Vesicular stomatitis virus G-pseudotyped lentivirus vectors mediate efficient apical transduction of polarized quiescent primary alveolar epithelial cells

We investigated the use of lentivirus vectors for gene transfer to quiescen t alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) gr own on plastic or as polarized monolayers on tissue culture-treated polycar bonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the v esicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by co nfocal microscopy and quantified by fluorescence-activated cell sorting, wa s dependent on the dose of vector, ranging from 4% at a multiplicity of inf ection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a c omparable titer and MOI, transduction of these cells by a similarly pseudot yped murine leukemia virus vector was similar to 30-fold less than by the l entivirus vector. Importantly, comparison of lentivirus-mediated gene trans fer from the apical or basolateral surface of confluent AEC monolayers (R-t > 2 k Omega . cm(2); MOI = 10) revealed efficient transduction only when V SV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment wi th EGTA to increase access to the basolateral surface did not increase tran sduction of apically applied virus, indicating that transduction was primar ily via the apical membrane domain. In contrast, differentiated tracheal ep ithelial cells were transduced by apically applied lentivirus only in the p resence of EGTA and at a much lower overall efficiency (similar to 15-fold) than was observed for AEC. Efficient transduction of AEC from the apical c ell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that diff erences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.