Lentiviral vectors (LVs) are attractive vehicles for liver-directedgene therapy by virtue of their ability to stably integrate in thegenome of target cells and the lack of pre-existing immunityagainst vector components in most humans. Over the past years,we have developed a LV platform that can achieve stable transgeneexpression in the liver, induce transgene-specific immune toleranceand establish correction of hemophilia in mouse models uponsystemic administration. This LV is designed to stringently targettransgene expression to hepatocytes through transcriptional andmicroRNA-mediated regulation. We then investigated the efficacyand safety profile of portal vein administration of LVs expressingwild-type, codon-optimized (c.o.) or c.o. and hyperactive factor IX(FIX) in a canine model of hemophilia B. We produced large-scalebatches of LVs qualified for in vivo administration and treated adulthemophilia B dog by portal vein administration. We observed longtermstable reconstitution of canine FIX activity up to 1% of normaland significant amelioration of the clinical phenotype in 3 treated dogs(>9 years cumulative follow up). LV infusion was associated withtransient signs of inflammation and mild hepatotoxicity, which couldbe abrogated by pretreatment with anti-inflammatory drugs. There wasno detectable long-term toxicity or development of FIX inhibitors.In the perspective of clinical translation and to increase therapeuticefficacy, we next treated an 11-kg, hemophilia B dog by peripheralvein administration of LVs expressing the c.o. and hyperactive canineFIX at a 5-fold higher dose than those previously administered. At thecurrent follow-up (3 months after gene therapy) FIX activity is 6-9%of normal. Intravenous LV administration, coupled with a 1-day antiinflammatoryand anti-histamine pre-treatment, induced mild and selflimitingleukopenia and elevation of aminotransferases. Treatmentof more hemophilia B dogs is underway to confirm and extend theseresults. Overall, our studies, which suggest comparable efficacy ofLVby both portal and peripheral vein administration, position LVmediatedliver gene therapy for further pre-clinical development andclinical translation. LVs may thus complement other available vectorsto address some of the outstanding challenges posed by liver genetherapy of hemophilia and conceivably other diseases.