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Long-term, efficient inhibition of microRNA function in mice using rAAV vectors

Nature Methods volume 9, pages 403–409 (2012)Cite this article

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Abstract

Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA 'tough decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti–miR-122 or anti–let-7 TuDs depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti–miR-122 TuD but not anti–let-7 TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High-throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuDs induced miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.

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Figure 1: Comparison of miR-122 inhibitor strategies in cultured cells.
Figure 2: Real-time monitoring of endogenous miRNA activity using an miRNA sensor system.
Figure 3: Analysis of miRNA expression in liver from mice administered scAAV9 expressing TuDs.
Figure 4: Analysis of TuD-directed inhibition of miR-122 in HuH-7 cells.
Figure 5: Expression in TuD-treated mice of previously validated regulatory targets of miR-122 and let-7.
Figure 6: Change in cholesterol profiles of wild-type C57BL/6J mice after administration of scAAV expressing both the TuD targeting miR-122 and the GLuc reporter bearing miR-122–binding sites, relative to control GLuc reporter lacking the TuD and the miR-122 binding sites.

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Acknowledgements

This work was supported in part by a European Molecular Biology Organization long-term fellowship (ALTF 522-2008) and an Erwin Schrödinger-Auslandsstipendium (Austrian Science Fund FWF, J2832-B09) to S.L.A. and by grants from the US National Institutes of Health to P.D.Z. (GM62862 and GM65236), to P.D.Z. and G.G. (UL1RR031982), to the University of Massachusetts Mouse Metabolic Phenotypic Center (U24-DK093000), and to T.R.F. (P01 DK58327), and from the University of Massachusetts Medical School to G.G.; T.R.F., J.K., P.D.Z., and G.G. are members of the University of Massachusetts Diabetes and Endocrinology Research Core, which is supported by a grant from the US National Institute of Diabetes and Digestive and Kidney Diseases (P30 DK32520).

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Author notes

  1. Jun Xie and Stefan L Ameres: These authors contributed equally to this work.

Authors and Affiliations

  1. Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Jun Xie, Yu Zhang, Qing Xie, Li Zhong, Qin Su, Ran He, Mengxin Li, Huapeng Li, Xin Mu, Hongwei Zhang, Terence R Flotte & Guangping Gao

  2. Department of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Jun Xie, Mengxin Li, Huapeng Li, Hongwei Zhang, Terence R Flotte & Guangping Gao

  3. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Stefan L Ameres, Jennifer A Broderick & Phillip D Zamore

  4. Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria

    Stefan L Ameres

  5. Mouse Phenotyping Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Randall Friedline & Jason K Kim

  6. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Randall Friedline & Jason K Kim

  7. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Jui-Hung Hung & Zhiping Weng

  8. Bioinformatics Program, Boston University, Boston, Massachusetts, USA

    Jui-Hung Hung

  9. Department of Thoracic Cancer, Cancer Center, West China Hospital, West China School of Clinical Medicine, Sichuan University, Chengdu, China

    Yu Zhang

  10. Department of Microbiology, Peking University Health Science Center, Beijing, China

    Qing Xie

  11. Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Li Zhong

  12. Department of Dermatology, First Affiliated Hospital of Medical College of Xian Jiaotong University, Xian, China

    Xin Mu

  13. Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Terence R Flotte

  14. Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Phillip D Zamore

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  1. Jun Xie
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Contributions

J.X. created the miRNA inhibitor constructs, performed the experiments in cultured cells and mice. J.-H.H., S.L.A. and Z.W. analyzed high-throughput sequencing data. S.L.A. designed, conducted and analyzed the experiments to study how TuDs inhibit miRNAs. R.F. and J.K.K. measured cholesterol profiles and liver function in mice. Y.Z. contributed to most of the anti-miRNA TuD studies in mice. R.H. and Q.X. contributed to vector construction as well as in vitro characterization of miRNA inhibitors. L.Z., M.L., H.L. and X.M. contributed to the miRNA inhibitor studies in mice and tissue sample analyses. Q.S. and R.H. produced the AAV vectors. H.Z. assisted in in vitro characterization. J.A.B. designed and cloned anti-miRNA sponges. T.R.F. contributed to the development of rAAV delivered miRNA therapeutics for treating hyperlipidemia. J.X., S.L.A., P.D.Z. and G.G. conceived the research. S.L.A., J.X., P.D.Z. and G.G. wrote the manuscript. S.L.A. and P.D.Z. prepared the figures.

Corresponding authors

Correspondence to Phillip D Zamore or Guangping Gao.

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Competing interests

P.D.Z. is a member of the scientific advisory board of Regulus Therapeutics.

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Supplementary Figures 1–9, Supplementary Tables 1–3 and Supplementary Note 1 (PDF 783 kb)

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Xie, J., Ameres, S., Friedline, R. et al. Long-term, efficient inhibition of microRNA function in mice using rAAV vectors. Nat Methods 9, 403–409 (2012). https://doi.org/10.1038/nmeth.1903

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