163: Sequence-dependent off-target inhibition of TLR7/8 by synthetic microRNA inhibitors

Abstract

Anti-microRNA (miRNA) oligonucleotides (AMOs) are commonly used to study miRNA function and present clear therapeutic potential. 2′-O-methyl (2′Ome) modified AMOs are among the most popular AMOs and can be modified to achieve high potency, high specificity and low cytoxicity. Nonetheless, 2′Ome oligonucleotide modification has previously been associated with an inhibitory activity on the sensing of pathogenic RNAs by innate immune sensors Toll-like receptor 7 and 8 (TLR7/8). In this work, we report on the ability of 2′Ome AMOs to inhibit TLR7 sensing of RNA in a sequence-dependent manner, independent of their miRNA-targeting function. Through a screen of 30 AMOs targeting common miRNAs, we found a subset of highly inhibitory sequences in mouse macrophages. Interspecies conservation of inhibitory activity on TLR7/8 was confirmed in human peripheral blood mononuclear cells – with an IC 50 activity as low as 2 nmol/l. Critically, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 70 AMOs targeted to human miRNAs in miRBaseV20. DNA/LNA AMOs synthesised with a phosphorothioate backbone also inhibited TLR7 in a sequence-dependent manner, demonstrating that the off-target effects of AMOs on TLR7/8 are not restricted to 2′Ome AMOs. Collectively, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account when studying the role of miRNAs in immune responses.