We have constructed an oncolytic adenovirus expressing the Escherichia coli nitroreductase gene nfsB from an internal ribosome entry site (IRES) in the adenovirus L5 major late transcript. The virus (Tcf-NTR) has Tcf transcription factor-binding sites in the E1A, E1B, and E4 promoters, which restrict viral replication to cells that have activation of the Wnt signaling pathway. This virus was compared with an E1B-55K-deleted virus expressing nitroreductase (NTR) from a cytomegalovirus (CMV) promoter in the E1B-55K region [CRAd-NTR(PS1217H6)]. Both viruses express NTR in colorectal cancer cell lines and show increased cytopathic effect in the presence of the prodrug CB1954. Unlike the Tcf-NTR virus, the CMV-NTR virus expresses NTR in human lung fibroblasts and sensitizes these normal cells to CB1954. The in vivo activity of the viruses was tested in SW620 xenografts in nude mice by intravenous injection of 1,011 particles of virus followed 1 week later by intraperitoneal injections of CB1954. The CMV-NTR virus produced minimal effects in this model. The median time to form 1,000-mm(3) tumors in mice treated with the Tcf-NTR virus plus CB1954 was increased from 14 to 26 days (p=0.003), but this was due mainly to the direct oncolytic effect of the virus. Combination therapy with 3 x 10(11) particles of Tcf-NTR virus (given intravenously) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) (given orally) significantly improved survival (median, >50 days), and addition of CB1954 to this regimen further delayed tumor growth. These results show that the Tcf-NTR virus is more tumor selective and active than the CMV-NTR virus. At the level of transduction that can be achieved currently with oncolytic viruses given intravenously, drugs such as RAD001, which do not require activation by the virus, produce greater increases in efficacy than prodrugs such as CB1954.