Taking advantage of the proximity of bowel mucosa to luminal bacteria, we have attempetd to deliver a therapeutic gene to the colonic mucosa by oral administration of an invasive and non-pathogenic Escherichia coli. E. coli diamenopimelate (dap) auxotroph, harboring plasmid pGB2Ωinv-hly, express the inv gene from Yersinia pseudotubercolosis that confers the ability to invade nonprofessional phagocytic cells and the hly gene from Listeria monocytogenes that allows expression of lystreriolysin O, a perforin cytolysin able to perfore phagosomal membranes. This bacterial vector invades and transfers functional DNA to epithelial cells in vitro. We have shown that this strain carrying a therapeutic gene (pC1ΩTGF-β1) can significantly reduce the severity of experimental colitis in mice. However, as a consequence of mucosal barrier disruption during colitis, vector-specific mRNA transcripts could be recovered from the colon and also from extra-colonic tissues. We therefore replaced the constitutive CMV promoter in pC1ΩTGF-β1 by the inflammation-inducible interleukin-8 promoter generating plasmid pC1ΩTGF-β1IND. Plasmid-specific TGF-β1 mRNA transcripts were detectable in mouse CMT-93 epithelial cells incubated with E. coli BM2710/pGB2Ωinv-hly carrying pC1ΩTGF-β1IND following exposure to inflammatory cytokines. Furthermore, the transcripts were detectable only within inflamed tissues and the therapeutic effects were comparable to those in animals treated with E. coli BM2710/pGB2Ωinv-hly+pC1ΩTGF-β1. In summary, engineered enteric bacteria can efficiently deliver in vivo therapeutic genes to the intact intestinal mucosa and regulation expression of the therapeutic gene by an inflammation-inducible promoter prevents its dissemination during colitis.
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This work was supported in part by grants from the University of Padua to IC, MIUR to GP, and ‘Vaincre la Mucoviscidose’ to CGC.
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Castagliuolo, I., Beggiao, E., Brun, P. et al. Engineered E. coli delivers therapeutic genes to the colonic mucosa. Gene Ther 12, 1070–1078 (2005). https://doi.org/10.1038/sj.gt.3302493
- inflammatory bowel disease
- inducible promoter
- invasive bacteria
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