E2F acts as a transcriptional repressor when bound to unphosphorylated RB during the G(1) or G(0) phase. Upon phosphorylation, E2F is released from the E2F-RB complexes to activate transcription. Tumor cells are characterized by an increase in the level of "free" E2F as a consequence of the absence or hyperphosphorylation of RB. The E2F-1 promoter is a well-characterized E2F-responsive promoter, and it can be used to control adenovirus E1a gene expression as a strategy to achieve tumor-selective expression and replication of an adenovirus. ICOVIR-5 (Ad-DM-E2F-K-Delta24RGD) is an optimized oncolytic adenovirus that combines E1a transcriptional control by an insulated form of the E2F promoter with the Delta24 mutation of E1a to improve the therapeutic index of AdDelta24RGD. ICOVIR-5 also contains the Kozak sequence at the E1a start codon, which is important to restore E1a expression and viral replication to AdwtRGD levels in tumor cells. The unique combination of genetic elements in ICOVIR-5 allows the selectivity for cells with a deregulated E2F-RB pathway to be increased and potent anti-tumoral activity to be maintained. Dose-response toxicological and efficacy studies after a single systemic administration in pre-clinical models in mice are presented to demonstrate that this virus holds promise for treatment of disseminated cancer.