Targeting Repeated Regions Unique to a Gene Is an Effective Strategy for Discovering Potent and Efficacious Antisense Oligonucleotides.

Well-validated strategies for discovering potent and efficacious antisense oligonucleotides are central to realize the full therapeutic potential of RNA therapy. In this study, we focus on RNA targets where the same sequence of 16-20 nt is found in several regions across the RNA, and not in any other RNA. Targeting such unique repeated regions with oligonucleotides designed as gapmers and capable of recruiting RNase H has previously been proposed as a strategy for identifying potent gapmers. By sequence analysis of the human and monkey transcriptomes, we find that such unique repeated regions in RNA are often conserved between humans and monkeys, which allow pharmacodynamic effects to be evaluated in non-human primates before testing in humans. For eight potential RNA targets chosen in an unbiased fashion, we targeted their unique repeated regions with locked nucleic acid (LNA)-modified gapmers, and for six of them we identified gapmers that were significantly more potent and efficacious in vitro than non-repeat-targeting gapmer controls. We suggest a stochastic model for repeat-targeting gapmers that explains all effects observed so far and can help guide future work. Our results support the targeting of repeated regions as an effective strategy for discovering gapmer antisense oligonucleotides suitable for therapeutic development.