Ultrasound-mediated gene delivery to achieve therapeutic correction of hemophilia A

项目摘要 该提案的目标是通过非侵入性的方法实现血友病A（HemA）的长期治疗矫正。 在狗模型中超声（US）介导的因子FVIII（FVIII）质粒的基因递送（UMGD）方案。 A型血友病是一种遗传性疾病，其特征在于缺乏凝血因子VIII。患者接受急性治疗 或者通过蛋白质替代疗法进行治疗，这是非常昂贵和不方便的。基因治疗是高度 有希望通过将hFVIII转基因递送到靶细胞中以持续产生 治疗水平的FVIII蛋白。使用重组腺病毒进行HemA基因治疗的最新临床试验 相关病毒（rAAV）载体已经显示出非常有希望的结果。然而，在以下方面仍然存在重大障碍： 阻止对大部分患者的治疗，特别是具有高滴度抗AAV抗体的患者。 也禁止重复治疗。UMGD已经成为一种有效的基因转移方法， 临床相关性和翻译潜力。与病毒基因转移相比，UMGD转移质粒 更容易制备和更具成本效益的载体;它还具有更低的免疫应答和毒性， 特异性组织靶向，防止随机整合，并允许载体的重复递送。其他 非病毒基因递送方法如DNA包装的纳米颗粒遇到了跨越 核被膜用于DNA转录。我们已经建立了一个微创的，经血管的 一种有效地将质粒DNA（pDNA）/微泡（MB）混合物递送到靶肝叶中的方法， 在大型动物模型中的经皮超声应用。我们发现高水平的荧光素酶报告基因 在猪中实现了基因表达，在犬中检测到治疗水平的FVIII表达。 使用临床上可行的方案。仅观察到短暂的组织损伤并迅速修复， 在短时间内恢复正常。然而，为了将这项新技术应用于临床，我们认识到， 需要解决几个主要问题，（i）需要更高的FVIII表达水平来实现长期的- 术语治疗效果，（ii）需要评估和维持治疗性FVIII表达的持续性， (iii)需要在靶向肝组织上持续高效的US治疗， 高效转染。(iv)需要更好的功能性FVIII表达和减少的肝损伤。这可能是 通过靶向肝窦内皮细胞（LSEC）中的FVIII转染实现， 合成或通过使用新合成的纳米气泡（NB）。因此，我们建议继续改善 经皮UMCD仪器、换能器、US方案、FVIII质粒构建体和小鼠中的MB/NB 猪。此外，我们将使用最佳经皮UMGD方案结合 最佳的质粒构建体和MB/NB，以实现FVIII表达的持续和治疗水平， 正常和HemA狗。这些进展将促进这项新技术最终转化为 人类应用，为治疗A型血友病患者和潜在的其他遗传疾病带来显著益处。

Project Summary The goal of this proposal is to achieve long-term therapeutic correction of hemophilia A (HemA) via a noninvasive protocol of ultrasound (US) mediated gene delivery (UMGD) of factor FVIII (FVIII) plasmids in the dog model. HemA is a genetic disorder characterized by a deficiency of the blood clotting FVIII. Patients are treated acutely or prophylactically by protein replacement therapy, which is very costly and inconvenient. Gene therapy is highly promising for treating HemA patients by delivering hFVIII transgene into targeted cells to persistently produce therapeutic levels of FVIII protein. Recent clinical trials for HemA gene therapy using recombinant adeno- associated viral (rAAV) vectors have shown very promising results. However, significant obstacles remain to prevent treatment to a significant portion of patients especially patients who have high-titer anti-AAV antibodies. Repeated treatment is also prohibited. UMGD has emerged as an effective gene transfer approach with great clinical relevancy and translational potential. In comparison to viral gene transfer, UMGD transfers plasmid vectors that are easier to prepare and more cost-effective; it also elicits less immune response and toxicity due to specific tissue targeting, prevents random integration, and allows for repeated delivery of the vectors. Other nonviral gene delivery method such as DNA-packaged nanoparticle encounters the challenge of crossing the nuclear envelope for DNA transcription. We have established a minimally invasive, transhepatic venous approach to efficiently deliver plasmid DNA (pDNA)/microbubble (MB) mixture into the target liver lobe combined with transcutaneous US applications in large animal models. We showed that high levels of luciferase reporter gene expression were achieved in swine and therapeutic levels of FVIII expression was detected in canine0 using the clinically feasible protocol. Only transient tissue damages were observed and repaired quickly and returned to normal within short time. However, in order to translate this novel technology to clinics, we recognize that several major problems need to be solved, (i) higher FVIII expression levels are needed to achieve a long- term therapeutic effect, (ii) persistence of therapeutic FVIII expression needs to be evaluated and maintained, (iii) consistently high efficiency of US treatment on targeted liver tissue is needed to achieve reproducible and efficient transfection. (iv) Better functional FVIII expression and reduced liver damage are desired. This may be achieved by targeting FVIII transfection in liver sinusoidal endothelial cells (LSECs), the native site of FVIII synthesis or by using newly synthesized nanobubbles (NBs). Thus, we propose to continue improving the transcutaneous UMGD instrument, transducers, US protocols, FVIII plasmid constructs, and MBs/NBs in mice and swine. Furthermore, we will deliver FVIII gene using the best transcutaneous UMGD protocol combined with optimal plasmid constructs and MBs/NBs to achieve persistent and therapeutic levels of FVIII expression in normal and HemA dogs. These progresses will promote the eventual translation of this novel technology into human application, bringing significant benefit for treating HemA patients, and potentially other genetic diseases.