694. Involvement of Glycosaminoglycans in VSV-G Pseudotyped Lentiviral Vector Mediated Gene Transfer into Airway Epithelial Cells

HIV-1-derived lentiviral (LV) vectors are considered promising vehicles for Cystic Fibrosis (CF) gene therapy since they integrate in the host genome, transduce non-dividing cells and ensure long-term expression of the transgene. Although some knowledge on the infection of respiratory epithelial cells has been accumulated, the receptors involved in the transduction of these cells by VSV-G-pseudotyped LV vectors are not well known. Previous studies have shown that heparin inhibited infection of HT1080 cells by Moloney Murine Leukemia retrovirus pseudotyped with VSV-G (Guibinga, 2002), indicating that interaction with glycosaminoglycans (GAGs) of cellular membrane plays a role in infectivity of viral particles with VSV-G envelope. Our study investigated the involvement of heparan sulfate (HS) and chondroitin sulfate B in gene transfer mediated by a VSV-G pseudotyped LV vector in a respiratory epithelial cell line of tracheal origin (CFT1-C2). The HIV-1 derived LV vector PPT-GFP carries the Green Fluorescence Protein (GFP) reporter gene under the control of the CMV promoter (Follenzi, 2000). To evaluate the interaction between GAGs expressed on the apical surface of respiratory epithelial cells and lentiviral particles, the LV vector (3.2×10E8 TU, corresponding to MOI 2000) was pre-incubated with heparin or chondroitin sulfate B at different concentrations (10-250 micrograms/ml) for 60 minutes in binding buffer and then added to polarized CFT1-C2 cells (polarization was evaluated by the measure of transepithelial resistance and apical expression of ZO-1). The virus-containing solution was incubated with cells for 24 hours and GFP expression was analyzed by flow cytometry 72 hours after. Heparin and chondroitin sulfate B determined a dose-dependent inhibition of transduction efficiency up to 86% and 83% of untreated vector, respectively. HS expression in CFT1-C2 cells was analyzed by flow cytometry and confocal microscopy. HS was expressed by 85-88% of cells, the expression being much stronger on the basolateral side of polarized cells. Cells were incubated with 12 mM EGTA before LV administration to disrupt the epithelial tight junctions and allow the lentiviral particles to access the basolateral side of the epithelium. In the absence of EGTA, 60% of cells resulted GFP positive, whereas the pre-treatment with EGTA increased the percentage of GFP-expressing cells up to 80.5%. Our data strongly indicate that the little amount of GAGs restricted to the apical s