We have evaluated the potential of liver-directed, helper-dependent adenoviral (HDAd) vector-mediated gene therapy in the hemophilia B dog. Two dogs were injected intravenously with HDAd (3 x 10(12) VP/kg) bearing a liver-restricted canine coagulation factor IX (FIX) expression cassette. After injection, the whole blood clotting time for both dogs declined from >60 min to </=20 min for at least 604 and 446 days, respectively. Peak FIX activities of 34.1 and 129.2% were detected at 12x14 days and then slowly declined to 2 to 5% by 120 days and stabilized at these therapeutic levels for at least 418 and 257 days. For one dog, a peak FIX level of 500 ng/ml was achieved and stabilized at >170 ng/ml for at least 256 days. For the other dog, a peak FIX level of 1258 ng/ml was achieved and stabilized at >400 ng/ml for at least 213 days. Inhibitor formation was not evident in either animal. Importantly, whereas untreated hemophilia B dogs suffer five or six spontaneous bleeds per year, the treated dogs suffered no such bleeds postinjection. Significantly, this study is the first to demonstrate long-term phenotypic correction of a genetic disorder in a large animal with HDAd. Although no evidence of chronic toxicity was observed in either animal, systemic vector administration at 3 x 10(12) VP/kg was accompanied by acute, albeit transient and variable laboratory abnormalities (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine phosphokinase, and platelet counts). The results of this study highlight both the potential benefit and the risk associated with systemic intravascular delivery of high-dose HDAd for liver-directed gene therapy.