Adenovirus type 5 viral particles pseudotyped with mutagenized fiber proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells

A major limitation of adenovirus type 5 (AdS)-based gene therapy, the inabi lity to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-a denovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domai n have been shown to alter the tropism of the virus. We have developed a no vel system to rapidly evaluate the function of modified fiber proteins in t heir most relevant context, the adenoviral capsid. This transient transfect ion-infection system combines transfection of cells with plasmids that expr ess high levels of the modified fiber protein and infection with Ad5.beta g al.DeltaF, an E1-, E3-, and fiber-deleted adenoviral vector encoding beta - galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were in corporated via mutagenesis into the fiber expression plasmid, and the resul ting fiber proteins were subsequently incorporated onto adenoviral particle s. Mutations Located in the fiber knob AB and CD loops demonstrated the gre atest reduction in fiber-mediated gene transfer in HeLa cells. We also obse rved effects on transduction efficiency with mutations in the FG loop, indi cating that the binding site may extend to the adjacent monomer in the fibe r trimer and in the HI loop. These studies support the concept that modific ation of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneous ly with novel ligands for the development of a systemically administered, t argeted adenoviral vector.