Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs1. To ensure the success of 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. We previously observed liver toxicity—including hepatocyte turnover, loss of gene repression and lethality2—in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-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 the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122–knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.
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This work was supported by NIH grants R01DK078424 (M.A.K.), R01AI071068 (M.A.K.) and R01CA193244 (K.G.) and a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (P.N.V.). We would like to thank all members of the Kay laboratory for input and suggestions, and H. Vogel for EM image analysis. Dicer1-knockout cells were kindly provided by B. Cullen (Duke University). We would like to thank the Stanford Functional Genomics Facility and the Stanford Center for Genomics and Personalized Medicine for high-throughput sequencing services.
The authors declare no competing financial interests.
Supplementary Figures 1–8 and Supplementary Tables 1, 6–7 (PDF 2789 kb)
Total small RNA read counts per million mapped microRNAs (XLSX 97 kb)
Ago2 immunoprecipitated small RNA read counts per million mapped microRNAs (XLSX 88 kb)
RNA-seq FPKM values for U6-rAAV-shRNA mouse livers relative to control and H1-rAAV-shRNA livers (XLSX 987 kb)
RNA-seq FPKM values for additional U6-rAAV-shRNA mouse livers relative to control liver (XLSX 1241 kb)
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Valdmanis, P., Gu, S., Chu, K. et al. RNA interference–induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice. Nat Med 22, 557–562 (2016). https://doi.org/10.1038/nm.4079
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