Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

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.

Accession codes


Sequence Read Archive


  1. 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).

    Article  CAS  Google Scholar 

  2. Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P. & Burge, C.B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).

    Article  CAS  Google Scholar 

  3. Hutvagner, G., Simard, M.J., Mello, C.C. & Zamore, P.D. Sequence-specific inhibition of small RNA function. PLoS Biol. 2, E98 (2004).

    Article  Google Scholar 

  4. Meister, G., Landthaler, M., Dorsett, Y. & Tuschl, T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10, 544–550 (2004).

    Article  CAS  Google Scholar 

  5. Krutzfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).

    Article  Google Scholar 

  6. Elmen, J. et al. LNA-mediated microRNA silencing in non-human primates. Nature 452, 896–899 (2008).

    Article  CAS  Google Scholar 

  7. Lanford, R.E. et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327, 198–201 (2010).

    Article  CAS  Google Scholar 

  8. Esau, C. et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 3, 87–98 (2006).

    Article  CAS  Google Scholar 

  9. Ebert, M.S., Neilson, J.R. & Sharp, P.A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat. Methods 4, 721–726 (2007).

    Article  CAS  Google Scholar 

  10. Loya, C.M., Lu, C.S., Van Vactor, D. & Fulga, T.A. Transgenic microRNA inhibition with spatiotemporal specificity in intact organisms. Nat. Methods 6, 897–903 (2009).

    Article  CAS  Google Scholar 

  11. Gentner, B. et al. Stable knockdown of microRNA in vivo by lentiviral vectors. Nat. Methods 6, 63–66 (2009).

    Article  CAS  Google Scholar 

  12. Luikart, B.W. et al. miR-132 mediates the integration of newborn neurons into the adult dentate gyrus. PLoS ONE 6, e19077 (2011).

    Article  CAS  Google Scholar 

  13. Berns, K.I. & Giraud, C. Biology of adeno-associated virus. Curr. Top. Microbiol. Immunol. 218, 1–23 (1996).

    CAS  PubMed  Google Scholar 

  14. Gao, G.P. et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 99, 11854–11859 (2002).

    Article  CAS  Google Scholar 

  15. Krol, J. et al. Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell 141, 618–631 (2010).

    Article  CAS  Google Scholar 

  16. Zhu, Q. et al. Sponge transgenic mouse model reveals important roles for the microRNA-183 (miR-183)/96/182 cluster in postmitotic photoreceptors of the retina. J. Biol. Chem. 286, 31749–31760 (2011).

    Article  CAS  Google Scholar 

  17. Haraguchi, T., Ozaki, Y. & Iba, H. Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells. Nucleic Acids Res. 37, e43 (2009).

    Article  Google Scholar 

  18. Rayner, K.J. et al. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 328, 1570–1573 (2010).

    Article  CAS  Google Scholar 

  19. Ameres, S.L. et al. Target RNA-directed trimming and tailing of small silencing RNAs. Science 328, 1534–1539 (2010).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Tannous, B.A. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat. Protoc. 4, 582–591 (2009).

    Article  CAS  Google Scholar 

  22. Sen, C.K., Gordillo, G.M., Khanna, S. & Roy, S. Micromanaging vascular biology: tiny microRNAs play big band. J. Vasc. Res. 46, 527–540 (2009).

    Article  CAS  Google Scholar 

  23. McCarty, D.M. Self-complementary AAV vectors; advances and applications. Mol. Ther. 16, 1648–1656 (2008).

    Article  CAS  Google Scholar 

  24. Grimson, A. et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007).

    Article  CAS  Google Scholar 

  25. Kutay, H. et al. Downregulation of miR-122 in the rodent and human hepatocellular carcinomas. J. Cell. Biochem. 99, 671–678 (2006).

    Article  CAS  Google Scholar 

  26. Coulouarn, C., Factor, V.M., Andersen, J.B., Durkin, M.E. & Thorgeirsson, S.S. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 28, 3526–3536 (2009).

    Article  CAS  Google Scholar 

  27. Tsai, W.C. et al. MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma. Hepatology 49, 1571–1582 (2009).

    Article  CAS  Google Scholar 

  28. Griffiths-Jones, S., Grocock, R.J., van Dongen, S., Bateman, A. & Enright, A.J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34, D140–D144 (2006).

    Article  CAS  Google Scholar 

  29. Gao, G. et al. Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc. Natl. Acad. Sci. USA 100, 6081–6086 (2003).

    Article  CAS  Google Scholar 

  30. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T. & Sato, J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 42, 3858–3863 (1982).

    CAS  PubMed  Google Scholar 

  31. Gao, G. et al. Biology of AAV serotype vectors in liver-directed gene transfer to nonhuman primates. Mol. Ther. 13, 77–87 (2006).

    Article  CAS  Google Scholar 

Download references


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).

Author information

Authors and Affiliations



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.

Ethics declarations

Competing interests

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

Supplementary information

Supplementary Text and Figures

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

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing