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Short hairpin RNA–expressing bacteria elicit RNA interference in mammals

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

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  • DOI: https://doi.org/10.1038/nbt1211

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