Noxopharm collaborators at Hudson confirm the relevance of TLR7 activation in Lupus validating the great potential of Noxopharm’s in-licenced molecules, comprising a new class of TLR7 inhibitors.

27 April 2022 by Pharmorage

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Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2′-O-methyl gapmer oligonucleotides

8 November 2021 by Pharmorage

Valentin R, Wong C, Alharbi AS, Pradeloux S, Morros MP, Lennox KA, Ellyard JI, Garcin AJ, Ullah TR, Kusuma GD, Pépin G, Li HM, Pearson JS, Ferrand J, Lim R, Veedu RN, Morand EF, Vinuesa CG, Behlke MA, Gantier MP. Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2′-O-methyl gapmer oligonucleotides. Nucleic Acids Res. 2021 Jun 21;49(11):6082-6099. DOI: 10.1093/nar/gkab451

Abstract:

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2_-O-methyl (2_OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2_OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2_OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2_OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.

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Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists

8 November 2021 by Pharmorage

Alharbi AS, Garcin AJ, Lennox KA, Pradeloux S, Wong C, Straub S, Valentin R, Pépin G, Li HM, Nold MF, Nold-Petry CA, Behlke MA, Gantier MP. Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists. Nucleic Acids Res. 2020 Jul 27;48(13):7052-7065. DOI: 10.1093/nar/gkaa523.

Abstract:

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2′-methoxyethyl (2′MOE) modifications. Through a screen of 192 2′OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2′OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.

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Inhibition of STING Signaling with Flavonoid Compounds

8 November 2021 by Pharmorage

T R Ullah1, 2, RL Ambrose1, 2, O Laczka3, KR Balka4, 5, 6, D De Nardo4, 5, 6, MP Gantier1, 2

1. Molecular and translational Science, Monash University

2. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria

3. Noxopharm Limited , Gordon NSW 2072 Australia, Sydney

4. Department of Anatomy and Developmental Biology , Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria

5. Department of Medical Biology, The University of Melbourne

6. The Walter and Eliza Hall Institute of Medical Research, Inflammation division, Parkville, Victoria, Australia

Abstract Content

The cGAS-STING pathway plays a major role in aberrant immune responses seen in autoinflammatory diseases including interferonopathies such as the Aicardi-Goutieres syndrome. Critically, this pathway can be amplified through the propagation of cyclic GMP-AMP (cGAMP), the product of cGAS, between cell: cell interactions, known as gap junctions. Flavonoid compounds have been suggested to impact the activity of gap junction, but whether this can alter propagation of cGAS-STING signalling is not known. In co-cultures of cGAMP donor cells with STING expressing recipient cells, Genistein was the only flavonoid tested to decrease adjacent cell transactivation. This effect was concurrent with a decreased gap junction intercellular communication. Critically, Genistein pre-treatment of STING expressing recipient cells abolished cellular transactivation, indicating a direct effect on STING signalling. Accordingly, Genistein significantly decreased STING signalling and its antiviral effect against Semliki Forest Virus, upon activation by its agonist. Short pre-treatment with Genistein impacted STING phosphorylation, along with that of IRF3 and IKKe, confirming a direct effect on STING signalling. Further, Idronoxil, a synthetic variant of Genistein with great safety profile in clinical trials over >800 patients, was a >30 fold more potent inhibitor of STING signalling than Genistein.

These findings establish a converging inhibitory activity of Genistein on cGAS-STING signalling, acting on preventing cGAMP transfer between adjacent cells, and preventing STING activation. In addition, select flavonoid compounds such as Idronoxil may present novel therapeutic opportunities to inhibit the cGAS-STING pathway involved in a growing number of auto-inflammatory diseases.

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Characterization of 2’-O-methyl oligonucleotides that potently inhibit cGAS and TLR9 DNA sensing

8 November 2021 by Pharmorage

R Valentin, C Wong1, AS Alharbi1, KA Lennox2, MA Behlke2, MP Gantier1, 3

1. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Australia

2. Integrated DNA Technologies Inc, Coralville, United States

3. Department of Molecular and Translational Science, Monash University, Clayton, Australia

Abstract Content: Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2’-O-methyl (2’OMe) antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2’OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2’OMe ASOs and sequence mutants, we characterized key features regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. This novel cGAS inhibiting oligonucleotide limited the basal interferon gene signature seen in primary macrophages from TREX1-mutant mice, and also decreased senescence of primary human cells. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the 2’OMe ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2’OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development. In addition, our works establish proof of principle that oligonucleotides inhibiting cGAS, with strong therapeutic potential in select interferonopathies, can be rationally designed.

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