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Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of αv β3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.

Limited Entry of Adenovirus Vectors into Well-Differentiated Airway Epithelium Is Responsible for Inefficient Gene Transfer