Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectors.
OBJECTIVE
While mature enterocytes are resistant to transduction by adenovirus type 5 (Ad5) vectors, undifferentiated cells are transduced much more efficiently. Our purpose was to study enterocyte transduction in models of intestinal wound healing.


METHODS
Transduction was studied ex vivo using cultures of endoscopic biopsies and in vitro utilizing Caco-2 cells in models of mucosal wound healing. Vectors carried either the LacZ or the luciferase gene. CAR (coxsackievirus and adenovirus receptor) and integrins were studied with transduction inhibition and immunofluorescent staining.


RESULTS
Increased transduction efficiency was observed for a subset of enterocytes with a flattened de-differentiated phenotype present at the edge of cultured biopsies. In the in vitro systems, expanding Caco-2 cell monolayers exhibited increased transducibility that was time- and dose-dependent, reaching virtually 100% in cells along the leading edge at high viral load. Bioluminescence activity of transduced expanding monolayers was up to 3-fold greater than that of non-expanding monolayers ('fence' system, 48 h, MOI 1000, p < 0.05). Mitomycin C pre-treatment did not affect levels of transduction in expanding monolayers. At the highest viral load tested, CAR or integrin blocking prior to virus application resulted in 39.4% and 45.4% reduction in transduction levels (p < 0.05). Immunofluorescence revealed altered expression of CAR on the migrating edge of the Caco-2 cultures and the expression of CAR on the apical membrane of biopsy enterocytes.


CONCLUSIONS
Increased CAR and integrin accessibility in migrating enterocytes mediates increased transduction by Ad5 vectors. This subset of enterocytes provides a target for the delivery of genes of interest for both research and gene therapy applications.
Kesisoglou
Filippos
F
Department of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, Ann Arbor, MI 48109-1065, USA.
Schmiedlin-Ren
Phyllissa
P
Fleisher
David
D
Roessler
Blake
B
Zimmermann
Ellen M
EM
D000256:Adenoviridae / Q000145:classification* / Q000235:genetics*; D018938:Caco-2 Cells; D003106:Colon / Q000166:cytology* / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D014161:Transduction, Genetic