MUC1 and other sialoglycoconjugates inhibit adenovirus-mediated gene transfer to epithelial cells.

Recombinant adenoviruses are currently being evaluated as gene transfer vectors for the treatment of airway diseases. Recent evidence indicates that gene transfer to differentiated airway epithelial cells is inefficient. We hypothesized that apical membrane glycoconjugates, such as the transmembrane mucin MUC1, reduce the efficiency of adenovirus-mediated gene transfer. To address this, studies were performed in primary bronchial epithelial and Madin Darby canine kidney (MDCK) cells transduced to express human MUC1. Colocalization of MUC1 and an adenoviral lacZ transgene in the bronchial epithelial cells revealed that at several multiplicities of infection, the percentage of cells expressing lacZ was five-fold less in MUC1-expressing cells. Moreover, lacZ expression was three- to eight-fold lower in MUC1-expressing than in control MDCK cells, demonstrating that MUC1 interferes with gene transfer and is not merely a phenotypic marker of a cell that is refractory to adenovirus infection. Neuraminidase pretreatment of cells to remove sialic acid residues prior to viral adsorption increased the efficiency of gene transfer two- to five-fold in human airway and MDCK cells, and in a xenograft model of human airway. This effect was also observed in cultured cells that do not express MUC1, suggesting that other sialylated glycoconjugates impact on the efficiency of gene transfer. An inhibitory effect of negatively charged glycoconjugates on adenovirus binding was further supported by the finding that adsorption of adenovirus with a polycation significantly increased gene transfer efficiency. These data demonstrate for the first time that sialoglycoconjugates on epithelial cells reduce the efficiency of adenovirus-mediated gene transfer.

Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo.

Airway epithelium is subject to injury during inflammation and exposure to a variety of inhaled and infectious agents. Little is known about the expression of integrins during human airway epithelial regeneration and differentiation after injury. We therefore characterized integrin subunit expression after mechanical injury in an in vivo xenograft model of human bronchial epithelium. On the migrating cells at the edges of surface epithelial wounds, there was increased expression of the alpha v-, beta 5-, beta 6-, and alpha 5-integrin subunits. During the later phase of repair, the increased expression of alpha v-, beta 5-, and beta 6-subunits persisted, but the expression of the beta 8-subunits was restricted to basal cells. In addition, there was a redistribution of the alpha 2- and alpha 6-collagen/laminin-binding integrins to suprabasal epithelial layers. There was no expression of the beta 3- or alpha 4-integrin subunit on reparative epithelium. A similar upregulation of alpha v-, beta 5-, and beta 6-integrin receptor subunits was observed in areas of undifferentiated airway from cystic fibrosis patients. Injured epithelium was found to be significantly more susceptible to gene transfer with a recombinant adenovirus, suggesting that the increased integrin expression has implications for the acquisition of adenovirus infections and for lung-directed gene therapy.

Polycations increase the efficiency of adenovirus-mediated gene transfer to epithelial and endothelial cells in vitro.

Recombinant adenoviruses are being developed for gene therapy for cystic fibrosis and other lung diseases, and for prevention and treatment of vascular thrombosis. A major limitation to the clinical utility of adenoviruses is the low efficiency of gene transfer achieved in vivo. In addition, little is known about the initial interactions between adenoviruses and the target cell. To address the hypothesis that the negative charge presented by membrane glycoproteins reduces the efficiency of adenovirus-mediated gene transfer, primary cultures of human airway, Madin-Darby canine kidney cells, an immortalized cystic fibrosis airway epithelial cell line, and primary cultures of sheep pulmonary artery endothelium were infected with recombinant adenovirus containing the E. coli lacZ reporter gene (Ad2 beta gal2) in the presence of various polyions. For each cell type, adsorption of Ad2 beta gal2 in the presence of the polycations polybrene, protamine, DEAE-dextran, and poly-L-lysine significantly increased the percentage of cells that express lacZ. The polyanion heparin did not significantly alter gene transfer efficiency, but completely abrogated the effects of polycations. These data provide evidence that negatively charged moieties on the cell surface reduce the efficiency of adenovirus-mediated gene transfer, and that alteration of the charge interaction between adenoviruses and the cell surface may improve the potential clinical application of these vectors.