Nonviral Gene Medicine for Hemophilia A

这项研究的目标是开发一种安全、有效、临床可行的非病毒 血友病A的基因治疗策略目前血友病A患者 通过反复输注蛋白质浓缩物进行治疗，这既昂贵又 不方便。此外，约30%的血友病A患者出现抑制 蛋白质替代治疗后的抗因子VIII(FVIII)抗体。我们有 先前证明了肝脏特异的FVIII质粒的非病毒基因转移 在免疫缺陷患者中产生持续的超生理/治疗水平的FVIII 使用基于流体力学的递送方法的小鼠。然而，强健的FVIII特定于 免疫活性HEMA小鼠发生免疫应答并消除功能 FVIII基因转移后两周。这个小鼠模型允许我们开发出一种 有效的免疫抑制方案与非病毒基因治疗相结合 达到长期的治疗效果。9种单一或联合免疫抑制药 实验证明，阻断共刺激途径的最佳策略是 用CTLA4-Ig和抗鼠CD40配体单抗(MR1)联合诱导的长时间淋巴细胞亚群 小鼠对因子VIII的长期耐受性。此外，由于流体动力基因传递是 在目前的形式下不适合在人类身上使用，我们也追求并获得了 超声(US)介导的基因传递技术的初步成功 体内和细胞通透性多肽(CPP)介导的体外基因转移。为了方便 将这些方法直接翻译成人工应用程序，我们建议进一步 优化超声介导的质粒DNA或CPP/DNA的基因传递效率 复合体，评估最有效和最少的免疫调节疗法 血友病A小鼠模型中的毒性免疫抑制方案。经过优化的 给药方法也将在#年在正常狗身上进行的初步放大实验中进行测试 为血友病A犬模型的进一步评价做准备。 我们将检验以下假设：1)安全且临床可行的非病毒基因传递 可以建立方法，主要是美国介导的基因治疗，以允许有效的 小鼠肝脏内的质粒DNA转移；2)低毒免疫抑制 单独使用或联合使用免疫抑制剂的方案可以开发成 预防和/或调节基因治疗后的转基因特异性免疫反应； 3)这些结合的方法将导致疾病的长期纠正 血友病A小鼠模型；4)超声介导的基因传递方法可以 在普通的狗实验中被放大。

The goal of this study is to develop a safe, efficient, and clinically feasible nonviral gene therapy strategy for the treatment of hemophilia A. Currently hemophilia A patients are treated with repeated infusions of protein concentrates, which is both costly and inconvenient. Furthermore, ~30% of hemophilia A patients developed inhibitory antibodies against factor VIII (FVIII) following protein replacement therapy. We have previously demonstrated that nonviral gene transfer of a liver-specific FVIII plasmid produced persistent, supra-physiological/therapeutic levels of FVIII in immunodeficient mice using a hydrodynamics-based delivery method. However, robust FVIII-specific immune responses occurred in immunocompetent HemA mice and eliminated functional FVIII two weeks following gene transfer. This murine model permitted us to develop an effective immunosuppressive regimen in combination with nonviral gene therapy to achieve a long-term therapeutic effect. Nine single or combined immunosuppressive regimens have been tested, and the best strategy of blocking the co-stimulation pathway using a combination of Ctla4-Ig and anti-murine CD40 ligand mAb (MR1) induced long- term tolerance to factor VIII in mice. Furthermore, since hydrodynamic gene delivery is unsuitable for use in humans in its current form, we have also pursued and gained preliminary success in the development of ultrasound (US)-mediated gene delivery in vivo and cell permeable peptide (CPP)-mediated gene transfer in vitro. To facilitate direct translation of these methods to human applications, we propose to further optimize the efficiency of the US-mediated gene delivery of plasmid DNA or CPP/DNA complexes, and evaluate immunomodulation therapy for the most effective and least toxic immunosuppressive regimen in the hemophilia A mouse model. The optimized delivery method will also be tested in preliminary scale-up experiments in normal dogs in preparation for further evaluation in the hemophilia A dog model. We will test the hypotheses that: 1) Safe and clinically feasible nonviral gene delivery methods, mainly US-mediated gene therapy, can be established to allow efficient plasmid DNA transfer into the mouse liver; 2) Minimally toxic immunosuppressive regimens using immunosuppressive agents alone or in combination can be developed to prevent and/or modulate transgene-specific immune responses following gene therapy; 3) These combined approaches will lead to long-term correction of disease in a hemophilia A murine model; and 4) The ultrasound-mediated gene delivery method can be scaled up in normal dog experiments.