Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

Adenoviral vector systems for gene therapy can be much improved by targetin g vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affini ty in the viral capsid. We reasoned that these requirements could be fulfil led by deleting the entire knob domain of the adenovirus fiber protein and replacing it with two distinct moieties that provide a trimerization functi on for the knobless fiber and specific binding to the target cell, respecti vely. To test this concept, we constructed adenoviral vectors carrying knob less fibers comprising the ct-helix trimerization domain from MoMuLV envelo pe glycoprotein. Two mimic targeting ligands, a Myc-epitope and a 6His-tag, were attached via a flexible linker peptide. The targeted knobless fiber m olecules were properly expressed and imported into the nucleus of adenoviru s packaging cells, where they were incorporated as functional trimers into the adenovirus capsid. Both ligands were exposed on the surface of the viri on and were available for specific binding to their target molecules. Moreo ver, the knobless fibers mediated gene delivery into cells displaying recep tors for the coupled ligand. Hence, these knobless fibers are prototype sub strates for Versatile addition of targeting ligands to generate truly targe ted adenoviruses.