RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs1,2. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics3,4. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.
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We thank J. Wilson for providing the AAV8 packaging plasmid, P. Sarnow for the gfp fusion and miR-122 expression plasmids, H. Doege and A. Stahl for the H1-driven shRNA cassettes, I. Macara for the exportin-5 vector, D. Haussecker and B. Garrison for critically reading the manuscript, and J. S. Lee and H. Xu for technical assistance. This work was supported by grants from the National Institutes of Health (to M.A.K.) and the Anna Ng Charitable Foundation (to M.A.K.). Author contributions D.G. performed and designed (with M.A.K.) most of the included studies. K.L.S. maintained the hAAT-transgenic mice and performed all injections and histological liver analyses. C.L.J. performed crucial steps of the small RNA Northern blot analyses and provided the gfp plasmids used in Fig. 4a. T.A.S. and K.P. generated the sdsAAV8 preparations and helped with the DNA, RNA and protein analyses. C.R.D. performed the complete mouse pathologies. P.M. and F.S. provided and maintained the HBV-transgenic mice. M.A.K. supervised the research project, and assisted in the experimental design. All authors discussed the experimental results and had input into the writing of the final manuscript.
This figure exemplifies the in vivo transduction efficiency of the novel sdsAAV8 vector, when used to express Firefly luciferase in mice. It also documents the first findings of toxicity and lethality with a subset of the five anti-luciferase shRNAs tested in this study.
The data in this figure document two of the key findings in this paper — that shRNA-induced lethality does not require an actual shRNA target, and that it does not involve a general shutdown of liver protein synthesis.
The Western blots in this figure document the evidence that the observed toxicity/lethality was not caused by an activation of the interferon pathway, or by perturbation of the cell cycle.
The Northern blots in this figure document that liver regeneration, induced by shRNA overexpression or by surgical injury, is not generally accompanied by changes in cellular mRNA or rRNA transcription.
This figure displays a scheme of the cellular shRNA and miRNA processing pathways (nucleus and cytoplasm), and highlights the potential overlap between the two which could explain the findings in this study.
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