Stereo-random oligonucleotides enable efficient recruitment of ADAR in vitro and in vivo

Abstract

Abstract Site-directed RNA editing is a promising and potentially safer alternative to genome editing. Previous methods have been developed that recruit the endogenously and ubiquitously expressed ADAR enzymes to initiate site-specific A-to-I edits, but often suffer from low efficacy or dependency on viral delivery. Chemically modified oligonucleotides may be a promising alternative, but the approach still lacks systematic in-depth studies. Furthermore, the best characterized platform uses stereo-pure backbone chemistry, which is not widely used, commercially unavailable and challenging to manufacture. Here, we report on single-stranded oligonucleotides of 30-60 nt length, which are fully chemically stabilized by applying commercially available, classical RNA drug modifications, like 2´-O-methyl, 2´-fluoro, and DNA on a stereo-random phosphate/phosphorothioate backbone. We demonstrate our so-called RESTORE 2.0 oligonucleotides to induce the correction of pathogenic point mutations, efficacy after GalNAc-mediated uptake into human primary hepatocytes, and proof of in-vivo efficacy in mice upon lipid nanoparticle-mediated delivery. The discovered design principles may increase the accessibility of site-directed RNA base editing to expand and support further research in this field.