A Phase I Clinical Trial Testing Feasibility of Hematopoietic Stem Cell Gene Therapy Using Platelet Factor VIII to Safely Improve Hemostasis for Severe Hemophilia A with Inhibitory Antibodies

项目摘要/摘要 严重甲型血友病(PWHA)患者凝血因子明显缺乏(1%正常) (女八)，经常引起反复自发性出血发作，导致显着的发病率和 死亡率。供体血浆FVIII冷沉淀物输注治疗HA的常规治疗 可通过血液传播病毒疾病(包括艾滋病毒和丙型肝炎)而复杂化。使用 重组FVIII(RFVIII)产品已在很大程度上取代了人源性FVIII的使用(因为rFVIII 防止病毒传播的风险)；然而，血浆FVIII在整个过程中仍然是HA的宝贵资源 世界。FVIII的预防治疗需要每周静脉注射FVIII 2-3次 在病人的一生中。不幸的是，反复静脉注射，顺应性低，突破 尽管采取了FVIII预防措施，但仍可能发生出血和关节损伤。此外，≈30%的HA开发 对FVIII替代产品的同种抗体，抑制其恢复止血的能力。因此，治疗 在这些患者中，出血涉及到昂贵的“搭桥”药物治疗(即，FVIII与 免疫抑制和/或FVIIa)。通过诱导对FVIII的免疫耐受已经取得了成功 通过几次严格的FVIII输注(通常在预防的情况下)，≈60%的HA带有抑制性抗体 尽管FVIII抑制剂的治疗对HA患者来说仍然是一个关键问题。 由于其单基因的性质，HA是基因替代治疗的理想候选者，具有潜在的 更正HA。有希望的方法包括通过以下方式将人FVIII靶向表达到肝脏 静脉输注裸DNA构建新型腺相关病毒载体 免疫原性较低的外壳蛋白，以及含有FVIII小活性形式的载体(符合 4.4kb的AAV包装容量)。然而，这些策略排除了携带抑制性抗体的个体。 对FVIII(≈30%PWHA)、2)预先存在的甲型肝炎病毒抗体(≈40%人类)和3)慢性肝损伤。 为了解决这个具有相当临床意义的问题，我们提出了一项人类第一阶段临床试验。 采用利用慢病毒基因转移载体的造血干细胞(HSC)基因治疗策略 巨核细胞特异性ITGA2B基因启动子转录调控下的人FVIII编码 靶向FVIII基因在巨核细胞中的表达，导致异位合成、储存和 精确地在血管损伤部位从激活的血小板的α颗粒中调节释放因子VIII。 这一建议得到了临床前研究的支持，这些研究表明，血小板FVIII得到了安全有效的改善 在无FVIII和FVIII抑制性抗体产生的HA小鼠和犬模型中止血 即使在小鼠体内存在针对FVIII的预先存在的抑制性抗体。总而言之，拟议的审判 使用抗FVIII的抑制性抗体应可降低PWHA严重出血的风险 使用因子旁路制剂、耐受疗法和其他遗传疗法的策略是不够的。 1

PROJECT SUMMARY/ABSTRACT Patients with severe hemophilia A (PWHA) have a significant deficiency (<1% normal) in coagulation factor VIII (FVIII) that frequently causes recurrent spontaneous bleeding episodes leading to significant morbidity and mortality. Conventional therapy for HA employing the infusion of donor plasma FVIII cryoprecipitate product can be complicated by blood-borne transmission of viral illnesses (including HIV & hepatitis C). Use of recombinant FVIII (rFVIII) products has largely replaced the use of human-derived FVIII (because rFVIII prevents risk of viral transmission); however, plasma FVIII remains a valuable resource for HA throughout the world. Prophylactic therapy with FVIII requires the intravenous administration of FVIII 2-3 times weekly throughout a patient's lifetime. Unfortunately, recurrent intravenous access, low compliance, break-through bleeding, and joint-damage can occur despite FVIII prophylaxis. Additionally, ≈30% of HA develop allo-antibodies to FVIII replacement products that inhibit its ability to restore hemostasis. Thus, treatment of bleeding in these patients involves the administration of a costly “bypass” agent therapy (i.e., FVIII with immune suppression and/or FVIIa). Success has been achieved by inducing immune tolerization to FVIII in ≈60% of HA with inhibitory antibodies by several rigorous infusions of FVIII (often in the setting of prophylaxis with bypassing agents) although treatment for FVIII inhibitors remains a critical issue for HA patients. Due to its monogenic nature, HA is an ideal candidate for gene replacement therapy with the potential for correction of HA. Promising approaches include the targeted expression of human FVIII to the liver by intravenous infusion of naked DNA, the generation of a novel adeno-associated viral (AAV) vector equipped with less immunogenic coat proteins, and vectors incorporating small active forms of FVIII (that conform to the 4.4 kb packaging capacity of AAV). However, these strategies exclude individuals with 1) inhibitory antibodies to FVIII (≈30% PWHA), 2) pre-existing antibodies to the AAV (≈40% humans) and 3) chronic liver damage. To address this problem of considerable clinical significance, we propose a first-in-human phase I clinical trial employing a hematopoietic stem cell (HSC) gene therapy strategy that utilizes a lentiviral gene transfer vector encoding human FVIII under the transcriptional control of the megakaryocyte-specific ITGA2B gene promoter that targets expression of the FVIII gene in megakaryocytes causing ectopic synthesis, storage and regulated-release of factor VIII from α-granules of activated platelets precisely at the site of vascular injury. This proposal is supported by pre-clinical studies that showed platelet FVIII safely and efficiently improved hemostasis in murine and canine models of HA without the development of inhibitory antibodies to FVIII and even in the presence of pre-existing inhibitory antibodies to FVIII in mice. In summary, the proposed trial should reduce the risk of severe bleeding in PWHA with inhibitory antibodies to FVIII for whom current strategies employing factor bypassing agents, tolerizing therapy, and other genetic therapies are inadequate. 1