Abstract
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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References
Kay, M.A. State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 12, 316–328 (2011).
Grimm, D. et al. Fatality in mice due to oversaturation of cellular microRNA–short hairpin RNA pathways. Nature 441, 537–541 (2006).
Lares, M.R., Rossi, J.J. & Ouellet, D.L. RNAi and small interfering RNAs in human disease therapeutic applications. Trends Biotechnol. 28, 570–579 (2010).
Maguire, A.M. et al. Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial. Lancet 374, 1597–1605 (2009).
Rodino-Klapac, L.R., Chicoine, L.G., Kaspar, B.K. & Mendell, J.R. Gene therapy for Duchenne muscular dystrophy: expectations and challenges. Arch. Neurol. 64, 1236–1241 (2007).
Nakai, H. et al. Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver. J. Virol. 79, 3606–3614 (2005).
Grimm, D. et al. Argonaute proteins are key determinants of RNAi efficacy, toxicity and persistence in the adult mouse liver. J. Clin. Invest. 120, 3106–3119 (2010).
Sekine, S. et al. Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis. Gastroenterology 136, 2304–2315.e1-4 (2009).
Hand, N.J., Master, Z.R., Le Lay, J. & Friedman, J.R. Hepatic function is preserved in the absence of mature microRNAs. Hepatology 49, 618–626 (2009).
Hsu, S.H. et al. Essential metabolic, anti-inflammatory and antitumorigenic functions of miR-122 in liver. J. Clin. Invest. 122, 2871–2883 (2012).
Tsai, W.C. et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J. Clin. Invest. 122, 2884–2897 (2012).
Gu, S. et al. The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo. Cell 151, 900–911 (2012).
Lagos-Quintana, M. et al. Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739 (2002).
Chang, J. et al. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high-affinity cationic amino acid transporter CAT-1. RNA Biol. 1, 106–113 (2004).
Landgraf, P. et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007).
Katoh, T. et al. Selective stabilization of mammalian microRNAs by 3′ adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2. Genes Dev. 23, 433–438 (2009).
Cross, N.C., Tolan, D.R. & Cox, T.M. Catalytic deficiency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation. Cell 53, 881–885 (1988).
Luna, J.M. et al. Hepatitis C virus RNA functionally sequesters miR-122. Cell 160, 1099–1110 (2015).
Castoldi, M. et al. The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice. J. Clin. Invest. 121, 1386–1396 (2011).
Esau, C. et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 3, 87–98 (2006).
Gatfield, D. et al. Integration of microRNA miR-122 in hepatic circadian gene expression. Genes Dev. 23, 1313–1326 (2009).
Krützfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).
Xie, J. et al. Long-term, efficient inhibition of microRNA function in mice using rAAV vectors. Nat. Methods 9, 403–409 (2012).
Hommes, F.A. & Draisma, M.I. The development of L- and M-type aldolases in rat liver. Biochim. Biophys. Acta 222, 251–252 (1970).
Numazaki, M., Tsutsumi, K., Tsutsumi, R. & Ishikawa, K. Expression of aldolase isozyme mRNAs in fetal rat liver. Eur. J. Biochem. 142, 165–170 (1984).
Bhattacharyya, S.N., Habermacher, R., Martine, U., Closs, E.I. & Filipowicz, W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 125, 1111–1124 (2006).
Kandathil, A.J. et al. Use of laser-capture microdissection to map hepatitis C virus–positive hepatocytes in human liver. Gastroenterology 145, 1404–13.e1-10 (2013).
Elmén, J. et al. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to upregulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res. 36, 1153–1162 (2008).
Helwak, A., Kudla, G., Dudnakova, T. & Tollervey, D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153, 654–665 (2013).
Loeb, G.B. et al. Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting. Mol. Cell 48, 760–770 (2012).
Betel, D., Koppal, A., Agius, P., Sander, C. & Leslie, C. Comprehensive modeling of microRNA targets predicts functional nonconserved and noncanonical sites. Genome Biol. 11, R90 (2010).
Jenkins, D.D. et al. Donor-derived, liver-specific protein expression after bone marrow transplantation. Transplantation 78, 530–536 (2004).
Lewis, B.P., Burge, C.B. & Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Anders, S. & Huber, W. Differential expression analysis for sequence-count data. Genome Biol. 11, R106 (2010).
Selitsky, S.R. et al. Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C. Sci. Rep. 5, 7675 (2015).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).
Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Huang, W., Sherman, B.T. & Lempicki, R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).
Bartonicek, N. & Enright, A.J. SylArray: a web server for automated detection of miRNA effects from expression data. Bioinformatics 26, 2900–2901 (2010).
Paraskevopoulou, M.D. et al. DIANA-microT web server v5.0: service integration into miRNA functional-analysis workflows. Nucleic Acids Res. 41, W169–W173 (2013).
Lu, J., Zhang, F. & Kay, M.A. A mini-intronic plasmid (MIP): a novel robust transgene expression vector in vivo and in vitro. Mol. Ther. 21, 954–963 (2013).
Bogerd, H.P., Whisnant, A.W., Kennedy, E.M., Flores, O. & Cullen, B.R. Derivation and characterization of Dicer- and microRNA-deficient human cells. RNA 20, 923–937 (2014).
Acknowledgements
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.
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P.N.V., S.G., L.L. and M.A.K. conceived the project and designed the experiments. P.N.V., S.G., K.C., L.J., F.Z., Y.H. and L.L. performed the experiments. Y.Z. provided bioinformatics support. H.K. and K.G. provided the Mir122-knockout mice. P.N.V., S.G., E.M.M. and L.L. analyzed the data. P.N.V. and M.A.K. wrote the manuscript with critical review from S.G., E.M.M., K.G. and L.L., and input from all other coauthors.
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Supplementary Text and Figures
Supplementary Figures 1–8 and Supplementary Tables 1, 6–7 (PDF 2789 kb)
Supplementary Table 2
Total small RNA read counts per million mapped microRNAs (XLSX 97 kb)
Supplementary Table 3
Ago2 immunoprecipitated small RNA read counts per million mapped microRNAs (XLSX 88 kb)
Supplementary Table 4
RNA-seq FPKM values for U6-rAAV-shRNA mouse livers relative to control and H1-rAAV-shRNA livers (XLSX 987 kb)
Supplementary Table 5
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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DOI: https://doi.org/10.1038/nm.4079
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