Vector targeting and purification

The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres is lacking. Likewise, direct comparison of one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for re-targeting.

Metabolic biotinylation allows one to tag a number of capsomere proteins with biotin acceptor peptides and then combine these with any biotinylated ligand. This provides a unique platform to compare the functions of a given virus's proteins for targeting and purification.

As a first example of this approach, the lab is utilizing metabolic biotinylation to directly compare targeted transduction of adenovirus as mediated by its fiber, protein IX and hexon capsomeres using a variety of biotinylated ligands. These results clearly demonstrate that cell targeting with a variety of high-affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells.

These data suggest that vector targeting by genetic incorporation of high-affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of adenoviral vectors using the metabolic biotinylation system is most effective through capsomeres such as protein IX and hexon.

The team predicts the same rules may apply when targeting ligands are applied on other virus proteins that are not evolved to release from the virion during cell entry. Likewise, it is probable that chemical conjugation approaches that cross-link ligands to nonshedded proteins may also confront these problems. Work is underway to determine exactly how these modified vectors fail, how the affinity of the ligands affects this process, and how one might use or avoid this biology to maximize vector specificity.