The utility of conditionally replicative adenoviruses (CRAds) in cancer gene therapy is based on their ability to destroy tumor cells by oncolysis. However, in order to achieve adequate therapeutic response, CRAds have to spread through the tumor tissue by replication. Thus, to study the potency of these viruses to replicate and penetrate in tumors, we created a herpes simplex virus type 1 thymidine kinase-green fluorescent protein fusion gene (TK-GFP) encompassing CRAd (Ad5Δ24TK-GFP), whose tumor selectivity is mediated by a retinoblastoma (Rb)-binding site mutation. In addition, we evaluated the oncolytic efficacy of Ad5Δ24TK-GFP in combination with the TK/ganciclovir (GCV) system. Based on our results, Ad5Δ24TK-GFP replicates in cancer cells resulting in oncolysis and can efficiently penetrate into tumors. Additionally, the combination of GCV with Ad5Δ24TK-GFP augmented cell death in vitro but this was not observed in vivo: tumor growth was significantly reduced by oncolysis when compared to non-replicative virus (p<0.001), but administration of GCV did not significantly enhance oncolysis. This suggests that in certain conditions, TK/GCV-mediated cell killing may be counterproductive to replication and oncolysis, which on the other hand might be useful feature for clinical trials in case of replication-associated toxicity.

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