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

项目摘要 这项提议的目标是通过一种非侵入性的方法实现血友病A(HEMA)的长期治疗纠正 超声(US)介导的FVIII(FVIII)基因表达载体(UMGD)在犬模型中的实验方案。 HEMA是一种遗传性疾病，其特征是凝血FVIII缺乏。病人得到了敏锐的治疗 或者通过蛋白质替代疗法来预防，这是非常昂贵和不方便的。基因治疗是非常重要的 将hFVIII转基因导入靶细胞持续生产有望治疗HEMA患者 治疗水平的FVIII蛋白。重组腺病毒载体用于HEMA基因治疗的临床研究进展 相关病毒载体(RAAV)已经显示出非常有希望的结果。然而，仍然存在重大障碍， 防止对相当一部分患者，特别是具有高效价抗AAV抗体的患者进行治疗。 重复治疗也是禁止的。UMGD已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转移的是质粒 更容易制备和更具成本效益的载体；它还会引起较少的免疫反应和毒性 靶向特定组织，防止随机整合，并允许重复递送载体。其他 非病毒基因传递方法，如DNA包装的纳米颗粒，遇到了跨越 用于DNA转录的核膜。我们已经建立了一种微创的经肝静脉 一种高效靶向靶肝DNA(PDNA)/微泡(MB)混合物的方法 经皮美国在大型动物模型中的应用。我们发现了高水平的荧光素酶报告 在猪中实现了基因表达，在狗0中检测到了FVIII的治疗水平 使用临床上可行的方案。只有短暂的组织损伤被观察到并迅速修复 在短时间内恢复正常。然而，为了将这项新技术转化为临床，我们认识到 有几个主要问题需要解决，(I)需要更高的FVIII表达水平才能实现长期的- 长期疗效，(Ii)治疗性FVIII表达的持续性需要评估和维持， (3)需要对目标肝组织进行持续高效的超声治疗，以实现可重复性和可重复性 高效的转染法。(4)更好的功能性FVIII表达和减少肝损伤是理想的。这可能是 通过靶向FVIII在肝窦内皮细胞(LSECs)中的表达而实现的 合成或使用新合成的纳米气泡(NBS)。因此，我们建议继续改善 经皮UMGD器械，转导，US方案，FVIII质粒构建，和小鼠的MBS/NBS 还有猪。此外，我们将使用最好的经皮UMGD方案结合 获得持续和治疗水平的FVIII表达的最佳质粒构建和MBS/NBS 普通犬和赫马犬。这些进展将推动这项新技术最终转化为 人类应用，为治疗HEMA患者以及潜在的其他遗传病带来重大好处。

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.