Endoscopic-mediated hydrodynamic gene therapy for hemophilia B through the biliary system

基因疗法已被探索用于治疗血友病，但凝血表达水平下降

Gene therapy has been explored for cure of hemophilia, but declining expression levels of clotting factors from AAV vectors in clinical trials over-time represents a significant limitation. For hemophilia B, the long-term goal is to establish a redosable gene therapy that could maintain human Factor IX (hFIX) production throughout a patient’s lifetime. The overall objective is to use bioengineering principles to develop a non-viral, hydrodynamic strategy in large animals that would not be limited by any neutralizing capsid antibodies or T cell responses hampering AAV vectors. The central hypothesis is that an endoscopic procedure mediating hydrodynamic injection through the biliary system could mediate effective delivery system for hFIX into the liver of large animals with clinically relevant expression. The rationale is that biliary system effectively contacts all hepatocytes, while possessing much less intrahepatic volume than the vascular system (30 mL vs. 600 mL), avoiding limitations of previous, inefficient vascular hydrodynamic delivery approaches with balloon catheters. The hypothesis is supported by preliminary studies in pigs, which show that the procedure is well- tolerated with hepatocyte transfection levels exceed AAV transduction levels in the liver of primates. The central hypothesis will be tested by pursuing two specific aims: 1) Safety and parameters of biliary hydrodynamic injection will be evaluated to address concerns over physiologic disturbance. Maximum volumes and flow rates during endoscopic injection will be tested to understand tolerability, followed by correlation of these parameters and fluid pressure with reporter gene expression. Biochemical and hematologic side effects will be examined during plasmid DNA injection, and distribution of plasmid DNA within the liver and other pig tissues will be assessed to categorize off- target risks. 2) hFIX gene therapy will be optimized for therapeutic levels against hemophilia B. hFIX DNA will be injected through biliary hydrodynamic delivery in pigs and expression levels quantified. The percentage of pig hepatocytes expressing hFIX and their localization within the liver lobule will be assessed. Biliary hydrodynamic injection will also be modeled in non-human primates to assess tolerability in this animal model, with goal of achieving similar hFIX plasma levels to AAV vectors in primates. Repeated hydrodynamic injection of hFIX pDNA vector will be performed to increase hFIX levels and validate redosing. The research proposal is innovative, in the applicant’s opinion, because hydrodynamic delivery was thought to be clinically unfeasible and inefficient in large animals versus viral vectors, so this research solves this major gap. The proposed research is significant because non-viral, plasmid based gene therapy is safer and magnitudes less expensive than viral vectors, paving the way for redosable gene therapy for other monogenic liver diseases beyond hemophilia.