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

Abstract

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2′-O-Methyl (2′OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2′OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2′OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application.

Clinical potential of oligonucleotide-based therapeutics in the respiratory system

“…Advantageously, nucleoside analogues (Table 1 and incorporated in RNAi mediators act as a TLR7/8 antagonists (Robbins et al, 2007;Sarvestani et al, 2015;Sioud, Furset, & Cekaite, 2007), with at least one of these, 2'-O-methyl, altering RISC off-target effect profiles (Fedorov et al, 2006;Jackson et al, 2006). The impact of other modifications on RISC off-target effects has not been publicly disclosed.…”

miRNA Targeting Drugs: The Next Blockbusters?

“…Cells of the innate immune system express Toll-like receptors ( TLRs ), a type of pattern recognition receptor (PRR), those recognize molecules from pathogens and, therefore, play a central role in microbial defense. Several TLRs (TLR3, TLR7 , TLR8 , and TLR9 ) have been identifi ed that induce interferon alpha (IFN-α) response after binding a short, both single-or double-stranded nucleic acid [ 32 ]. Because of this, the interaction of a potential anti-miR drug is routinely investigated in vitro and in vivo in hope to avoid receptor activation.…”

Assessing the Off-Target Effects of miRNA Inhibitors on Innate Immune Toll-Like Receptors

“…pNF-kB-Luc4 reporter, expressing the NF-kB response element (5′-GGGAATTTCCGGGAATTTCCGG GAATTTCCGGGAATTTCC-3′) upstream of the Firefl y luciferase reporter (Clontech). 7. miR-16 AMO LNA/ DNA [ 31 ] (also referred to as "miR-16 DNA/LNA PS" [ 16 ]; 5′-c*g* ± C *c*a* ± A *t*a* ± T *t*t* ± A *c*g* ± T *g*c* ± T *g*c* ± T *a-3′, where lowercase is for DNA, ± underlined for LNA, with the asterisks for phosphorothioate linkages (Integrated DNA Technologies). Stock solution at 4 μM in sterile RNase-free TE buffer (Life Technologies).…”