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

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.

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

“…Data shown are averaged from two independent experiments in biological triplicate (± s.e.m and ordinary one-way ANOVA with Tukey's multiple comparison tests to the ‘ISD70 only’ condition, or otherwise indicated pairs of conditions are shown). There was no basal effect of the ASOs on NT cells ( 21 ). ( C ) HT-29 cells pre-treated overnight with 125, 250 or 500 nM of indicated ASOs, were transfected or not (non-treated [NT]) with 2.5 μg/ml of ISD70 for 24h, and IP-10 levels in supernatants were determined by ELISA.…”

“…Unexpectedly, we demonstrated that the majority of the 2′OMe gapmer ASOs tested were potent inhibitors of TLR7, but that selected sequences harbouring 2′OMe ‘CUU’ motifs did not block TLR7 activation ( 21 ). Conversely, we found that TLR8 sensing of the low molecular weight synthetic ligand Resiquimod (referred to as R848 hereafter), was potentiated by select 2′OMe ASOs, in a sequence-dependent manner, an observation that aligns with previous findings with poly-dT PS ODNs ( 21–23 ). These findings prompted us to investigate the broader immunomodulatory effects of 2′OMe ASOs on nucleic sensing, with the aim of defining the molecular patterns controlling these activities.…”

“…We recently investigated the TLR7 and TLR8 immunomodulatory effect of RNase-H1-recruiting gapmer 2′OMe ASOs, which have a central DNA core of 10 nt flanked by 5 nt of 2′OMe, on a PS backbone ( 21 ). Unexpectedly, we demonstrated that the majority of the 2′OMe gapmer ASOs tested were potent inhibitors of TLR7, but that selected sequences harbouring 2′OMe ‘CUU’ motifs did not block TLR7 activation ( 21 ).…”

“…We recently investigated the TLR7 and TLR8 immunomodulatory effect of RNase-H1-recruiting gapmer 2′OMe ASOs, which have a central DNA core of 10 nt flanked by 5 nt of 2′OMe, on a PS backbone ( 21 ). Unexpectedly, we demonstrated that the majority of the 2′OMe gapmer ASOs tested were potent inhibitors of TLR7, but that selected sequences harbouring 2′OMe ‘CUU’ motifs did not block TLR7 activation ( 21 ). Conversely, we found that TLR8 sensing of the low molecular weight synthetic ligand Resiquimod (referred to as R848 hereafter), was potentiated by select 2′OMe ASOs, in a sequence-dependent manner, an observation that aligns with previous findings with poly-dT PS ODNs ( 21–23 ).…”

Low compositions of human toll-like receptor 7/8-stimulating RNA motifs in the MERS-CoV, SARS-CoV and SARS-CoV-2 genomes imply a substantial ability to evade human innate immunity

“…In the past, many studies have focused on developing TLR7 agonists to activate host inflammatory responses to cope with RNA virus infections ( Alharbi et al, 2020 ; Shah et al, 2016 ). For example, TLR7 agonists were shown to provide protection against influenza A virus-induced morbidity in mice ( To et al, 2019 ).…”