting peptide.

[0149] In a further embodiment, 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. (2018)).Uses

[0150] Oligonucleotides of the invention are designed to be administered to an animal. For this purpose, the oligonucleotide can be administered in combination with another molecule, such as a further nucleic acid (e.g., a mRNA molecule, a short interfering RNA, an antisense oligonucleotide, a CRISPR guide RNA, etc), a peptide, a carrier agent, a therapeutic agent, and the like. In an embodiment, the oligonucleotide can be conjugated with the other molecule.

[0151] Typically, the oligonucleotide is used to modify a trait of an animal, more typically to treat or prevent a disease or condition. In a preferred embodiment, the disease or condition will benefit from the animal not being able to mount a RIG-I response following administration of the oligonucleotide.

[0152] In one aspect, there is provided use of an oligonucleotide or composition according to the first or second aspect for inhibiting RIG-I activity.

[0153] In one aspect, there is provided a method of inhibiting RIG-I activity in a cell, the method comprising contacting the cell with an oligonucleotide, or a composition according to the first or second aspect, thereby inhibiting RIG-I activity in the cell.

[0154] In one aspect, there is provided a method of inhibiting RIG-I activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, or a composition according to the first or second aspect, thereby inhibiting RIG-I activity in the subject.

[0155] In one aspect, there is provided a method of treating or preventing a disease, disorder or condition in a subject that is responsive to RIG-I inhibition, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, or a composition according to the first or second aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0156] In one aspect, there is provided use of an oligonucleotide or a composition according to the first or second aspect for inhibiting RIG-I activity in a cell, the use comprising contacting the cell with the oligonucleotide or the composition, thereby inhibiting RIG-I activity in the cell.

[0157] In one aspect, there is provided use of an oligonucleotide or a composition according to the first or second aspect for inhibiting RIG-I activity in a subject, the use comprising administering to the subject a therapeutically effective amount of the oligonucleotide, or the composition according to the first or second aspect, thereby inhibiting RIG-I activity in the subject.

[0158] In one aspect, there is provided use of an oligonucleotide or a composition according to the first or second aspect for treating or preventing a disease, disorder or condition in a subject that is responsive to RIG-I inhibition, the use comprising administering to the subject a therapeutically effective amount of an oligonucleotide, or a composition according to the first or second aspect, thereby treating or preventing the disease, disorder or condition in the subject.

[0159] In one aspect, there is provided use of an oligonucleotide according to the first or second aspect in the manufacture of a medicament for inhibiting RIG-I activity in a subject.

[0160] In one aspect, there is provided use of an oligonucleotide according to the first or second aspect in the manufacture of a medicament for treating or preventing a disease, disorder or condition in a subject that is responsive to RIG-I inhibition.

[0161] Preferably, the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 3 or 4. More preferably, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC (SEQ ID NO: 46) or mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0162] The disease, disorder or condition is selected from the group consisting of: immune inflammation-related diseases, allergic diseases, infections, cancers and autoimmune diseases relying on auto-antibodies.

[0163] Examples of 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.

[0164] Examples of the infections can include diseases caused by infections by viruses (such as a single-stranded RNA virus, a double-stranded RNA virus, a singlestranded DNA virus, and a double-stranded DNA virus).

[0165] Examples of single stranded RNA viruses include Encephalomyocarditis virus, Coxsackie B3 virus, Rhinovirus, West Nile virus, Dengue virus, Japanese encephalitis virus, Hepatitis C virus, SARS-CoV-2, Zika virus, Sindbis virus, Ebola, Rabies virus, Vesicular stomatitis virus, Measles virus, human Metapneumovirus, Sendai Virus, Influenza A virus, Influenza B virus, Newcastle disease virus, Respiratory syncytial virus, Nipah, human parainfluenza 5, Lassa, Lymphocytic, choriomeningitis virus and Rift Valley fever virus.

[0166] Examples of double stranded RNA viruses include Reovirus and Rotavirus.

[0167] Examples of double stranded DNA viruses include Herpes simplex virus 1, Hepatitis B virus, Epstein-Barr virus and Myxoma virus.

[0168] 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 metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, salivary gland carcinoma, kidney or renal cancer, prostate cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, oesophageal cancer, tumors of the biliary tract, as well as head and neck cancer.

[0169] In some embodiments, the subject has hyperinflammation associated with, or caused by, RIG-I activation.

[0170] In one embodiment, here is provided a method of reducing or minimising a symptom associated with diseases, disorders and conditions responsive to RIG-I inhibition. Symptoms associated with diseases, disorders and conditions responsive to RIG-I inhibition include inflammation, fever, muscle aches, fatigue. In a preferred embodiment, the condition responsive to RIG-I inhibition is mRNA administration.

[0171] In another embodiment, the oligonucleotides of the first or 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. In particular, the oligonucleotides described herein may be used in the prevention or inhibition of inflammation mediated RIG-I during or following administration of the therapeutic RNA. It is envisaged that the inflammation may involve or include any cells, tissues or organs of the body. In particular embodiments, the inflammation is or comprises hepatic inflammation. To this end, 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.

[0172] In certain examples, the oligonucleotides of the first or second aspect exhibit RIG-I -inhibitory activity, and may be utilised to prevent or inhibit a RIG-I -dependent inflammatory response associated with the administration of a therapeutic RNA selectedfrom 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. More particularly, the therapeutic RNA may be part of RNA-based therapeutic agent, such as an mRNA vaccine. In this regard, the oligonucleotide can at least partly inhibit the engagement or sensing of these therapeutic RNA molecules by RIG-1. The oligonucleotides of the first or 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.

[0173] As such, the oligonucleotides of the first or second aspect may be a component or included within an immunogenic composition, such as an RNA or mRNA vaccine composition, as are known in the art. The term “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.

[0174] In another embodiment of the first or second aspect, the oligonucleotide is functionalised. Preferably, the functionalised oligonucleotide comprises a compound selected from the group consisting of: amino, maleimide, NHS ester, polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclyl, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclyl, substituted arylalkyl, hydrophobic lipid, and peptide. Preferably, the hydrophobic lipid is selected from cholesterol, tocopherol and variants thereof.Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, tocopherol and variants thereof.

[0175] In one embodiment, the compound is conjugated directly to the oligonucleotide. In another embodiment, the compound is conjugated to the oligonucleotide via a linker. The linker may be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0176] Preferably, the compound is conjugated to a terminal nucleotide of the sequence. The compound may be conjugated to the terminal 5'- nucleotide, the terminal3'- nucleotide, or a combination thereof preferably the terminal 3'- nucleotide.Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'- position.

[0177] Examples of a functionalised oligonucleotide according to the present disclosures, include TEG-amino, TEG-alkyl-maleimide, 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-a-tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3'-position via a monophosphate group.

[0178] Functionalised sequences may comprise functionalised nucleotides selected from the group consisting of: dX-TEG.

[0179] In a particular form, the present invention provides an immunogenic composition, such as a vaccine composition, comprising a therapeutic RNA and an oligonucleotide of the first or second aspect. Preferably, 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. Preferably, 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. Suitably, the oligonucleotide of the immunogenic composition exhibits RIG-I inhibitory activity as described herein. In certain embodiments, the oligonucleotide of the immunogenic composition exhibits RIG-I inhibitory activity. In some embodiments, the oligonucleotide of the immunogenic composition exhibits RIG-I 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.

[0180] It will be appreciated that mRNA vaccines provide unique therapeutic alternatives to peptide- or DNA-based vaccines. When the mRNA vaccine is delivered 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. To this end, the oligonucleotideof the first or second aspect may be included as a separate or discrete component and / or conjugated with a therapeutic RNA of the vaccine composition. Preferably the therapeutic RNA is selected from RNA or mRNA. Even more preferably, the therapeutic RNA is self-amplifying mRNA. With respect to such embodiments, the therapeutic RNA of the RNA vaccine may be unmodified or substantially unmodified (e.g., does not include any modified bases). Alternatively, 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).

[0181] Additionally, 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. For example, the therapeutic RNA of the immunogenic composition may be encapsulated or complexed in nanoparticles, and more particularly lipid nanoparticles. According to various embodiments, 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.

[0182] In some embodiments, the oligonucleotide of the first or second aspect is included in the immunogenic composition separate from the carrier system. In other embodiments, the oligonucleotide of the first or second aspect 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.

[0183] In some embodiment, the oligonucleotide of the first or second aspect may be applied to the surface of an implantable biomaterial, such as a prosthetic.

[0184] In particular examples, therapeutically effective amounts of the therapeutic RNA and the oligonucleotide of the first or second aspect may be administeredsimultaneously, concurrently, sequentially, successively, alternately or separately in any particular combination and / or order.Use of oligonucleotide sequences

[0185] As stated above, more broadly, the inventors have identified a series of oligonucleotides, including previously disclosed oligonucleotides, that surprisingly inhibit RIG-I and have therefore discovered new uses for these oligonucleotides as RIG-I inhibitors. There is provided use of novel oligonucleotides described herein as RIG-I inhibitors, as well as a new use of previously disclosed oligonucleotides as RIG-I inhibitors.

[0186] Accordingly, in a third aspect, there is provided use of an oligonucleotide for inhibiting RIG-I, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

[0187] In one embodiment of the third aspect, there is provided an oligonucleotide comprising the following sequence:mG*mC*XA*Xc*XD*XE,whereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mU or dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, mA is 2'-0Me adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

[0188] Preferably, when the sequence is mG*mC*XA*XC*XD*XE, XAis mG, dG or +G.

[0189] Preferably, the sequence is mG*mC*mG*XC*XD*XE. More preferably, the sequence is mG*mC*mG*[mG / mU]*XD*XE. Even more preferably, the sequence is mG*mC*mG*mG*XD*XE. Even more preferably, the sequence is mG*mC*mG*mG*[mG / mU]*XE. Even more preferably, the sequence is mG*mC*mG*mG*mU*XE. Most preferably, the sequence is mG*mC*mG*mG*mU*[dA / dG].

[0190] In one embodiment of the third aspect, the oligonucleotide comprises the sequence mG*mC*XA*Xc*XD*XE, wherein the sequence is located at the 5’ terminal end, middle or 3’ terminal end of the oligonucleotide. Preferably the sequence is located at the 5’ terminal end.

[0191] In a fourth aspect there is provided use of an oligonucleotide for inhibiting RIG-I, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XB;whereinXAis mG, dG or +G; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is LNA guanosine, and +T is LNA thymidine; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

[0192] In a preferred embodiment of the third or fourth aspect, each internucleotide linkage is a phosphorothioate linkage, more preferably a 3’-5’ phosphorothioate linkage.

[0193] In a preferred embodiment of the third or fourth aspect,, the oligonucleotide is between about 3 and about 50 nucleotides, between about 3 and 20 nucleotides, including any value therein, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0194] In one embodiment of the third or fourth aspect, the oligonucleotide consists of the sequence, such that the oligonucleotide is 3 nucleotides in length. In such embodiments the oligonucleotide consists of a sequence selected from the group consisting of: mG*mC*mG, mG*mC*dG, mG*mC*+G, mG*mU*mU, mG*mU*dT, and mG*mU*+T. More preferably, the oligonucleotide consists of a sequence selected from the group consisting of: mG*mC*mG, mG*mC*dG, mG*mC*+G and mG*mU*mU. More preferably, the oligonucleotide consists of a sequence defined by mG*mC*+G.

[0195] In another embodiment of the third or fourth aspect, the oligonucleotide is about 10 to about 20 nucleotides in length, including any value therein, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, most preferably about 15 to about 20 nucleotides in length, even more preferably about 15 nucleotides in length.

[0196] The sequence may be located at any position within the oligonucleotide, including at the 5’ terminal end, the middle region, or the 3’ terminal end.

[0197] In a preferred embodiment of the third or fourth aspect, the oligonucleotide comprises a sequence defined by mG*mC*XA.

[0198] In one embodiment of the third or fourth aspect, the oligonucleotide comprises the sequence mG*mC*mG. In such embodiments, the sequence mG*mC*mG is preferably located at the 5’ terminal end of the oligonucleotide.

[0199] In one embodiment of the third or fourth aspect, the oligonucleotide comprises the sequence mG*mC*mG*mG. Preferably, the sequence mG*mC*mG*mG is located at the 5’ terminal end of the oligonucleotide.

[0200] In one embodiment of the third or fourth aspect, the oligonucleotide comprises the sequence mG*mC*mG*mG*mU. Preferably, the sequence mG*mC*mG*mG*mU is located at the 5’ terminal end of the oligonucleotide.

[0201] In one embodiment of the third or fourth aspect, the oligonucleotide comprises the sequence mG*mC*mG*mG*mU*dA or mG*mC*mG*mG*mU*dG. Preferably, the sequence mG*mC*mG*mG*mU*dA or mG*mC*mG*mG*mU*dG is located at the 5’ terminal end of the oligonucleotide.

[0202] In one embodiment of the third or fourth aspect, the 3’ terminal end of the oligonucleotide comprises the following sequence:mX*[mX / dX]*[dX / mX]*[dX / mX]*[dX / mX / +X];wherein mX is a nucleotide comprising a 2’-OMe modification, dX is a deoxyribonucleotide and +X is an LNA nucleotide, and* is independently an internucleotide linkage.

[0203] Preferably, the 3’ terminal end of the oligonucleotide comprises the sequence mC*[mA / dA]*[dG / mG]*[dC / mC]*[dC / mC / +C].

[0204] Preferably, the last two nucleotides at the 3’ terminal end of the oligonucleotide are not mC*+C.

[0205] In one embodiment of the third or fourth aspect, there is provided an oligonucleotide comprising or consisting of a sequence selected from the group of oligonucleotides in Figures 4, 7, 17 and 18.

[0206] In one embodiment of the third or fourth aspect, there is provide use of an oligonucleotide for inhibiting RIG-1, wherein the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 5.Table 5. Oligonucleotides that are inhibitors of RIG-1 sensing, “m” indicates 2'-OMe base, * denotes a 3’-5’ phosphorothioate linkage, “d” indicates DNA base, “+” indicates LNA base. Sequence is from

[0207] In one embodiment of the third or fourth aspect, there is provided use of an oligonucleotide for inhibiting RIG-1, wherein the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 6.Table 6. Oligonucleotides that are inhibitors of RIG-1 sensing, “m” indicates 2'-OMe base, * denotes a 3’-5’ phosphorothioate linkage, “d” indicates DNA base, “+” indicates LNA base. Sequence is from

[0208] In a preferred embodiment of the third or fourth aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mU*mG*mU*dT*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*mG*mC (SEQ ID NO: 4) or mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*dC*dA*dG*dG*dC (SEQ ID NO: 9).

[0209] In another preferred embodiment of the third or fourth aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dG*dG*dG*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 70), mG*mC*mG*mG*mll*dA*dG*dG*dG*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*Dc (SEQID NO: 72) mG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC (SEQ ID NO: 46), or mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0210] In another preferred embodiment of the third or fourth aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC (SEQ ID NO: 46) or mG*mC*mG*mG*mll*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*Dc (SEQ ID NO: 42).

[0211] In another preferred embodiment of the third or fourth aspect, the oligonucleotide comprises or consists of the sequence mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

[0212] In one embodiment of the third or fourth aspect, the use comprises inhibiting RIG-I activity in a cell. In such an embodiment, there is provided a method of inhibiting RIG-I activity in a cell, the method comprising contacting the cell with an oligonucleotide, or a composition according to the third or fourth aspect, thereby inhibiting RIG-I activity in the cell.

[0213] In one embodiment of the third or fourth aspect, the use comprises inhibiting RIG-I activity in a subject. In such embodiments, there is provided a method of inhibiting RIG-I activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, or a composition according to the third or fourth aspect, thereby inhibiting RIG-I activity in the subject.

[0214] In one embodiment of the third or fourth aspect, the use comprises treating or preventing a disease, disorder or condition in a subject responsive to increased RIG-I signalling. In such embodiments, there is provided a method of treating or preventing a disease, disorder or condition in a subject responsive to increased RIG-I signalling, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide, or a composition according to the third or fourth aspect.

[0215] In one embodiment of the third or fourth aspect, the use comprises use of an oligonucleotide, or a composition according to the third or fourth aspect, in the manufacture of a medicament for inhibiting RIG-I activity in a subject.

[0216] In one embodiment of the third or fourth aspect, the use comprises use of an oligonucleotide, or a composition according to the third or fourth aspect, in themanufacture of a medicament for treating or preventing a disease, disorder or condition in a subject that is responsive to RIG-1 inhibition.

[0217] Preferably, the oligonucleotide according to the third or fourth aspect is selected from the group of oligonucleotides in Table 6.

[0218] In another embodiment of the third or fourth aspect, the oligonucleotide is functionalised. Preferably, the functionalised oligonucleotide comprises a compound selected from the group consisting of: amino, maleimide, NHS ester, polyethylene glycol, alkyl, alkenyl, alkynyl, heterocyclyl, arylalkyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted heterocyclyl, substituted arylalkyl, hydrophobic lipid, and peptide. Prefearbly, the hydrophobic lipid is selected from cholesterol, tocopherol and variants thereof.Preferably, the compound is selected from the group consisting of: polyethylene glycol, cholesterol, tocopherol and variants thereof.

[0219] In one embodiment, the compound is conjugated directly to the oligonucleotide. In another embodiment, the compound is conjugated to the oligonucleotide via a linker. The linker may be cleavable or non-cleavable. Preferably, the linker is a non-cleavable linker.

[0220] Preferably, the compound is conjugated to a terminal nucleotide of the sequence. The compound may be conjugated to the terminal 5'- nucleotide, the terminal 3'- nucleotide, or a combination thereof, preferably the terminal 3'- nucleotide.Preferably, the compound is conjugated to the terminal 3'-nucleotide at the 3'- position.

[0221] Examples of a functionalised oligonucleotide according to the present disclosures, include TEG-amino, TEG-alkyl-maleimide, 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-a-tocopheryl)-triethyleneglycol-1-yl]-glyceryl group covalently linked to the 3'-position via a monophosphate group.

[0222] According to the third or fourth aspect, the disease, disorder or condition responsive to increased RIG-I signalling includes any disease, disorder or conditionresponsive to increased RIG-1 signalling that is not responsive to increased TLR7 and / or TLR8 signalling. The disease, disorder or condition is selected from the group consisting of: Aicardi-Goutieres syndrome (AGS), systemic lupus erythematosus (SLE), Singleton-Merten syndrome (SMS), Singleton-Merten syndrome 2 (SGMRT2), Selective IgA deficiency, dilated cardiomyopathy, psoriasis, Mitochondrial DNA (mtDNA) depletion syndrome, fulminant type 1 diabetes and slowly progressive insulin-dependent diabetes mellitus (SPIDDM).Administration

[0223] In one embodiment, the oligonucleotide is administered systemically.

[0224] As used herein “systemic administration” is a route of administration that is either enteral, intranasal, inhalation or parenteral.

[0225] As used herein “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.

[0226] As used herein, the term "intranasal" refers to administration to the nasal cavity of a subject such that a therapeutic agent is delivered directly to one or more epithelium located in the nose. In certain embodiments, intranasal administration is achieved using a liquid preparation (e.g., an aqueous preparation), an aerosolized preparation, or a dry powder preparation, each of which can be administered via an externally propelled or self-propelled (e.g., via inhalation) non-invasive nasal delivery device, or via a gel, cream, ointment, lotion, or paste directly applied to one or more nasal epithelium (e.g., olfactory epithelium or nasal respiratory epithelium).

[0227] As used herein, the term “inhalation” refers to intake of air to the alveoli. In specific examples, intake can occur by self-administration of a formulationof the invention while inhaling, for example, aerolised particles containing a therapeutic agent, or by administration via a respirator.

[0228] As used herein “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), 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.

[0229] In one embodiment, administration of the pharmaceutical composition is subcutaneous.

[0230] Preferably, administration of the pharmaceutical composition is intravenously.

[0231] 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.

[0232] In one embodiment, administration is 1 to 3 times per week, or once every week, two weeks, three weeks, four weeks, or once every two months.

[0233] In one embodiment, administration is once weekly.

[0234] In one embodiment, 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.

[0235] 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. In one embodiment doses are administered once per week. Thus in one embodiment, 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. In one embodiment 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.ExamplesMethodsCell Culture and Stimulation

[0236] p125 HEK 293 cells (Rehwinkel et al., Cell, 2010; 140(3): 397-408] naturally expressing RIG-I and stably expressing and IFN-b luciferase reporter 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). 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, and rather used in suspension. hTERT immortalised foreskin fibroblasts (Pepin et al., Nucleic Acids Res, 2017; 45(1): 198-205) 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) and sodium pyruvate. All the cells were cultured at 37°C with 5% CO₂. Cell lines were passaged 2-3 times a week and tested for mycoplasma contamination using Mycostrip (Invivogen).

[0237] PBMCs were purified from healthy donors with gradient purification as previously reported in Gantier & Williams (In: Min, WP., Ichim, T. (eds) RNA Interference. Methods in Molecular Biology, 2010; 623. Humana Press) and were grown in complete RPMI.

[0238] Indicated cells were treated with indicated concentration of oligonucleotides -20-60 min, prior to 3phpRNA (Invivogen) stimulation. 3phpRNA was delivered with lipofectamine 2000 at 50 ng / ml (final concentration), using a ratio of 40 ng 3phpRNA per 1 ul of lipofectamine 2000. Desalted trimer and longer oligonucleotides were synthesised by Integrated DNA Technologies (IDT), Syngenis Pty Ltd, or Wuxi Apptec and resuspended in RNase-free TE buffer, pH 8.0 (Thermo Fisher Scientific). 2’OMe is mX, DNA is dX, and phosphorothioate inter-nucleotide linkages are denoted with a *.Luciferase Assays

[0239] p125 HEK 293 cells were plated the day before 3phpRNA stimulation, and treated overnight with oligos and 3phpRNA. The next day, the cells were lysed in 40 µl (for a 96-well plate) of 1X Glo Lysis buffer (Promega) for 10 min at room temperature.15 µl of the lysate was then subjected to firefly luciferase assay using 40 µl of Luciferase Assay Reagent (Promega). Luminescence was quantified with a Fluostar OPTIMA (BMG LABTECH) luminometer.Detection of Cytokines

[0240] Human IP-10 levels were measured using supernatants from the different cultures and were quantified using IP-10 (BD Biosciences, #550926) ELISA kit, according to the manufacturers’ protocol. Tetramethylbenzidine substrate (Thermo Fisher Scientific) was used for quantification of the cytokines on a Fluostar OPTIMA (BMG LABTECH) plate-reader.

[0241] IL-8 levels were measured using supernatants from the different cultures and were quantified using IL-8 (BD Biosciences, # 555244) ELISA kit, according to the manufacturers’ protocol.Statistical Analyses

[0242] Statistical analyses were carried out using Prism 10 (GraphPad Software Inc.). Every experiment was repeated a minimum of two independent times (except the oligo screens and hTERT data). One-way analyses of variance (ANOVA) with uncorrected Fisher’s LSD tests were used when comparing groups of conditions.Example 1 - Antagonistic activities of 2'-OMe 20 mer ASOs on RIG-I sensing

[0243] The inventors demonstrated antagonism of cytosolic cGAS by 2'-O-methyl (2-OMe) 20-mer gapmer phosphorothioate (PS) antisense oligonucleotides (ASOs) and hypothesised that 2'-OMe ASOs could also modulate cytosolic RNA sensing by RIG-I. An unbiased screen of a set of 882'-OMe PS ASOs was completed (Plate 1). For this purpose, HEK 293 cells stably expressing an IFN-p promoter-luciferase (referred to as p125 HEK 293 cells) were pre-treated with 50 nM of ASOs for 1 h, prior to stimulation of RIG-I using a selective commercial RIG-I agonist (3phpRNA - Invivogen). 52 of the 88 ASOs inhibited RIG-I sensing by >50%, with 14 of the 88 ASOs inhibiting RIG-I sensingby >80%, clearly indicating of sequence-selective RIG-1 antagonism by 2'-OMe gapmer ASOs (Figure 1).

[0244] The top two ASOs (H2 and F10) were validated in the same cells to confirm the robustness of the observations (Figure 2). In agreement with the screen data, both 20-mer ASOs significantly blunted RIG-I sensing of 3phpRNA, with H2 being the most potent (>70% inhibition of RIG-I sensing).

[0245] To gain insights into potential motifs at play in RIG-I antagonism, sequence alignment analyses using the MEME tool was performed (Figure 3).

[0246] These in silico analyses suggested a common ATCAA motif between the two top antagonists (the motif being partially overlapping the 3'-end 2'-OMe region of H2, while being located in the central DNA region of F10). Noting that a closely related motif was present in another ASO, C2P2-Mut1 (also referred to as C2Mut1), a set of oligonucleotides derived from C2P2-Mut1 were tested (Figure 4 - the putative antagonistic motif in H2 / F10 is underlined in C2P2-Mut1).

[0247] These analyses of the C2P2-Mut1 variants confirmed the trends from the ASO screen, with clear sequence specific effects on the potency of RIG-I antagonism.Critically, changing two bases in C2P2-Mut1 significantly enhanced antagonism in C2P2-Mut1v1 (changing the ATCCA DNA motif to TTCCA being potentially at play). Surprisingly, replacing the entire central DNA region of C2P2-Mut1 to a string of dC bases rather increased antagonism as seen with C2P2-Mut1-10DNA (Figure 4), suggesting further a role for the central DNA region (or 2'-OMe / DNA junctions) in the antagonism. Nonetheless, the 5'- and 3'-end 2'-OMe wing regions were also clearly at play in the RIG-I antagonistic activity of the ASOs. As such, modification of the 3'-end 2'-OMe region in C2P2-Mut1-3Mod1 and 3Mod2 ASOs also significantly increased antagonism.

[0248] Counterintuitively given the above points, shortening C2P2-Mut1 to a 15-mer oligo, and thereby removing the 3'-end 2'-OMe region rather increased antagonism -establishing that 15 bases were sufficient to engage with RIG-I antagonism.

[0249] These analyses also revealed that C2Mut1-dC, a variant of C2P2-Mut1 with low sequence complexity reproducing a section of the 5'-end of C2P2-Mut1, only had modest antagonistic activity on RIG-I. In contrast, appending a mGmC motif at the 5'-end of C2Mut1-dC strongly increased RIG-I antagonism - compared to both C2Mut1-dC and C2P2-Mut1 (Figure 4). This specific finding suggested that 5'-end mGmCmG, which is naturally present in all the C2P2-Mut1 ASOs tested here, is directly at play in RIG-I antagonism.

[0250] These responses were also confirmed in monocytic TH P-1 cells, which produce IP-10 upon stimulation with RIG-I agonists. As shown in Figure 5, RIG-I antagonism of C2P2-Mut1 was limited in this set up but strongly enhanced for C2P2-Mut1v1, C2P2-Mut1-3Mod1, C2P2-Mut1-10DNA and C2Mut1-dC-5GC. Critically, C2P2-Mut1-15 was significantly more antagonistic than C2P2-Mut1 suggesting that shortening to a 15-mer was actually beneficial to the antagonism in this sequence context.

[0251] The levels of interferon beta (IFN-P), which is produced down-stream of RIG-I activation by 3phpRNA in THP-1 cells, were also analysed. As shown in Figure 6, these results mirrored those obtained with IP-10, with modification of several key bases of C2P2-Mut1 significantly hampering IFN-p levels (e.g. C2P2-Mut1-v1, C2P2-Mut1-GGG, C2P2-Mut1-3DNA, C2P2-Mut1-3Mod1, Mut1-3Mod2, C2P2-Mut1-10DNA and C2P2-Mut1-15 being more potent than C2P2 and C2P2Mut1).

[0252] Similarly, the addition of 5'-end mGmC to C2Mut1-dC (in C2Mut1-dC-5GC) strongly increased its antagonistic function on IFN-p production.

[0253] Given its relative low sequence complexity, the motifs underpinning the inhibitory activity of C2Mut1-dC-5GC on RIG-I sensing were investigated further. For this purpose, 3 new sequence variants were generated. The first variant, C2Mut1-dC-5GC-DNA, was synthesised using 5'-dGdC instead of mGmC, thereby allowing assessment of the impact of these 2'-OMe moieties in the antagonistic activity on RIG-I. Two additional variants, where the mG*mC*mG*mG*mU*dA*dT motif was positioned in the middle or the 3'-end of the 20-mers, were also synthesised to assess whether the motif location affects RIG-I antagonism (Figure 7).

[0254] As shown in Figure 7, a significant decrease of RIG-I antagonism was observed when the 5'end bases were converted to DNA bases (dGdC in C2Mut1-dC-5GC-DNA), indicating an important role for 2'-OMe residues at these positions in this antagonistic activity. In addition, the location of the antagonistic motif within the 20-meralso was at play, as evidenced by the decreased RIG-1 antagonism seen with the central motif (C2Mut1-dC-5GC-2) or at the 3'end (C2Mut1-dC-5GC-3).

[0255] To further validate the antagonistic activity of some of the best oligos studied above, their antagonistic effect on other agonists of RIG-I was tested. High molecular weight poly(IC), which activates both RIG-I and MDA-5, was first tested in THP-1 cells (Figure 8A). The antagonistic effects of C2P2-Mut1v1 on the RIG-I response to a T7 synthesised mRNA, which was only partially capped thereby containing 5'-triphosphate moieties which activate RIG-I, was also tested (Figure 8B).

[0256] Similar to what was observed with the canonical RIG-I agonist, 3phpRNA, both high molecular weight poly(IC) and T7 mRNA strongly induced IP-10 production in undifferentiated THP-1 cells, and this was decreased with the RIG-I antagonists (C2P2-Mut1-v1 and C2Mut1-3Mod1 for pIC, and C2P2-Mut1-v1 for mRNA sensing).

[0257] Interestingly, C2Mut1-3Mod1 was slightly more potent than C2P2-Mut1-v1 to block pIC sensing, suggesting it may also act on another sensor such as MDA5 since both exhibited similar potency on detection of the RIG-I agonist 3phpRNA.

[0258] Collectively, these experiments established the capacity of the oligonucleotides to block RIG-I activation in a sequence / motif specific manner.Example 2 - Antagonistic activities of short 2'-OMe oligonucleotides on RIG-I sensingThe data suggested that a 15-mer oligonucleotide could significantly antagonise RIG-I -as seen with C2P2Mut1-15 (Figures 4, 5 and 6). Having previously demonstrated the capacity of 2'-O-Methyl containing 3-mer oligonucleotides to antagonise TLR7 and TLR8, we investigated whether such short oligonucleotides could also affect RIG-I sensing.

[0259] For this purpose, a screen of 64 2'-OMe PS 3-mers was conducted in THP-1 cells stimulated with 3phpRNA. A concentration of 5 µM of the 3-mers, as a highest dose that was considered to be biologically relevant for the other receptors, was used (Figure 9).

[0260] This screen identified several 3-mer sequences with strong inhibitory activity on RIG-1, including mGmUmU and mGmCmG (Figure 9). The activities of these selective 3-mer oligonucleotides were validated in THP-1 cells (Figure 10).

[0261] These experiments confirmed that mGmCmG and mGmUmU were antagonistic of RIG-I (Figure 10). It should be noted that the antagonistic activity of mGmCmG is directly aligned with the fact that this motif is missing at the 5'end of C2Mut1-dC and is reconstituted in C2Mut1-dC-5GC - thereby suggesting that it is the region of C2Mut1-dC-5GC driving the interaction with RIG-I to block its activation. Aligning with this, however, the antagonistic motif worked better at the 5'-end of the oligonucleotide (as seen with a loss of activity with C2Mut1-dC-5GC-2 and C2Mut1-dC-5GC-3), indicative that the interaction with RIG-I is hampered by 5'-end extension of this mGmCmG motif. These results therefore indicate that the optimal location for this mGmCmG motif appears to be at the 5'-end of an oligonucleotide.

[0262] Nonetheless, other sequence requirements can also be involved in RIG-I antagonism, and it is worth noting that mGmUmU is present in the middle of C2P2-Mut1-v1 as a mGmUdT motif - which is uniquely present in this long sequence (Figure 4).

[0263] Finally, the activity of mGmCmG was tested in two different clones of hTERT immortalised fibroblasts stimulated with 3phpRNA (Figure 11).

[0264] These analyses confirmed the robust antagonistic activity of mGmCmG on RIG-I sensing, in human fibroblasts (Figure 11).

[0265] Based on the concept that mGmUdT may be at play in the antagonistic activity of C2P2-Mut1-v1, we also tested a series of GCG 3-mers with mixed base modifications we previously found could improve activities on TLR7 or TLR8 antagonism. As shown in Figure 12, 3'-end LNA-modification of mGmC+G increased antagonism of 2'-OMe GCG 3-mers in these experiments, indicating that 3'-end modification may also increase antagonism of RIG-I sensing (Figure 12).

[0266] Similarly, 3'-end LNA-modification of mGmU+T increased antagonism of 2'-OMe GUU 3-mers (Figure 13).Example 3 - Activity of C2Mut1 -variants in hTERT-immortalised fibroblasts

[0267] Next, the inventors compared the activity of C2P2-Mut1-v1, C2Mut1-dC-5GC, C2Mut1-dC and C2Mut1-3Mod1 in hTERT immortalised foreskin fibroblasts. The cells were pre-treated with 200 nM of the oligonucleotides, C2Mut1-dC acting as a negative control (due to its limited inhibitory activity in the previous THP-1 cell based assays), prior to RIG-I stimulation with transfected 3phpRNA (Figure 14).

[0268] Consistent with the analyses in THP-1 cells, C2Mut1-3mod1 was the most potent inhibitor and was the only oligo blunting IL-8 production by the cells in these assays, suggesting its broad activity on both IRF3 and NF-kB branches of inflammation down-stream of RIG-I activation.Example 4 - Activity of C2Mut1-3Mod1 on primary human blood cells

[0269] Having found that C2Mut1-3Mod1 (also referred to as C2P2Mut1-3Mod1 herein) was a potent RIG-I antagonist and could inhibit pIC sensing in THP-1 cells, the inventors next tested its effect in the context of human peripheral blood mononuclear cells (PBMCs).

[0270] In PBMCs, C2Mut1-3Mod1 pre-treatment halved the production of IP-10 induced by pIC transfection, at the dose used, confirming the relevance of its antiinflammatory effect on RIG-I Like Receptor (RLR) signalling in primary cells (Figure 15). Example 5 - Activity of C2Mut1-3Mod1 on inhibition of self-amplifying RNA sensing

[0271] Similar to the observations in Example 2 in relation to T7 mRNA, transfection of a commercial self-amplifying (sa)RNA induced IP-10 production in undifferentiated THP-1 cells. IP-10 production was significantly decreased with pre-treatment with C2Mut1-3Mod1 and C2Mut1-dC-5GC (Figure 16).Example 6 - Activity of C2Mut1-3Mod1 variants

[0272] Given C2Mut1-3Mod1’s broad inhibitory effect on RNA sensing (e.g. 3phpRNA, pIC, saRNA) in different cell systems (HEK, THP-1, hTERT fibroblasts and PBMCs), and its only difference from the parental C2Mut1 oligonucleotide being two 3'-end terminal bases, the inventors tested the impact of single base variations to assess itsstructure-activity relationship (SAR). These included sugar modifications to the 3'-end (V1-V8) and modification of the 5-base motif towards the 5'-end.

[0273] While 3mod1-v4 showed some variability, suggesting a negative impact of the last two bases being mC and +C (LNA-C) on antagonism, the only modification significantly dampening the inhibitory activity of the parental C2P2-Mut1-3mod1 at this concentration was in 3mod1-v8, although 3mod1-8 was still able to inhibit RIG-I sensing (Figure 17). This demonstrates-that all the variants tested exhibited similar antagonistic activity on RIG-I sensing at this dose and time, with the exception of v8 that had lower antagonistic activity on RIG-I sensing.

[0274] The inventors next investigated single base modifications at the 5'-end of C2Mut1-3Mod1 (V12-V16), modifications to a 5-base motif (V17-V20), incorporation of consecutive guanosine bases to increase TLR9 antagonism (V21-V27), and inclusion of TEG linker at the 3'-end via a 3'-phosphorothioate group (V28). Similar to the C2Mut1-3Mod1-v1 to v11, all of the C2Mut1-3Mod1-v12 to v28 variants retained inhibitory activity on RIG-I after 6 h incubation (Figure 18 - top panel). However, when using a longer incubation time after RIG-I stimulation, select modifications were seen to impair the activity of the parental molecule (Figure 18 - bottom panel).

[0275] As such, the significant decrease of RIG-I antagonism seen with both 3mod1-v12 and v17 underlines the importance of the mG residue at the 3rd position in the sequence (from 5'-end) - since mil at this position dampens antagonism for both oligonucleotides. Similarly, the mGmU residues at the 4th and 5th positions in the sequence (from 5'-end) are also important as seen in the decreased activity of the 3mod1-v19 variant where these bases were changed to DNA. Interestingly, however, positions 6-14 appear more flexible and can be changed to dGdGdG triplets without significantly impacting function (as suggested by 3mod1-v21 to v26). Similarly, 3'-end modification from mGdCdC to dGdGdG did not have an impact (see 3mod1-v27).

[0276] Further, the inventors also investigated whether 3'-end extension of C2P2-Mut1-3mod1 with a TEG group could impair function. As seen in Figure 18, this extension did not impact activity suggesting that the oligonucleotide could be functionalised at the 3'-end without impacting RIG-I antagonism.Conclusions

[0277] These analyses establish the capacity of 2'-OMe modified PS oligonucleotides to antagonise RIG-1 sensing of its agonists. It is considered that very selective 2'-OMe motifs underpin this activity, and these motifs can be as short as 3-base long. The demonstration that 3-bases were sufficient to lead a biologically relevant antagonistic activity is indicative of broad natural antagonism driven by catabolism of abundant 2'-OMe RNA fragments to maintain steady state.

[0278] These analyses further establish the capacity of 3mod1 and its variants to inhibit RIG-I, while underlining the importance of the 5'-end mGmCmG motif.

Claims

CLAIMS1. An oligonucleotide comprising a sequence defined by:mG*mC*XA*Xc*XD*XEwhereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mU or dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, mA is 2'-0Me adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 2.

2. The oligonucleotide according to claim 1, wherein the internucleotide linkage is a phosphorothioate linkage.

3. The oligonucleotide according to claim 2, wherein the phosphorothioate linkage is a 3’-5’ phosphorothioate linkage.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide is between about 3 and about 50 nucleotides.

5. The oligonucleotide according to any one of claims 1 to 4, wherein the sequence is located at the 5’ terminal end of the oligonucleotide.

6. The oligonucleotide according to any one of claims 1 to 5, wherein the sequence is definedmG*mC*mG*mG*[m7. The oligonucleotide according to any one of claims 1 to 6, wherein the oligonucleotide is about 4 to about 20 nucleotides in length, the sequence is defined by mG*mC*XA*Xc*XD*XE, and wherein the sequence is located at the 5’ terminal end of the oligonucleotide.

8. The oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 3.

9. The oligonucleotide according to claim 8, wherein the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 4.

10. The oligonucleotide according to claim 9, wherein the oligonucleotide comprises or consists of the sequence:mG*mC*mG*mG*mll*dA*dT*dG*dG*dG*dT*dG*dT*dC*dC*mC*mA*mG*dC*Dc (SEQ ID NO: 70), mG*mC*mG*mG*mU*dA*dG*dG*dG*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 71), mG*mC*mG*mG*mU*dG*dG*dG*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 72) mG*mC*mG*mG*mU*dA*dT*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*dC*Dc (SEQ ID NO: 46), or mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

11. The oligonucleotide according to claim 10, wherein the oligonucleotide comprises or consists of the sequence:mG*mC*mG*mG*mU*dA*dT*dC*dC*dA*dT*dG*dT*dC*dC*mC*mA*mG*dC*dC (SEQ ID NO: 42).

12. The oligonucleotide according to any one of claims 1 to 11, wherein the oligonucleotide inhibits TLR7 and / or TLR8.

13. An oligonucleotide comprising a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is LNA thymidine; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 1.

14. An oligonucleotide comprising a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG or +G; andXBis mU, dT or +T;wherein mG is 2'-0Me guanosine, mC is 2'-OMe cytidine, mil is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is LNA guanosine, and +T is LNA thymidine; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-1 activity; andwherein the oligonucleotide does not consist of a sequence selected from the group of oligonucleotides in Table 1.

15. A composition comprising an oligonucleotide according to any one of claims 1 to 14.

16. An immunogenic composition comprising an oligonucleotide according to any one of claims 1 to 14, and a therapeutic RNA.

17. A method of inhibiting RIG-I activity in a cell, the method comprising contacting the cell with an oligonucleotide according to any one of claims 1 to 14, or a composition according to claim 15 or 16, thereby inhibiting RIG-I activity in the cell.

18. A method of inhibiting RIG-I activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 14, or a composition according to claim 15 or 16, thereby inhibiting RIG-I activity in the subject.

19. A method of treating or preventing a disease, disorder or condition in a subject that is responsive to RIG-I inhibition, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 14, or a composition according to claim 15 or 16, thereby treating or preventing the disease, disorder or condition in the subject.

20. A method of reducing or minimising a symptom associated with diseases, disorders and conditions responsive to RIG-I inhibition in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 14, or a composition according to claim 15 or 16, thereby reducing or minimising a symptom associated with diseases, disorders and conditions responsive to RIG-I inhibition in the subject.

21. The method according to claim 20, wherein the symptoms associated with diseases, disorders and conditions responsive to RIG-I inhibition are one or more of: inflammation, fever, muscle aches, and fatigue.

22. The method according to any one of claims 19 to 21, wherein the condition responsive to RIG-I inhibition is mRNA administration.

23. A method of preventing or inhibiting inflammation associated with administration of a therapeutic RNA in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 14, or a composition according to claim 15 or 16, thereby preventing or inhibiting inflammation associated with administration of a therapeutic RNA in the subject.

24. A method of inhibiting RIG-I activity in a cell, the method comprising contacting the cell with an oligonucleotide comprising a sequence defined by:mG*mC*XA*Xc*XD*XEwhereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mU or dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, mA is 2'-OMe adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

25. A method of inhibiting RIG-1 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide comprising a sequence defined by:mG*mC*XA*Xc*XD*XEwhereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mU or dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, mA is 2'-0Me adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-1 activity26. A method of treating or preventing a disease, disorder or condition in a subject responsive to increased RIG-1 signalling, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide comprising a sequence defined by:mG*mC*XA*Xc*XD*XEwhereinXAis mG, dG, +G or mU;Xcis mG, dG or mU;XDis mG, mU or dU; andXEis dA, dU, mA, dG, dT or dC;wherein mG is 2'-0Me guanosine, mC is 2'-OMe cytidine, mil is 2'-OMe uridine, mA is 2'-OMe adenosine, dG is 2 '-deoxyribose guanosine, dU is 2 '-deoxyribose uridine, dT is 2'-deoxyribose thymidine, dC is 2'-deoxyribose cytidine and +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and * is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

27. The method according to claim 26, wherein the disease, disorder or condition responsive to increased RIG-1 signalling is a disease, disorder or condition responsive to increased RIG-1 signalling that is not responsive to increased TLR7 and / or TLR8 signalling.

28. The method according to any one of claims 24 to 27, wherein each internucleotide linkage is a phosphorothioate linkage.

29. The method according to claim 28, wherein each phosphorothioate linkage is a 3’-5’ phosphorothioate linkage.

30. The method according to any one of claims 24 to 29, wherein the oligonucleotide is between about 3 and about 50 nucleotides.

31. The method according to any one of claims 24 to 30, wherein the sequence is located at the 5’ terminal end of the oligonucleotide.

32. The method according to any one of claims 24 to 31, wherein the oligonucleotide comprises or consists of a sequence selected from the group of oligonucleotides in Table 5.

33. The method according to claim 32, wherein the oligonucleotide comprises or consists of a sequence selected the group of oligonucleotides in Table 6.

34. The method according to claim 33, wherein, the oligonucleotide consists of a sequence selected from the group consisting of: mG*mC*mG, mG*mC*dG, mG*mC*+G, mG*mU*mU, mG*mU*dT, and mG*mU*+T.

35. The method according to claim 34, wherein the oligonucleotide consists of a sequence selected from the group consisting of: mG*mC*mG, mG*mC*dG, mG*mC*+G and mG*mU*mU.

36. A method of inhibiting RIG-1 activity in a cell, the method comprising contacting the cell with an oligonucleotide for inhibiting RIG-1, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

37. A method of inhibiting RIG-1 activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide for inhibiting RIG-1, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

38. A method of treating or preventing a disease, disorder or condition in a subject responsive to increased RIG-1 signalling, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide for inhibiting RIG-1, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XBwhereinXAis mG, dG, +G or mU; andXBis mU, dT or +T;wherein mG is 2'-0Me guanosine, mC is 2'-0Me cytidine, mU is 2'-0Me uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is a guanosine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group, and +T is a thymidine analogue comprising a ribofuranose sugar comprising a 2’-O,4’-C bridging group; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

39. The method of any one of claims 36 to 38, wherein the oligonucleotide comprises a sequence selected from the group consisting of:mG*mC*XA; andmG*mU*XB;whereinXAis mG, dG or +G; andXBis mU, dT or +T;wherein mG is 2'-OMe guanosine, mC is 2'-OMe cytidine, mU is 2'-OMe uridine, dG is 2’-deoxyribose guanosine, dT is 2’-deoxyribose thymidine, +G is LNA guanosine, and +T is LNA thymidine; and* is independently an internucleotide linkage;wherein the oligonucleotide inhibits RIG-I activity.

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