Cystic fibrosis (CF) is a lethal inherited disorder of which the pulmonary complications are the most morbid and life limiting. Recombinant adenoviruses are being evaluated with the goal of reconstituting expression of the Cf gene in the human airway. The therapeutic potential of this approach is limited in part by the relative inefficiency by which adenovirus infects the proximal and conducting human airway. The central hypothesis of this proposal is that inefficient adenovirus infection of luminally exposed airway cells is due to the absence of the adenovirus internalization receptor (alphav integrins) on these cells. This hypothesis is supported by data in this application showing that adenovirus infection of airway cells in a human bronchial xenograft model directly correlates with the availability of an alphavbeta5 integrin. This integrin receptor is predominately localized to basal cells, and thus not accessible to an intraluminally delivered adenovirus. Adenovirus binds to target cells by virtue of its fiber protein and accomplishes cell entry by means of its penton base capsid protein. Many adenovirus serotypes appear to bind cellular alphav integrins through an RGD (Arg-Gly-Asp) motif present in the adenovirus penton base. Little is known about the role that fiber-mediated attachment and penton base- alphav integrin binding play in promoting adenovirus infection in the lung. This application proposes to study the role of these initial molecular events in the lung and to pursue strategies to improve adenovirus-mediated gene transfer to the airway epithelium. The first specific aim of this proposal is to determine the importance of fiber- mediated binding and host-cell alphav integrins in adenovirus infection of airway epithelium. This will be accomplished by testing adenovirus capsid proteins, soluble RGD peptides, and function-blocking monoclonal antibodies directed against alphav integrins for their ability to inhibit an adenovirus infection in a xenograft model of a human airway. Our second specific aim is to upregulate the surface expression of alphav integrins in luminally exposed airway cells using cytokines and growth factors and determine if this improves adenovirus infection of these epithelia. In addition, we will infect lung epithelial cells used to generate bronchial grafts with retroviruses that carry alphav-containing integrins. We hypothesize that the increased surface expression of alphav integrins in these grafts will increase their susceptibility to an adenovirus infection. Our final specific aim is to develop an adenoviral vector that efficiently targets airway epithelium through an alphav integrin independent pathway. Chimeric adenoviruses will be engineered to express novel ligands that will redirect the pathway of adenovirus, and thereby improve gene transfer to the airway epithelium. In summary, our studies will provide insight into the biology of adenovirus infection in the human airway, the regulation of airway alphav integrins, and have implications for successful lung-directed gene therapy.
