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

基因治疗已被探索用于治愈血友病，但降低凝血因子的表达水平， 随着时间的推移，临床试验中来自AAV载体的因子代表了显著的限制。血友病 B，长期目标是建立一种可恢复的基因疗法， （hFIX）的产生。总体目标是利用生物工程 在大型动物中开发非病毒的流体动力学策略的原则， 任何阻碍AAV载体的中和衣壳抗体或T细胞应答。中央 假设是通过胆道介导流体动力学注射的内窥镜手术 系统可以介导hFIX进入大型动物肝脏的有效递送系统， 相关的表达。基本原理是胆道系统有效地接触所有肝细胞， 肝内体积远小于血管系统（30 mL vs. 600 mL）， 先前使用球囊导管的低效血管流体动力学递送方法的局限性。 这一假设得到了猪的初步研究的支持，这些研究表明，该程序是良好的- 在灵长类动物的肝脏中，肝细胞转染水平的耐受性超过AAV转导水平。 将通过追求两个特定目标来检验中心假设：1）安全性和参数 将评价胆汁流体动力学注射，以解决生理紊乱的问题。 将测试内窥镜注射期间的最大体积和流速，以了解耐受性， 然后将这些参数和流体压力与报告基因表达相关。 在质粒DNA注射期间将检查生化和血液学副作用， 将评估质粒DNA在肝脏和其他猪组织中的分布， 目标风险。2)hFIX基因治疗将针对血友病B的治疗水平进行优化。hfix 将通过猪的胆汁流体动力学递送注射DNA，并定量表达水平。 表达hFIX的猪肝细胞的百分比及其在肝小叶内的定位将被确定。 评估。还将在非人灵长类动物中对胆汁流体动力学注射进行建模，以评估 在该动物模型中的耐受性，目标是在小鼠中实现与AAV载体相似的hFIX血浆水平。 灵长类动物将进行hFIX pDNA载体的重复流体动力学注射以增加hFIX 水平和验证再给药。申请人认为，该研究提案具有创新性，因为 流体动力学递送被认为在大型动物中临床上不可行且效率低， 病毒载体，所以这项研究解决了这个主要的空白。这项研究意义重大，因为 非病毒的、基于质粒的基因治疗比病毒载体更安全且更便宜， 为血友病以外的其他单基因肝病的可恢复基因治疗铺平了道路。

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.