Genetic labeling of adenovirus with red fluorescent proteins on pIX for vector detection

Adenoviral vectors offer great promise for gene therapy of various diseases. Recent development in targeting gene delivery calls for a more powerful vector detection system to assess vector functionality. Current vector imaging techniques-including reporter gene expression, vector labeling, and viral component detection-are limited in several ways. The physical biodistribution of vectors cannot be accurately represented by post-infection reporter gene expression; vector labeling with chemical substrates may affect the infectivity of virions; and detection of viral components requires extensive preparation and strong accessible signal. When applied in the field of virotherapy, the drawbacks of current vector detection systems are even more evident. Due to the dynamic nature of replicative agents, current static detection techniques based on biopsy examination cannot effectively confirm replication and spread of these viruses, two key functions of the ideal replicative agent. To address the former issue of vector detection in targeting situations, we sought to develop a vector labeling approach to directly visualize adenoviral particles. We conceived of a genetically based labeling system that would also fulfill the latter purpose of dynamic monitoring of adenoviral replication and spread. We hypothesized that an adenoviral structural protein genetically fused to an imaging ligand would enable 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 had minimal effect on viral function and conferred 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 make red fluorescent adenoviruses. One reason for designing red fluorescent adenoviruses is that a better signal-to-noise ratio can be achieved in the red spectral range, yielding improved vector detection in tissue which typically contains high green autofluorescence. In addition, red fluorescence would provide better tissue penetration allowing deeper noninvasive detection of these red fluorescent proteins. We generated red fluorescent adenoviruses by incorporating monomeric (mRFP1) and tandem dimer (tdimer2(12)) red fluorescent proteins onto the pIX locale. After propagation and CsCl centrifugation of both viruses, red mature (bottom) and empty capsid (top) viral bands were observed. Fractionation studies validated our visual observation by revealing that both the bottom and top bands of the two viruses contained red fluorescence which co-migrated with viral DNA. Abundant purified Ad-IX-mRFP1 and Ad-IX-tdimer2(12) viral particles could be detected by fluorescence microscopy. We are characterizing these vectors further and are evaluating their general utility as well as potential for noninvasive detection in mice. Red fluorescent adenoviruses generated with our genetic labeling system will have great impact in vector targeting studies and can potentially be used to monitor adenovirus replication and spread in virotherapy applications.