We conducted a phase 1 trial for single-dose intravenous Ad5CRT, a replication-defective adenovirus vector expressing HSVtk (herpes simplex virus thymidine kinase) modulated by a specific trans-splicing ribozyme that targets human telomerase reverse transcriptase (hTERT)-encoding RNAs. Dose-limiting toxicities (DLTs) were evaluated in 15 patients at dose levels of 0.1-2 × 1012 virus particles. Patients well tolerated study treatment. During the DLT evaluation period, none of the 15 patients developed any grade 4 toxicities or treatment discontinuation that was related to agents investigated by this trial. The most frequent treatment-related adverse event was fever/chill (26.7%). Of the 18 patients, no patients achieved a partial or complete response, and the median progression-free survival for 18 patients was 1.1 months (95% CI, 1.0-1.3) and the results suggest no clinical benefit from this treatment. Ad5CRT's circulating virus half-life was approximately 10 min. Maximum tolerated dose was 2 × 1012 virus particles. Single-dose intravenous Ad5CRT was feasible and well tolerated in patients with gastrointestinal cancer liver metastasis. Ad5CRT did not provide meaningful clinical benefit, and the reason for the lack of efficacy was not entirely clear because no pharmocodynamic assessment was made.