Generation and selection of targeted adenoviruses embodying optimized vector properties.

The utility of adenovirus serotype 5 (Ad5)-based vectors for gene therapy applications would be improved by cell-specific targeting. However, strategies to redirect Ad5 vectors to alternate cellular receptors via replacement of the capsid fiber protein have often resulted in structurally unstable vectors. In view of this, we hypothesized that the selection of modified adenoviruses during their rescue and propagation would be a straightforward approach that guarantees the generation of functional, targeted vectors. Based on our first generation fiber-fibritin molecule, several new chimeric fibers containing variable amounts of fibritin and the Ad5 fiber shaft were analyzed via a new scheme for Ad vector selection. Our selected chimera, composed of the entire Ad5 fiber shaft fused to the 12th coiled-coil segment of fibritin, is capable of efficient capsid incorporation and ligand display. Moreover, transduction by the resultant vector is independent of the expression of the native Ad5 receptor. The incorporation of the Fc-binding domain of Staphylococcus aureus protein A at the carboxy terminus of this chimeric fiber facilitates targeting of the vector to a variety of cellular receptors by means of coupling with monoclonal antibodies. In addition, we have concluded that Ad5 vectors incorporating individual targeting ligands require individual optimization of the fiber-fibritin chimera, which may be accomplished by selecting the optimal fiber-fibritin variant at the stage of rescue of the virus in cells of interest, as described herein.

Genetic targeting strategies for adenovirus.

Adenovirus serotype 5 (Ad5) continues to be regarded as a gene delivery vehicle of high utility for a variety of clinical applications. However, targeting of the virus to alternate, non-native receptors has become a mandate for many gene therapy approaches, as inefficient viral transduction of target tissues has proven detrimental to the utility of Ad5. Thus, various targeting strategies have been endeavored to the end of highly specific cellular transduction, including that of genetic manipulation of the viral capsid. Modification of the tropism-determining fiber protein and other capsid locales has allowed vectorologists to develop vectors that are highly superior to the first-generation adenoviruses employed for gene therapy. Herein, the various genetic targeting strategies for Ad5 are reviewed, and the various schemas in which targeted transduction has been achieved with tropism-modified vectors are outlined.

A single-component CD40-targeted adenovirus vector displays highly efficient transduction and activation of dendritic cells in a human skin substrate system.

Dendritic cell (DC) based tumor vaccination usually involves the administration of ex vivo generated autologous DC. Transduction of DC by viral vectors in vivo has been proposed as a more standardized and easily clinically applicable approach. Previously, we have reported that an Ad5 vector targeted to CD40 via genetic capsid incorporation of CD40L achieves selective transduction of DC in vitro. In the present study, we evaluate the ability of this vector to deliver transgenes in a stringent human substrate system. We report the capacity of this CD40-targeted vector to infect, with high efficiency, cutaneous DC resident in human skin explants, while simultaneously inducing their activation and maturation. This latest generation of single-component, fully targeted vectors should make feasible the clinical testing of in vivo DC-targeted vaccines.