737. RNAi Induced Hepatotoxicity Results from a Functional Depletion of the First Synthesized Isoform of miR-122

Abstract

To ensure success of RNA interference (RNAi) therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function or causing liver toxicity. Using several recombinant adeno-associated viral (rAAV) vectors expressing shRNAs followed by small RNA sequencing, we determined that hepatic toxicity arises when exogenous shRNA levels exceed 12% of liver microRNAs. High shRNA expression specifically reduced miR-122-5p without affecting any other microRNAs ultimately resulting in functional de-repression of miR-122 target mRNAs. Furthermore, we found that only one isoform of miR-122-5p, 22 nucleotides in length, is displaced in toxic liver samples and that this isoform is the first to be synthesized from miR-122. A causative link between miR-122 reduction and toxicity was established when delivery of an AAV-shRNA expressing miR-122-5p could circumvent toxicity despite reaching 70% of microRNA reads. Consistent with these results, toxicity was not observed in miR-122 knockout mice -which in part adapt to an absence of miR-122 reduction - regardless of the level or sequence of shRNA. Together these results establish the limit to expendable miRNA/RNAi machinery and providing new paradigms for the role of miR-122 in liver homeostasis.