Engineered E. coli delivers therapeutic genes to the colonic mucosa.
Taking advantage of the proximity of bowel mucosa to luminal bacteria, we have attempted 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 pGB2Omegainv-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 (pC1OmegaTGF-beta1) 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 pC1OmegaTGF-beta1 by the inflammation-inducible interleukin-8 promoter generating plasmid pC1OmegaTGF-beta1IND. Plasmid-specific TGF-beta1 mRNA transcripts were detectable in mouse CMT-93 epithelial cells incubated with E. coli BM2710/pGB2Omegainv-hly carrying pC1OmegaTGF-beta1IND 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/pGB2Omegainv-hly+pC1OmegaTGF-beta1. 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.
Castagliuolo
I
I
Department of Histology, Microbiology and Medical Biotechnologies, University of Padua, Padua, Italy.
Beggiao
E
E
Brun
P
P
Barzon
L
L
Goussard
S
S
Manganelli
R
R
Grillot-Courvalin
C
C
Palù
G
G
D000284:Administration, Oral; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics; D003092:Colitis / Q000378:metabolism / Q000628:therapy*; D003106:Colon; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000382:microbiology*; D008089:Listeria monocytogenes / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics; D053773:Transforming Growth Factor beta1; D015011:Yersinia pseudotuberculosis
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C038705:hlyA protein, Listeria monocytogenes