RGD inclusion in the hexon monomer provides adenovirus type 5-based vectors with a fiber knob-independent pathway for infection

Hypervariable region 5 (HVR5) is a hydrophilic, serotypically nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. W e have replaced the HVR5 sequence of Ad5 with that of heterologous peptides and studied their effects on virus viability. and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine "isogenic" ,viruses that differed in their flanking spacers. Whereas virus productivit y was not profoundly altered by any of these modifications, immunoprecipita tion experiments under nondenaturing conditions demonstrated that epitope r ecognition by its cognate monoclonal antibody (C3 MAb) was strongly linker dependent and correlated perfectly with the ability of C3 MAb to inhibit tr ansgene delivery and expression. An alpha(v)-specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-mod ified capsids,would enhance the transduction of cells displaying limiting a mounts of the virus attachment receptors. Interestingly, although hexon has never been implicated in Ad entry, the modified virus significantly increa sed the transduction of human vascular smooth muscle cells in vitro. Compet ition experiments with 293 cells saturated with recombinant knob further in dicated that the hexon-modified virus could use an additional, knob-indepen dent pathway for entry. We concluded that genetic engineering of the Ad5 he xon monomer constitutes a novel and feasible approach to equip the virus wi th additional targeting ligands.