Specific research projects in my lab focus on tumor targeting using adenoviral vector technology. The knob domain of the adenovirus fiber protein is the primary viral attachment protein for the mammalian cell-surface CAR receptor. Recombinant protein and viral vector technologies are used to modify that interaction and enable us to develop specific protein adaptor molecules and adenoviral vectors capable of serving as molecular imaging and therapeutic reagents that will target the binding of the adenovirus fiber to receptors other than CAR. Peptides and monoclonal antibodies are used as proven methods of targeting tumors, and new technologies are under development. Both cell culture and well-characterized in vivo tumor models will be used to demonstrate tumor cell binding and infection by adenovirus.
Short peptides are inserted into the knob domain of the fiber protein into either the HI surface loop of at the C-terminus. Tumor targeting peptides including RGD, NGR and ASL tumor-specific motifs have been utilized.
Chemical retargeting of adenovirus using bispecific antibodies directed against both the fiber capsid protein and tumor-specific cell surface molecules present on either endothelial cells or tumor cells has been used. Targets include negatively-charged phospholipids and vegf:vegfR complexes.
Transcriptional targeting using hypoxia-responsive promoters
The use of hypoxia-responsive promoters to drive transgene expression in adenovirus vectors resulted in selective gene expression in tumors by comparison to liver, and generally performed better than the other promoters with respect to overall expression and tumor selectivity. We attribute this to augmented expression from stress-responsive promoters in the hypoxic and inflammatory tumor microenvironment.
MicroRNA target-mediated reduction of off-target expression
We have utilized miR122 target sites cloned downstream from the luciferase transgene in adenovirus vectors to reduce the liver expression of luciferase by 30-50 fold, while maintaining high level expression in tumors in vivo. Combining this reduction in off-target expression with hypoxia-responsive promoters was even more effective and produced vectors with even greater tumor-selectivity.
Tumors are routinely imaged by optical detection of bioluminescence, which is correlated with standard biochemical and molecular detection methods for gene delivery and expression. High resolution detection of gene expression is under development using novel reporter genes.