Eradication of FVIII Inhibitors using Gene-Based Therapy

项目总结（见说明）： 针对人因子VIII（h-fVIII）的抑制性抗体的开发仍然是最重要的。 与血友病A治疗相关的显著临床并发症，是基因治疗的关键障碍。 治疗方法。与大多数单基因疾病治疗中发生的情况相似， 在蛋白质替代疗法中，20 - 30%的血友病A患者对输注的蛋白质产生免疫应答。 使治疗无效的FVIII产品。因此，新方法的发现和发展， 需要诱导免疫耐受性或低反应性。造血干细胞移植 造血干细胞移植（HSCT）方案在临床试验中显示出治疗各种人类自身免疫性疾病的前景。 紊乱此外，至少在动物模型中，HSCT联合基因治疗已显示出， 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说， 将含有高表达猪（p）-fVIII转基因的HSC导入血友病A小鼠， 产生高水平的循环fVIII活性。没有受体小鼠产生抗-fVIII抗体 在HSCT基因治疗后，即使在非清髓性和非放射性的背景下， 条件反射随后，我们研究了携带anfi-hfVIII的血友病A小鼠的HSCT基因治疗， 抑制性抗体滴度。尽管大多数小鼠体内含有交叉反应的循环抗体 在体外具有并抑制p-fVIII活性的情况下，将表达p-fVIII III的遗传修饰的HSC移植到 骨髓清除的血友病A小鼠恢复了循环FVIII活性。治疗性p-fVII I活性水平持续存在 并且在所有小鼠中，抗h-FVIII抗体滴度在整个研究过程中稳定下降。而反观 使用非清髓性全身照射（TBI）仅部分成功地促进了 移植的基因修饰的细胞，并导致抗-fVIII抗体滴度的延迟降低。 因此，预先存在的免疫性对HSCT基因治疗提出了额外的障碍。这些数据表明 尽管掺入p-fVIII的基于HSCT的基因疗法可以成功地用于治疗 难治性抗hfVIII抑制剂患者，1）更积极的移植预处理是必要的， 实现疾病矫正，2）使用正向抗原（或其它非特异性抗原）转基因产物， 关键的，和3）可以实现持久的耐受性/低反应性。本提案的重点是 开发HSCT基因治疗策略，用于治疗伴有抑制剂的血友病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.