Letter | Published:

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways

Nature volume 441, pages 537541 (25 May 2006) | Download Citation



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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    RNA interference. Nature 418, 244–251 (2002)

  2. 2.

    & The RNAi revolution. Nature 430, 161–164 (2004)

  3. 3.

    & The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu. Rev. Med. 56, 401–423 (2005)

  4. 4.

    & Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378 (2004)

  5. 5.

    et al. Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice. J. Virol. 79, 214–224 (2005)

  6. 6.

    , & Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 8, 1248–1254 (2001)

  7. 7.

    & From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Curr. Gene Ther. 3, 281–304 (2003)

  8. 8.

    & Noise amidst the silence: off-target effects of siRNAs? Trends Genet. 20, 521–524 (2004)

  9. 9.

    et al. Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J. Virol. 75, 6969–6976 (2001)

  10. 10.

    MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004)

  11. 11.

    The functions of animal microRNAs. Nature 431, 350–355 (2004)

  12. 12.

    , , , & Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science 309, 1577–1581 (2005)

  13. 13.

    et al. Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739 (2002)

  14. 14.

    , , , & Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs. RNA 11, 220–226 (2005)

  15. 15.

    , , & Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011–3016 (2003)

  16. 16.

    , & Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10, 185–191 (2004)

  17. 17.

    , , , & Nuclear export of microRNA precursors. Science 303, 95–98 (2004)

  18. 18.

    Assembly and function of RNA silencing complexes. Nature Rev. Mol. Cell Biol. 6, 127–138 (2005)

  19. 19.

    , et al. in Frontiers in Viral Hepatitis (eds Schinazi, R. F., Rice, C. & Sommadossi, J.-P.) 197–209 (Elsevier Science, Amsterdam, 2002)

  20. 20.

    et al. Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438, 685–689 (2005)

  21. 21.

    & Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004)

  22. 22.

    , , , & DNA constructs designed to produce short hairpin, interfering RNAs in transgenic mice sometimes show early lethality and an interferon response. J. Appl. Genet. 46, 217–225 (2005)

  23. 23.

    , , & siRNA-mediated gene silencing in vitro and in vivo. Nature Biotechnol. 20, 1006–1010 (2002)

  24. 24.

    et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl Acad. Sci. USA 101, 10380–10385 (2004)

  25. 25.

    , , , & Local gene knockdown in the brain using viral-mediated RNA interference. Nature Med. 9, 1539–1544 (2003)

  26. 26.

    et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nature Med. 10, 816–820 (2004)

  27. 27.

    et al. Inhibition of hepatitis B virus in mice by RNA interference. Nature Biotechnol. 21, 639–644 (2003)

  28. 28.

    et al. RNA interference in adult mice. Nature 418, 38–39 (2002)

  29. 29.

    Production methods for gene transfer vectors based on adeno-associated virus serotypes. Methods 28, 146–157 (2002)

  30. 30.

    et al. Donor-derived, liver-specific protein expression after bone marrow transplantation. Transplantation 78, 530–536 (2004)

Download references


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.

Author information

Author notes

    • Konrad L. Streetz

    †Present address: Department of Medicine III, University-Hospital Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany


  1. Stanford University, School of Medicine, Departments of Pediatrics and Genetics,

    • Dirk Grimm
    • , Konrad L. Streetz
    • , Theresa A. Storm
    • , Kusum Pandey
    •  & Mark A. Kay
  2. Department of Microbiology and Immunology, and

    • Catherine L. Jopling
  3. Department of Comparative Medicine, 300 Pasteur Drive, Stanford, California 94305, USA

    • Corrine R. Davis
  4. Hepadnavirus Testing, Inc., 331H Sierra Vista, Mountain View, California 94043, USA

    • Patricia Marion
    •  & Felix Salazar


  1. Search for Dirk Grimm in:

  2. Search for Konrad L. Streetz in:

  3. Search for Catherine L. Jopling in:

  4. Search for Theresa A. Storm in:

  5. Search for Kusum Pandey in:

  6. Search for Corrine R. Davis in:

  7. Search for Patricia Marion in:

  8. Search for Felix Salazar in:

  9. Search for Mark A. Kay in:

Competing interests

Reprints and permissions information is available at www.npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Corresponding author

Correspondence to Mark A. Kay.

Supplementary information

Word documents

  1. 1.

    Supplementary Notes

    This file contains Supplementary Methods, Supplementary Table 1 (The 49 shRNA-expressing sdsAAV8 vectors used in this study) and the Supplementary Figure Legends.

Powerpoint files

  1. 1.

    Supplementary Figure 1

    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.

  2. 2.

    Supplementary Figure 2

    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.

  3. 3.

    Supplementary Figure 3.

    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.

  4. 4.

    Supplementary Figure 4

    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.

  5. 5.

    Supplementary Figure 5

    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.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.