Polarity Influences the Efficiency of Recombinant Adenoassociated Virus Infection in Differentiated Airway Epithelia

Abstract

To better understand mechanisms that limit rAAV transduction in the lung, we have evaluated several unique features of rAAV infection in polarized primary airway epithelial cultures. rAAV was found to transduce the basolateral surface of airway epithelia 200-fold more efficiently than the apical membrane. These differences in membrane infection correlated with the abundance of apical heparan sulfate proteoglycan (AAV-2 receptor) and virus binding. UV irradiation augmented rAAV transduction greater than 20-fold, only when virus was applied to the apical membrane. Ultrastructural analysis of UV-irradiated primary cultures demonstrated significant changes in microvilli architecture following exposure to 25 J/m² UV. Although virus binding and the abundance of heparan sulfate proteoglycan were not increased at the apical membrane following UV irradiation, increased receptor-independent endocytosis of fluorescent beads was seen at the apical membrane following UV irradiation. We hypothesize that endocytotic processes associated with apical membrane-specific pathways of viral entry, and/or processing of virus to the nucleus, may be altered following UV irradiation. Interestingly, UV irradiation had an inhibitory effect on rAAV transduction from the basolateral membrane, which correlated with a decrease in the abundance of heparan sulfate proteoglycan at the basal membrane. In summary, these findings suggest that independent pathways of viral transduction may occur in the apical and basolateral compartments of polarized airway epithelia.

Overview summary

Recombinant adenoassociated virus (rAAV) has been suggested as a potentially useful vector for gene therapy of cystic fibrosis. However, current application of this vector for gene therapy in the airway is limited by its low transduction efficiency in this tissue. A better understanding of the mechanism(s) responsible for this low efficiency will potentially lead to improved methods for rAAV gene delivery in the treatment of cystic fibrosis lung disease. In this article, we demonstrate a significant polarity of rAAV infection in differentiated airway epithelial cells, with transduction from the basolateral surface much greater than that from the apical surface. Polarized localization of the heparan sulfate proteoglycan AAV-2 receptor to the basal surfaces of differentiated airway epithelia appears, at least in part, to be responsible for this observed polarity of infection. However, UV irradiation preferentially induced rAAV transduction from the apical, but not the basolateral, side of epithelia, suggesting that additional rate-limiting steps distal to receptor binding may also exist. UV-associated alterations in the apical membrane cytoskeleton and increased receptor-independent apical membrane endocytosis suggest that UV irradiation may induce apical infection by augmenting endosomal processing of rAAV. These studies suggest that at least two independent mechanisms are responsible for limiting the efficiency of rAAV transduction in polarized airway epithelial cells.