Prior to the institution of a clinical trial for liver directed gene therapy in patients with partial ornithine transcarbamylase deficiency (OTCD), an inborn error of urea synthesis, preclinical studies were done in C3H mice and baboons to define a safety profile. A formal toxicology study was performed in male and female C3H mice to compare the safety of an E1-E4 deleted vector expressing the human OTC cDNA (H5.001CBhOTC) to two earlier generation vectors in which E1 is deleted, but E4 is left intact. Animals received a single intravenous dose of vector (1011 particles, infused via the tail vein), selected to achieve gene transfer in >50% of hepatocytes. Mice were necropsied 4, 11, 29 or 61 days later; the primary indices of toxicity were serum transaminases (ALT, AST) and histopathological analysis of liver tissue. The vehicle group had 1 animal of each gender at each time point while the vector infused groups had 3 animals of each gender at each time point. The two early generation (E4 intact) vectors were associated with a self-limited vector-induced hepatitis characterized by portal and lobular inflammation that peaked at day 11 and resolved by day 29. The E1-E4 deleted vector resulted in essentially no histological evidence of toxicity. The pattern and time course of toxicity was reflected in serum transaminases and other liver function tests, as well as histopathology. Substantial increases in AST and ALT to 1000-1500 IU (normal <50) were observed with the early generation vectors, peaking at day 11 and eventually returning to baseline. Following infusion of the E1-E4 deleted vector, 10-15-fold lower values for the liver transaminases were observed. The feasibility and safety of recombinant adenoviruses transduced into the hepatic artery of baboons was also studied. The E1-E4 deleted vector was introduced into the common hepatic artery at a dose of either 2×109 particles/kg, or 6×109 particles/kg. Serial sequential liver biopsies were performed at 8 and 29 days, and full necropsy was performed at 61 days after vector delivery. ALT and AST were essentially unchanged. The data show that adenoviral vectors deleted of both E1 and E4 offer significant safety advantages over corresponding E4-containing vectors in terms of hepatitis, and have acceptable safety profiles in doses selected for use in the proposed clinical trial.