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

基因治疗已被探索用于血友病的治愈，但凝血的表达水平下降 在临床试验中，来自AAV载体的因素随着时间的推移是一个显著的限制。治疗血友病 B，长期目标是建立一种可以维持人类凝血因子IX的可重复使用的基因疗法 (HFIX)患者一生中的产量。总体目标是利用生物工程 在大型动物中开发一种不受限制的非病毒、流体动力学策略的原则 任何中和衣壳抗体或T细胞反应阻碍AAV载体。中环 假说是内窥镜下通过胆道进行流体动力注射 系统可介导hFIX进入大动物肝脏的有效递送系统，临床上 相关表述。其基本原理是胆道系统有效地接触所有肝细胞，而 具有比血管系统小得多的肝内体积(30毫升对600毫升)，避免 以前使用球囊导管的低效血管流体动力学给药方法的局限性。 这一假说得到了对猪的初步研究的支持，这些研究表明，这种程序是很好的- 灵长类动物肝脏对肝细胞转导水平的耐受性超过AAV转导水平。 中心假设将通过追求两个具体目标来检验：1)安全和参数 将对胆道流体动力注射进行评估，以解决对生理障碍的担忧。 将测试内窥镜注射过程中的最大体积和流速，以了解耐受性， 然后对这些参数和流体压力与报告基因表达的相关性进行了研究。 在注射质粒DNA的过程中，将检查生化和血液学副作用，并 将对肝脏和其他猪组织中的质粒DNA分布进行评估，以将其归类为- 瞄准风险。2)hFIX基因治疗将针对血友病B的治疗水平进行优化。 DNA将通过胆道流体动力输送注入猪体内，并对表达水平进行量化。 表达hFIX的猪肝细胞百分比及其在肝小叶中的定位将是 评估过了。胆道流体动力注射也将在非人类灵长类动物中模拟，以评估 该动物模型的耐受性，目标是达到与AAV载体相似的hFIX血浆水平 灵长类动物。重复注射hFIX PDNA载体以增加hFIX 级别并验证重新设置。在申请人看来，这项研究提案具有创新性，因为 流体动力给药被认为在临床上是不可行的，在大型动物身上是低效的。 病毒载体，所以这项研究解决了这一重大差距。这项拟议的研究具有重要意义，因为 非病毒、基于质粒的基因治疗比病毒载体更安全、更便宜， 为血友病以外的其他单基因肝病的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.