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Short hairpin RNA–expressing bacteria elicit RNA interference in mammals

Nature Biotechnology volume 24, pages 697–702 (2006)Cite this article

Abstract

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.

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Acknowledgements

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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Author notes

  1. Shuanglin Xiang and Johannes Fruehauf: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, 02215, Massachusetts, USA

    Shuanglin Xiang, Johannes Fruehauf & Chiang J Li

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  1. Shuanglin Xiang
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  2. Johannes Fruehauf
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  3. Chiang J Li
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Correspondence to Chiang J Li.

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Competing interests

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). https://doi.org/10.1038/nbt1211

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