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Short hairpin RNA–expressing bacteria elicit RNA interference in mammals


RNA-interference (RNAi) is a potent mechanism, conserved from plants to humans for specific silencing of genes, which holds promise for functional genomics and gene-targeted therapies. Here we show that bacteria engineered to produce a short hairpin RNA (shRNA) targeting a mammalian gene induce trans-kingdom RNAi in vitro and in vivo. Nonpathogenic Escherichia coli were engineered to transcribe shRNAs from a plasmid containing the invasin gene Inv and the listeriolysin O gene HlyA, which encode two bacterial factors needed for successful transfer of the shRNAs into mammalian cells. Upon oral or intravenous administration, E. coli encoding shRNA against CTNNB1 (catenin β-1) induce significant gene silencing in the intestinal epithelium and in human colon cancer xenografts in mice. These results provide an example of trans-kingdom RNAi in higher organisms and suggest the potential of bacteria-mediated RNAi for functional genomics, therapeutic target validation and development of clinically compatible RNAi-based therapies.

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Figure 1: Expression of interfering shRNA by engineered bacteria and induction of potent and specific gene silencing in human colon cancer cells (SW480).
Figure 2: Specific cleavage of CTNNB1 mRNA in SW480 cells induced by trans-kingdom RNAi.
Figure 3: Trans-kingdom gene silencing requires bacterial entry of bacteria into mammalian cells in the presence of both bacterial Inv and Hly genes.
Figure 4: Trans-kingdom RNAi in vivo.


  1. Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998).

    CAS  Article  Google Scholar 

  2. May, R.C. & Plasterk, R.H. RNA interference spreading in C. elegans. Methods Enzymol. 392, 308–315 (2005).

    CAS  Article  Google Scholar 

  3. Fraser, A.G. et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325–330 (2000).

    CAS  Article  Google Scholar 

  4. Kamath, R.S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003).

    CAS  Article  Google Scholar 

  5. Ashrafi, K. et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268–272 (2003).

    CAS  Article  Google Scholar 

  6. Feinberg, E.H. & Hunter, C.P. Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301, 1545–1547 (2003).

    CAS  Article  Google Scholar 

  7. Xiang, R. et al. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res. 65, 553–561 (2005).

    CAS  PubMed  Google Scholar 

  8. Milligan, J.F. & Uhlenbeck, O.C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180, 51–62 (1989).

    CAS  Article  Google Scholar 

  9. Milligan, J.F., Groebe, D.R., Witherell, G.W. & Uhlenbeck, O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).

    CAS  Article  Google Scholar 

  10. Isberg, R.R., Voorhis, D.L. & Falkow, S. Identification of invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell 50, 769–778 (1987).

    CAS  Article  Google Scholar 

  11. Young, V.B., Falkow, S. & Schoolnik, G.K. The invasin protein of Yersinia enterocolitica: internalization of invasin-bearing bacteria by eukaryotic cells is associated with reorganization of the cytoskeleton. J. Cell Biol. 116, 197–207 (1992).

    CAS  Article  Google Scholar 

  12. Mathew, E., Hardee, G.E., Bennett, C.F. & Lee, K.D. Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes. Gene Ther. 10, 1105–1115 (2003).

    CAS  Article  Google Scholar 

  13. Grillot-Courvalin, C., Goussard, S., Huetz, F., Ojcius, D.M. & Courvalin, P. Functional gene transfer from intracellular bacteria to mammalian cells. Nat. Biotechnol. 16, 862–866 (1998).

    CAS  Article  Google Scholar 

  14. Kim, T.H., Xiong, H., Zhang, Z. & Ren, B. β-Catenin activates the growth factor endothelin-1 in colon cancer cells. Oncogene 24, 597–604 (2005).

    CAS  Article  Google Scholar 

  15. Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178 (2004).

    CAS  Article  Google Scholar 

  16. Bridge, A.J., Pebernard, S., Ducraux, A., Nicoulaz, A.L. & Iggo, R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet. 34, 263–264 (2003).

    CAS  Article  Google Scholar 

  17. Hornung, V. et al. Sequence-specific potent induction o f IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270 (2005).

    CAS  Article  Google Scholar 

  18. Roy, D. et al. A process for controlling intracellular bacterial infections induced by membrane injury. Science 304, 1515–1518 (2004).

    CAS  Article  Google Scholar 

  19. Battistoni, A. et al. Increased expression of periplasmic Cu,Zn superoxide dismutase enhances survival of Escherichia coli invasive strains within nonphagocytic cells. Infect. Immun. 68, 30–37 (2000).

    CAS  Article  Google Scholar 

  20. Rembacken, B.J., Snelling, A.M., Hawkey, P.M., Chalmers, D.M. & Axon, A.T. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 354, 635–639 (1999).

    CAS  Article  Google Scholar 

  21. Toso, J.F. et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J. Clin. Oncol. 20, 142–152 (2002).

    Article  Google Scholar 

  22. Bitko, V., Musiyenko, A., Shulyayeva, O. & Barik, S. Inhibition of respiratory viruses by nasally administered siRNA. Nat. Med. 11, 50–55 (2005).

    CAS  Article  Google Scholar 

  23. Zhao, M. et al. Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 102, 755–760 (2005).

    CAS  Article  Google Scholar 

  24. Narayanan, K. & Warburton, P.E. DNA modification and functional delivery into human cells using Escherichia coli DH10B. Nucleic Acids Res. 31, e51 (2003).

    Article  Google Scholar 

  25. Darji, A. et al. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 91, 765–775 (1997).

    CAS  Article  Google Scholar 

  26. Pawelek, J.M., Low, K.B. & Bermudes, D. Bacteria as tumour-targeting vectors. Lancet Oncol. 4, 548–556 (2003).

    Article  Google Scholar 

  27. Miliotis, M.D. Acridine orange stain for determining intracellular enteropathogens in HeLa cells. J. Clin. Microbiol. 29, 830–831 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

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We thank J.T. LaMont and Y.X. Yang (Harvard Medical School) for helpful discussions, Xiangao Sun and A.J. Wang for advice and C. Grillot-Courvalin for pGB2Ωinv-hly.

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Correspondence to Chiang J Li.

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C.J.L. holds stock in ArQule Inc. and CeQuent Inc.

Supplementary information

Supplementary Fig. 1

Structure of Transkingdom RNA Interference Plasmid (TRIP) and silencing sequences (PDF 18 kb)

Supplementary Fig. 2

Specific silencing of β-catenin in SW 480 cells (PDF 21 kb)

Supplementary Fig. 3

Silencing is caused by bacterial produced shRNA; TRIP plasmid transfection does not induce silencing. (PDF 39 kb)

Supplementary Fig. 4

GAPDH expression is not altered by E.coli expressing shRNA against β-catenin after oral dosing in mice (PDF 367 kb)

Supplementary Table 1

RT-PCR for marker genes of interferon pathway induction (PDF 13 kb)

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Xiang, S., Fruehauf, J. & Li, C. Short hairpin RNA–expressing bacteria elicit RNA interference in mammals. Nat Biotechnol 24, 697–702 (2006).

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