Genetic labeling of adenovirus with luciferase on pIX for vector detection

Adenoviral vectors have demonstrated great potential for gene therapy of many diseases. Significant effort has been devoted in this field to achieve targeted gene delivery to specific cells with the intention of improving the therapeutic index and safety. These targeting strategies require a powerful vector detection system to assess vector function. Present methods used to localize vectors are inadequate for evaluating targeting activity. Post-transduction reporter gene expression cannot accurately represent the physical biodistribution of vectors; vector labeling with chemical substrates may affect the infectivity of virions; and probing for viral components requires extensive preparation and strong accessible signal. The field of virotherapy has further challenged the effectiveness of current vector detection systems. Due to the dynamic nature of replicative agents, current static detection techniques cannot be used to monitor replication and spread of these viruses, two key functions of the ideal replicative agent. With respect to the former issue of vector detection in targeting schemes, we sought to develop a vector labeling approach to directly detect adenoviral particles. We designed a genetic labeling system that would also fulfill the latter goal of dynamic monitoring of adenoviral replication and spread. We hypothesized that an adenoviral structural protein genetically fused to an imaging ligand would allow vector detection and serve as a signature of viral replication and localization. Recently we successfully generated and characterized a recombinant adenovirus labeled with capsid fusion pIX-EGFP. The fluorescent label minimally perturbed viral function and provided an optical property useful for vector detection in tracking of cellular infection, flow cytometry, and tissue sections following systemic administration. We expanded our labeling strategy further to produce bioluminescent adenoviruses. The rationale for this pursuit revolves around the fact that bioluminescence can be detected much deeper in tissues than fluorescent proteins with much greater sensitivity and dynamic range as well as virtually no background luminescence. We generated bioluminescent adenoviruses by incorporating Renilla luciferase (RL) and firefly luciferase (FL) onto the pIX locale. Although these two proteins were significantly larger than EGFP, both Ad-IX-RL and Ad-IX-FL were rescued. After propagation and CsCl centrifugation of these two viruses, the viral gradients were fractionated and analyzed for viral DNA content and luciferase activity. Bioluminescence peaks corresponding with the viral DNA were detected for the mature (bottom) and empty capsid (top) viral bands of Ad-IX-RL and Ad-IX-FL, indicating the retained activity of both luciferases not only in the context of a fusion with pIX but also physical association with the virus capsid. We are characterizing these vectors further and are evaluating their general utility as well as potential for noninvasive detection in mice. Bioluminescent adenoviruses generated with our genetic labeling system will have great implications in vector targeting studies and can potentially be used to monitor adenovirus replication and spread in virotherapy applications.