Nonviral Gene Medicine for Hemophilia A

本研究的目的是开发一种安全，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 需要反复输注蛋白质浓缩物，这既昂贵， 不方便此外，约30%的血友病A患者出现抑制性 蛋白质替代疗法后抗因子VIII（FVIII）抗体。我们有 先前证明，肝脏特异性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对小鼠进行治疗。然而，强大的FVIII特异性 免疫活性的HemA小鼠发生免疫应答， 基因转移后两周的FVIII。这种小鼠模型使我们能够开发一种 有效免疫抑制方案与非病毒基因治疗组合， 达到长期治疗效果。9例单药或联合免疫抑制剂 方案已经过测试，阻断共刺激途径的最佳策略是 使用Ctla 4-IG和抗鼠CD 40配体mAb（MR 1）的组合诱导长- 小鼠对因子VIII的长期耐受性。此外，由于流体动力学基因递送是 目前的形式不适合用于人类，我们也追求并获得了 在超声（US）介导的基因传递的发展中取得了初步成功， 体内和体外细胞渗透肽（CPP）介导的基因转移。以促进 直接翻译这些方法，以人类的应用，我们建议进一步 优化US介导的质粒DNA或CPP/DNA基因递送的效率 复合物，并评估免疫调节治疗的最有效和最少 A型血友病小鼠模型中的毒性免疫抑制方案。优化 还将在正常狗的初步放大实验中测试递送方法， 制备用于在血友病A犬模型中进一步评价。 我们将检验以下假设：1）安全和临床可行的非病毒基因递送 方法，主要是US介导的基因治疗，可以建立， 质粒DNA转移到小鼠肝脏中; 2）最小毒性免疫抑制 可以开发单独或联合使用免疫抑制剂的方案 预防和/或调节基因治疗后的转基因特异性免疫应答; 3)这些综合方法将导致长期的疾病矫正， 血友病A鼠模型;和4）超声介导的基因递送方法可以 在正常的狗实验中被放大。

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.