Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that the deletion of a 48-60 aa region in the CR1 domain of E1A resulted in low levels of E1A protein, conditioning the replication of the mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CR1 (Delta-39), CR2 (Delta-24) regions or in both regions (Delta-24/39) of E1A protein. Analyses of cell viability showed a comparable cytopathic effect of Delta-24 and Delta-39, and viral replication studies revealed similar replication capability for both adenoviral constructs in glioma cells, although Delta-24/39 displayed more attenuated potency. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, immunoblotting analysis showed that E1A protein levels in cells infected with Delta-39 was remarkably downmodulated. Further, protein stability studies through inhibiting protein synthesis with anisomycin revealed enhanced degradation of CR1-mutant E1A proteins, and block of the proteasome activity with lactacystin resulted in the striking rescue of the E1A levels. Collectively, our data showed that, compared to Delta-24, the deletion in Delta-39 resulted in suboptimal expression level of E1A protein, leading to significant attenuation of the virus in normal astrocytes but still maintaining efficient replication in glioma cells. We conclude that the level of E1A protein is a critical determinant of the selectivity of oncolytic adenoviruses and propose a completely novel strategy for the design and construction of conditionally replicative adenovirus.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]