Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice

Abstract

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.

Evaluation of off‐target effects of gapmer antisense oligonucleotides using human cells

“…Our analysis indicated that not only the expression of genes with mis, but also those with ins or del, has the potential to be affected by off‐target effects induced by gapmer ASOs (Table ). This finding is agreement with previous reports showing off‐target effects of LNA gapmers (Hagedorn et al, ; Kamola et al, ). We further analyzed the proportion of off‐target genes to the off‐target candidate genes in mis = 1, ins = 1 and del = 1 groups, and found that a significant proportion of ins = 1 and del = 1 genes was down‐regulated to less than 50% of the control in a similar manner to that of mis = 1 genes (Table S1).…”

“…Some of the off‐target genes identified by this scheme could be downstream targets of the on‐target gene (not real off‐target genes). These downstream targets could be identified as commonly down‐regulated genes by microarray analysis using another ASO against the same on‐target gene as described previously (Hagedorn et al, ); however, in the light of prediction of gene expression change in human, we consider that the downstream targets are not needed to be excluded purposely from off‐target genes because they are also predicted to be down‐regulated in humans in a similar manner to the real off‐target genes.…”

Likelihood of Nonspecific Activity of Gapmer Antisense Oligonucleotides Is Associated with Relative Hybridization Free Energy

“…The correlation of mismatch destabilization with likelihood of reduction of an unintended transcript is similar to results reported by Lindow and colleagues [6,7]. Although they could not compare potency of intended and unintended targets, they report that unintended targets with observed reduction had significantly more favorable binding free energies than those that were not reduced.…”

“…Although early studies with mismatched ASOs showed antisense activity decreased with increasing mismatches [27], antisense activity at nearly matched sites has been reported [7,8]. These nearly complementary sites in the transcriptome can be identified in silico using sequence alignment tools [28][29][30][31][32].…”

“…Rukov et al tested two LNA gapmers for selective reduction of a library containing randomized 7-mer target sites [6], and demonstrated a correlation of binding score with activity at mismatched sites. Hagedorn et al tested four LNA gapmers for reduction of unintended transcripts in mouse liver [7]. They demonstrated fewer mismatches in the ASO:target duplex resulted in a higher chance of reduction of that transcript.…”