CHARACTERIZATION OF AN ADENOVIRUS VECTOR CONTAINING HETEROLOGOUS PEPTIDE EPITOPE IN THE HI LOOP OF THE FIBER KNOB

The utility of the present generation of recombinant adenovirus vector s for gene therapy applications could potentially be improved by desig ning targeted vectors capable of gene delivery to selected cell types in vivo. In order to achieve such targeting, we are investigating the possibilities of incorporation of ligands in the adenovirus fiber prot ein, which mediates primary binding of adenovirus to its cell surface receptor. Based on the proposed structure of the cell-binding domain o f the fiber, we hypothesized that the HI loop of the fiber knob can be utilized as a convenient locale for incorporation of heterologous lig ands. In this study, He utilized recombinant fiber proteins expressed in baculovirus-infected insect cells to demonstrate that the incorpora tion of the FLAG octapeptide into the HI loop does not ablate fiber tr imerization and does not disturb formation of the cell-binding site lo calized in the knob, We then generated a recombinant adenovirus contai ning this modified fiber and showed that the short peptide sequence en gineered in the knob is compatible with the biological functions of th e fiber, In addition, by using a ligand-specific antibody, we have sho wn that the peptide incorporated into the knob remains available for b inding in the contest of mature virions containing modified fibers, Th ese findings suggest that heterologous ligands can be incorporated int o the HI loop of the fiber knob and that this locale possesses propel ties consistent with its employment in adenovirus retargeting strategi es.