Point mutations underlie many genetic diseases. In this regard, while programmable DNA nucleases have been used to repair mutations, their use for gene therapy poses multiple challenges: one, efficiency of homologous recombination is typically low in cells; two, an active nuclease presents a risk of introducing permanent off-target mutations; and three, prevalent programmable nucleases typically comprise elements of non-human origin raising the potential of in vivo immunogenicity. In light of these, approaches to instead directly target RNA, and use of molecular machinery native to the host would be highly desirable. Towards this, we engineered and optimized two complementary approaches, referred together hereon as tRiAD, based on the use of tRNAs in codon suppression and adenosine deaminases in RNA editing. Specifically, by delivering modified endogenous tRNAs and/or the RNA editing enzyme ADAR2 and an associated guiding RNA (adRNA) via adeno-associated viruses, we enabled premature stop codon read-through and correction in the mdx mouse model of muscular dystrophy that harbors a nonsense mutation in the dystrophin gene. We further demonstrated inducible restoration of dystrophin expression by pyrolysyl-tRNA mediated incorporation of unnatural amino acids (UAAs) at the stop codon. Additionally, we also engineered ADAR2 mediated correction of a point mutation in liver RNA of the spfash mouse model of ornithine transcarbamylase (OTC) deficiency. Taken together, our results establish the use of suppressor tRNAs and ADAR2 for in vivo RNA targeting, and this integrated tRiAD approach is robust, genomically scarless, and potentially non-immunogenic as it utilizes effector RNAs and human proteins.
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