Development of clinically feasible Ultrasound-mediated gene therapy for hemophilia

 描述：这项建议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)的基因传递(UMGD)治疗血友病A(HEMA)。目前对HEMA患者的治疗包括昂贵和不方便的反复输注蛋白质浓度。在最近的一项临床试验中，腺相关病毒载体(AAV) 介导性基因治疗在治疗血友病B1-3方面显示出极好的前景。然而，AAV载体中容纳FVIII等大片段基因的局限性、对载体2、3及其相关转基因产物4的免疫应答、以及对AAV载体5、6预先存在的免疫重复治疗的限制，显著阻碍了有效的AAV介导的HEMA基因治疗的发展。先前我们证明了UMGD可以显著增强报告基因在小鼠7-9和大鼠肝脏10中的转移。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，在UMGD进入HEMA小鼠11之后，我们最近达到了FVIII的治疗水平。为了促进这些技术最终转化为人类应用，许多技术问题，包括治疗程序和方案，适当的甲基溴体积和类型，以及美国的参数和仪器，需要在大型动物模型中进行探索。我们已经成功地开发了原型美国系统，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MBS来促进基因转移14。目前的建议侧重于开发一种安全且临床可行的超声技术以及合适的手术技术，以在大型动物模型中实现高效的基因转移，从而导致FVIII基因的高水平表达。首先，我们将探索 最佳超声参数，可提高基因转移效率，并将对小鼠的组织损伤降至最低。此外，我们的数据表明，转基因主要是在UMGD进入肝脏后由肝细胞表达。我们建议在MIP质粒中构建携带高表达FVIII变异体基因的肝脏特异体，以进一步增加和延长FVIII基因在体内的表达，以实现HEMA小鼠的表型纠正。接下来，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的基因转移效率。重要的是，我们将开发微创介入放射学技术，将质粒DNA(PDNA)/MB混合物输送到靶区肝叶，并结合经皮超声治疗过程。我们将使用我们最有效的FVIII质粒和优化的治疗US方法，结合免疫调节在正常狗身上进行长期实验，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HEMA犬模型的表型纠正和 这项新技术最终将转化为人类应用，并可能从根本上改变HEMA患者的治疗方式，获得更好的患者结果。

 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.