Eradication of FVIII Inhibitors using Gene-Based Therapy

项目总结(见说明)： 针对人类因子V11(h-FVIII)的抑制性抗体的发展仍然是最多的 与血友病A治疗相关的显著临床并发症，是基因的关键障碍 治疗即将到来。与大多数单基因疾病治疗的情况并无不同 蛋白质替代疗法，20-30%的血友病A患者对输液产生免疫反应 使治疗无效的FVIII产品。因此，新方法的发现和发展将有助于 需要诱导免疫耐受或低反应性。造血干细胞移植 (HSCT)方案在治疗各种人类自身免疫疾病的临床试验中显示出希望 精神错乱。此外，至少在动物模型中，造血干细胞移植和基因治疗相结合已经被证明可以 促进新抗原或同种异体抗原的引入和耐受性，例如那些缺乏 像血友病A这样的单基因疾病我们的实验室最近报告了基因修饰的移植 含有高表达猪(P)-FVIII基因的HSCs进入血友病A小鼠导致 产生高水平的循环fV11活性。受体小鼠中没有一只产生抗fV11抗体 在HSCT基因治疗后，即使在非清髓性和非基于辐射的情况下 条件反射。随后，我们研究了携带ANFI-hfV11的血友病A小鼠的HSCT基因治疗。 抑制性抗体效价。即使大多数小鼠体内含有交叉反应的循环抗体 表达p-fV11基因修饰的HSCs在体外具有并抑制p-FVIII活性的移植入 去髓血友病A小鼠恢复了循环中的FVIII活性。治愈的p-fV11活性水平持续存在 在整个研究过程中，所有小鼠的抗h-FVIII抗体效价稳步下降。相比之下， 使用非清髓性全身照射(TBI)在促进 转基因细胞的植入导致了抗FVIII抗体效价的延迟下降。 因此，预先存在的免疫对HSCT基因治疗构成了额外的障碍。这些数据表明 虽然基于造血干细胞移植的基因疗法结合p-FVIII可以成功地用于治疗 使用难治性抗hfVIII抑制剂的患者，1)有必要进行更积极的移植调节 实现疾病矫正，2)使用同源(或其他不相同的抗原)转基因产品 关键，以及3)可以实现持久的容忍/低反应。这项提议的重点是 开发一种HSCT基因治疗策略，用于治疗合并有抑制剂的血友病A。

PROJECT SUMMARY (See instructions): The development of inhibitory antibodies directed against human factor Vlll (h-fVIII) remains the most signiflcant clinical complication associated with the treatment of hemophilia A and is a critical barrier to gene therapy approaches. Not dissimilar to what occurs in the majority of monogenic diseases treated using protein replacement therapy, 20 - 30% of hemophilia A patients develop an immune response to the infused fVIII product that renders treatment ineffective. Therefore, the discovery and development of new methods to induce immune tolerance or hypo-responsiveness are needed. Hematopoietic stem cell transplantation (HSCT) protocols are showing promise in clinical trials for the treatment of a variety of human autoimmune disorders. Furthermore, HSCT combined with gene therapy has been shown, at least in animal models, to facilitate the introduction of and tolerance to neo or allo-antigens, such as those proteins deficient in monogenic diseases like hemophilia A. Our laboratories recently reported that transplantation of geneticallymodified HSCs containing a high-expression porcine (p)-fVIII transgene into hemophilia A mice results in the production of high levels of circulating fVlll activity. None of the recipient mice developed anti-fVlll antibodies following HSCT gene therapy, even in the context of non-myeloablative and non-radiation-based conditioning. Subsequently, we studied HSCT gene therapy in hemophilia A mice harboring anfi-hfVlll inhibitory antibody titers. Even though the majority of mice contained circulating antibodies that cross-reacted with and inhibited p-fVIII activity in vitro, transplantation of genetically-modified HSCs expressing p-fVlll into myeloablated hemophilia A mice restored circulating fVIII activity. Curative p-fVlll activity levels persisted and anti-h-fVIII antibody titers steadily declined throughout the course of the study in all mice. In contrast, the use of non-myeloablative total body irradiation (TBI) was only partially successful in promoting the engraftment of genetically-modified cells and resulted in a delayed reduction of anti-fVIII antibody titers. Therefore, pre-existing immunity presents an additional barrier to HSCT gene therapy. These data suggest that, while HSCT-based gene therapy incorporating a p-fVIII can be utilized successfully for the treatment of patients with refractory anti-hfVIII inhibitors, 1) more aggressive transplant conditioning is necessary to achieve disease correction, 2) the use of an orthologous (or other non-idenfical antigen) transgene product is critical, and 3) enduring tolerance/hyporesponsiveness can be achieved. The focus of this proposal is to develop an HSCT gene therapy strategy for the treatment of hemophilia A when complicated by inhibitors.