title: Impact of lentiviral vector-mediated transduction on the tightness of a polarized model of airway epithelium and effect of cationic polymer polyethylenimine.
abstract: Lentiviral (LV) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP) gene as well as cytotoxicity and tight junction (TJ) integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER) and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25 kDa polyethylenimine (PEI). Transduction of cells with PEI/LV particles resulted in 2.5-3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1 x 10(7) PEI molecules/transducing units with 50 MOI LV) as compared to plain LV. At this dose PEI/LV transduction resulted in 6.5 +/- 2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness.
authors.affiliation: Department of Biomedical Sciences, University of Foggia, 71122 Foggia, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Castellani
authors.forename: Sante
authors.initials: S
authors.lastname: Di Gioia
authors.forename: Teresa
authors.initials: T
authors.lastname: Trotta
authors.forename: Angela Bruna
authors.initials: AB
authors.lastname: Maffione
authors.forename: Massimo
authors.initials: M
authors.lastname: Conese
mesh_terms: D002463:Cell Membrane Permeability / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D049452:Green Fluorescent Proteins; D016086:Lentivirus / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D062793:Occludin; D011094:Polyethyleneimine / Q000633:toxicity*; D011419:Propidium; D020545:Respiratory Mucosa / Q000166:cytology*; D019108:Tight Junctions / Q000187:drug effects; D018675:Toxicity Tests / Q000379:methods*; D014162:Transfection; D014771:Virion
chemical_list: D008565:Membrane Proteins; D062793:Occludin; D049452:Green Fluorescent Proteins; D011419:Propidium; D011094:Polyethyleneimine