Retroviral Vectors Efficiently Transduce Basal and Secretory Airway Epithelial CellsIn VitroResulting in Persistent Gene Expression in Organotypic Culture

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Abstract
Gene therapy of the lung requires the introduction and expression of a therapeutic gene in airway cells. Although retroviral vectors may be useful in this context, the ability of retroviruses to infect specific cell types in the airway is not known. In this study, we examined the ability of amphotropic recombinant retroviral vectors to transduce primary cultures of rabbit airway epithelial cell populations purified for basal or secretory cells. Transduction efficiencies in basal and secretory cell populations were found to be similar; about 27% after a single exposure to vector, and up to 77% after multiple exposures. The fate of genetically modified cells from the different populations was followed through terminal differentiation using organotypic cultures. The epithelium of the organotypic cultures generated from each population exhibited both pseudostratified and stratified morphology, produced mucin, and stained positively with antibodies specific for basal and ciliated cells. The mucociliary epithelium also showed co-localization of these phenotypic markers with the expression of the vector-encoded β-galactosidase gene. We conclude that retroviruses can efficiently transduce primary cultures of basal and secretory cells, and that both of these cell types can be progenitor cells of the airway epithelium. In vivo delivery of a retroviral vector containing a human placental alkaline phosphatase gene resulted in expression of the heterologous gene in rabbit tracheal epithelial cells. However, transduction efficiency was low and occurred only in the wounded trachea. We assessed the ability of retroviral vectors to transduce purified basal and secretory populations of airway epithelial cells and followed the fate of the retrovirally marked cells. We found that retroviral vectors efficiently delivered marker genes to both basal or secretory cell populations in monolayer cultures and that expression of the gene stably persisted in the epithelium generated in organotypic culture. Progeny basal, secretory, and ciliated cells in the epithelium generated from each population expressed the marker gene. Although retroviral transduction was efficient in vitro, the intact normal airway epithelium was resistant to retroviral transduction. However, a low transduction rate was observed in the wounded epithelium.