358. Gene Transfer to the Airway Epithelium Mediated by a Third Generation HIV-1 Based Vector: Efficiency and Role of Heparan Sulfate

Abstract

HIV-1 derived lentiviral (LV) vectors seem to be promising vehicles for gene transfer to the airway epithelium since they integrate in the host genome of non-dividing cells ensuring long-term expression of the transgene. Previous studies have shown that LV vectors could transduce the airway epithelium in vivo from the apical surface, only when tight junctions are disrupted or LV are pseudotyped with heterologous envelopes other than glycoprotein G of the vesicular stomatitis virus (VSV-G). Our study was aimed to investigate the efficiency of an improved VSV-G pseudotyped lentiviral vector, which contains the central polypurine tract (cPPT) and the Woodchuck post-translational regulatory elements, in human respiratory cells and in the murine lung. The vector PPT-GFP was injected intratracheally in C57BL/6 mice at the dose of 4.5|[times]|10E6 TU. After 48 hours, lungs were fixed in paraformaldehyde and cryosections stained with an antibody against GFP. Results show that PPT-GFP transduced the murine airway epithelium in the bronchial and alveolar compartments. Transgene expression was observed also after two weeks of infection with a LV dose of 1|[times]|10E8 TU. Hystological analysis showed no evidence of tissue damage in the lung, suggesting that this LV vector is not cytotoxic. Since heparan sulfate (HS) has been described to mediate infection by VSV-G-pseudotyped retroviral vectors and HIV-1, the expression of this glycosaminoglycan by human respiratory cells and its involvement in LV-mediated gene transfer were studied. Human bronchial epithelial cells (16HBE) and CF tracheal epithelial cells (CFT1) were analyzed for HS expression by flow cytometry and confocal microscopy. HS was expressed by 85-88% of cells, the expression being much stronger on the basolateral side in polarized epithelial cells. To correlate the efficiency of LV mediated transduction with HS expression, polarized CFT1 cells were infected with the lentivirus and some samples were pre-incubated with EGTA (Ethylene glycol-O,O-bis-[2-amino-ethyl]-N,N,N,N,-tetraacetic acid), which disrupts the epithelial tight junctions and allows the LV particles to access the basolateral side of the epithelium. Analysis of GFP expression by FACS indicates that the pre-conditioning increased the transduction efficiency, being the percentage of positive cells of 55%, when the infection was performed without pretreatment, and 71% when the cells were incubated with EGTA. To evaluate the interaction between glycosaminoglycans expressed on the apical surface and lentiviral particles, the LV vector was pre-incubated with heparin and then incubated with 16HBE cells. Heparin determined a dose-dependent inhibition of transduction efficiency up to 65% of untreated vector. Our study shows that 1) The PPT-GFP vector transduce murine airways and human polarized cells, without requirement of disruption of the epithelial barrier integrity and without cytotoxicy 2) HS might be a receptor involved in VSV-G-pseudotyped LV-mediated gene transfer.