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

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.

Roles of Emerging RNA-Binding Activity of cGAS in Innate Antiviral Response

“…Interestingly, a few ASOs can enhance cGAS sensing when used at low concentrations. The binding of these ASOs to the third DNA binding domain within cGAS was speculated to facilitate the formation of the cGAS oligomer and thus increases its enzymatic activity (70,71). These works suggest that RNA may compete for cGAS binding to dsDNA and inhibit cGAS activity, which has also been confirmed by a preprint study (25).…”

“…As mentioned earlier in the article, cGAS binds to DNA in a sequence-independent manner, which is mainly associated with the length and structures of DNA (21). However, for RNA, it is clear that short synthetic oligos can compete with DNA to bind cGAS in a sequence-dependent manner (70,79), suggesting that it is the sequence rather than the length of the RNA that may be important for the DNA binding activity of cGAS. In summary, a series of RNAs, such as synthetic oligos (70), tRNA (25), and circRNA (22), which all belong to ssRNA, can bind to cGAS and regulate its activity, whereas dsRNA can bind cGAS but does not affect the activity of cGAS (21).…”

“…Although cGAS has been widely regarded as a classical DNAbinding protein, some recent studies have uncovered or implied RNA-binding activity of cGAS. Recent in vitro and in vivo research has shown that 2′-O-methyl (2′-OMe) gapmer antisense oligonucleotides (ASOs) exhibit sequence-dependent inhibition of sensing by the RNA sensor TLR7 (69) and two major DNA sensors, cGAS and TLR9 (70). The 2'-OMemodified RNA motifs within synthetic oligonucleotides can inhibit the recognition of cGAS DNA in a sequence-dependent manner which is independent of cGAS mRNA targeting.…”

“…However, for RNA, it is clear that short synthetic oligos can compete with DNA to bind cGAS in a sequence-dependent manner (70,79), suggesting that it is the sequence rather than the length of the RNA that may be important for the DNA binding activity of cGAS. In summary, a series of RNAs, such as synthetic oligos (70), tRNA (25), and circRNA (22), which all belong to ssRNA, can bind to cGAS and regulate its activity, whereas dsRNA can bind cGAS but does not affect the activity of cGAS (21). These results indicate that ssRNA may be able to influence cGAS activity to a greater extent than dsRNA.…”

Pharmacological Targeting of STING-Dependent IL-6 Production in Cancer Cells

“…Conversely, ERK1/2 inhibition with SCH772984 strongly limited the expansion of MG-63 and TC-1 cells, and modestly impacted that of HOS cells ( Figures 4A,B ). Having previously shown that MG-63 had a functional cGAS-STING response ( Valentin et al, 2021 ), we reasoned that H151 may block canonical STING signaling basally engaged in these cells, normally restraining their growth. Accordingly, the basal expression of several ISGs ( RSAD2 , IFIT1 , IFIT2 , IFIT3 ) was significantly decreased by H151 treatment in MG-63 cells ( Figure 4D ).…”