Development of clinically feasible Ultrasound-mediated gene therapy for hemophilia

 产品说明：本提案的目标是开发一种临床可行的方案，用于超声（US）介导的因子VIII（FVIII）基因递送（UMGD）治疗血友病A（HemA）。目前对A型血友病患者的治疗涉及昂贵且不方便的蛋白质浓度的重复输注。在最近的临床试验中，腺相关病毒载体（AAV） 介导的基因疗法已显示出治疗血友病B1-3的极好前景。然而，在腺相关病毒载体中容纳大尺寸基因（例如凝血因子VIII）的局限性、对载体的免疫反应2，3和相关的转基因产物4，以及对腺相关病毒载体预先存在的免疫力重复治疗的局限性5，6显着阻碍了有效的腺相关病毒介导的基因治疗的发展A型血友病。以前我们证明了UMGD可以显著增强报告基因转移到小鼠7 -9和大鼠肝脏10中。这种非病毒基因转移策略可以绕过病毒基因治疗遇到的许多障碍。最重要的是，我们最近在UMGD进入A型血友病小鼠后达到了FVIII的治疗水平11。为了促进这些技术最终转化为人类应用，需要在大型动物模型中探索许多技术问题，包括治疗程序和方案、适当的甲基溴数量和类型以及超声参数和仪器。我们已经成功开发了原型US系统，包括几个非聚焦和半聚焦换能器，用于治疗犬12和猪13的大组织体积。我们还开发了几种新的中性和阳离子MB，以促进基因转移14。目前的提案侧重于开发一种安全且临床上可行的超声技术，沿着合适的手术技术，以在大型动物模型中实现有效的基因转移，从而导致高水平的FVIII基因表达。首先，我们将探讨 最佳的US参数，可以提高基因转移效率，最小的组织损伤的小鼠。此外，我们的数据表明，转基因主要是由肝细胞表达后UMGD进入肝脏。我们建议在MIP质粒中制备携带高表达FVIII变体基因的肝脏特异性构建体，以进一步增加和延长体内FVIII基因表达，从而实现A型血友病小鼠的表型校正。下一步，我们将改进我们的美国技术和手术技术，以优化大型动物模型中的基因转移效率。重要的是，我们将开发微创介入放射技术，将质粒DNA（pDNA）/MB混合物输送到猪的靶肝叶中，并结合经皮超声治疗程序。将在正常犬中进行长期实验，使用我们最有效的FVIII质粒和优化的治疗性US方法结合免疫调节，以实现持续和高水平的FVIII基因表达。如果成功，该项目将促进A型血友病犬模型的表型校正， 最终将这项新技术转化为人类应用，并可能从根本上改变A型血友病患者的治疗方式，获得更好的患者结局。

 DESCRIPTION: The goal of this proposal is to develop a clinically feasible protocol for ultrasound (US) mediated gene delivery (UMGD) of factor VIII (FVIII) to treat hemophilia A (HemA). Current treatment for HemA patients involves costly and inconvenient repeated infusions of protein concentrations. In a recent clinical trial, adenoassoicated viral vector (AAV) mediated gene therapy has shown excellent promise for treating hemophilia B1-3. However the limitation of accommodating large size gene such as FVIII in the AAV vector, the immune responses to the vector2, 3 and associated transgene products4, and limitation of repeated treatment with pre-existing immunity to AAV vector5, 6 significantly hinder the development of an effective AAV-mediated gene therapy treatment for HemA. Previously we demonstrated that UMGD can significantly enhance reporter gene transfer into the mouse7-9 and rat livers10. This nonviral gene transfer strategy can bypass many obstacles encountered by viral gene therapy. Most significantly, we have recently achieved therapeutic levels of FVIII following UMGD into HemA mice11. In order to facilitate the eventual translation of these technologies into human application, many technical issues, including treatment procedures and protocols, appropriate MB volumes and types, and US parameters and instrumentation require exploration in large animal models. We have successfully developed prototype US systems including several unfocused and semi-focused transducers to treat large tissue volumes in canine12 and swine13. We have also developed several new neutral and cationic MBs to facilitate gene transfer14. The current proposal focuses on the development of a safe and clinically feasible ultrasound technology along with suitable surgery techniques to achieve efficient gene transfer in large animal models, leading to high levels of FVIII gene expression. First, we will explore the best US parameters that can enhance gene transfer efficiency with minimal tissue damage in mice. In addition, our data indicate that transgene is principally expressed by hepatocytes following UMGD into the liver. We propose to make liver-specific constructs carrying a high-expressing FVIII variant gene in a MIP plasmid to further increase and prolong FVIII gene expression in vivo, in order to achieve phenotypic correction in HemA mice. Next, we will improve our US technology and surgery techniques to optimize gene transfer efficiencies in large animal models. Importantly, we will develop minimally invasive interventional radiologic techniques to deliver plasmid DNA (pDNA)/MB mixture into the target liver lobe combined with transcutaneous US treatment procedures in pigs. Long-term experiments will be performed in normal dogs using our most effective FVIII plasmid and the optimized therapeutic US method in combination with immunomodulation to achieve persistent and high-level FVIII gene expression. If successful, this project will facilitate the phenotypic correction in the HemA dog model and the eventual translation of this novel technology into human application, and could change fundamentally the way HemA patients are treated, with better patient outcomes.