RNAi induced hepatotoxicity results from loss of the first synthesized isoform of miR-122 in mice

Small RNAs can be used to target and eliminate expression of virtually any disease causing gene or infectious virus, resulting in their pre-clinical and clinical development for treating disease(1). To ensure success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity including hepatocyte turnover, loss of gene repression and lethality(2) in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs; however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs, we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of liver microRNAs. Once this threshold was surpassed, shRNAs specifically reduced the initial synthesized 22-nucleotide isoform of miR-122-5p without substantially affecting other microRNAs resulting in functional de-repression of miR-122 target mRNAs. Delivery of an rAAV-shRNA vector expressing miR-122 could circumvent toxicity despite accounting for 70% of microRNAs. Toxicity was also not observed in miR-122 knockout mice regardless of the level or sequence of shRNA. Our study establishes limits to the microRNA machinery available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.