T R Ullah^{1, 2}, RL Ambrose^{1, 2}, O Laczka³, KR Balka^{4, 5, 6}, D De Nardo^{4, 5, 6}, MP Gantier^{1, 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.

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.