INTRODUCTION: Gene therapy has the promise to provide curative therapies to many inherited monogenic liver diseases. Hemophilia B is an attractive target for gene therapy as a modest increase in the clotting factor activity can result in clinical benefits and attenuation of bleeding risk. Herein, we developed an alternative approach employing non-viral delivery of human factor IX (hFIX) DNA into the liver via hydrodynamic injection into the biliary tree. METHODS: We delivered hFIX DNA via biliary hydrodynamic injection into the pig liver, as accessed by endoscopic retrograde cholangio-pancreatography (ERCP) while using clinical equipment employed in treating human patients. The catheter was advanced into the common hepatic duct, and a balloon seal to prevent retrograde flow. The hydrodynamic injection consisted of 30 mL of saline solution containing 3–5.5 mg plasmid DNA injected at 2mL/sec. Four pigs in total were injected between 35 and 40 kg. RESULTS: The procedure was well tolerated in all four pigs. The safety profile was examined, including vital signs, liver function panel, and histology. hFIX DNA was integrated into the hepatocyte genome with co-injected piggyBac transposase plasmid and was found in all liver lobes by PCR, indicating global liver delivery. No human factor IX could be detected in the pig serum, but hFIX protein was detected in the liver tissue of all five lobes by western blot. Immunohistochemical staining revealed abundant hFIX-positive hepatocytes in all five lobes, with the most intense expression around the central vein. The transfection efficiency and pattern was reproduced in the three other pigs. CONCLUSION: We demonstrated non-viral endoscopic gene delivery successfully achieved hFIX expression in all liver lobes of the pig. No hFIX was detected in the pig serum, similar to reports in hFIX-transgenic pigs, suggesting pig hepatocytes may have a species-related secretory defect for hFIX protein. Future studies will continue to optimize gene delivery to develop this strategy as a therapy in human patients.Current clinical trials have focused on using adeno-associated viral (AAV) vectors to deliver hFIX into the liver to treat hemophilia B, but the resultant hFIX levels have been modest, and AAV vectors could not be re-dosed to increased hFIX expression.The advantages of a retrobiliary approach include the ability through one access point to reach the entire vast network of hepatocytes.Such a strategy would also be re-dosable in hemophilia B patients.Figure 1.: Immunohistochemistry staining showed hFIX-positive hepatocytes (orange-brown) with the most intense expression around the central vein.