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

项目总结/摘要 重度血友病A（PWHA）患者凝血因子明显缺乏（<1%正常） VIII（FVIII），经常引起复发性自发性出血发作，导致显著的发病率， mortality.采用供体血浆FVIII冷沉淀产物输注的HA常规治疗 血液传播的病毒性疾病（包括艾滋病毒和丙型肝炎）可能会使病情复杂化。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在整个治疗过程中仍然是HA的宝贵资源。 世界FVIII预防性治疗需要每周2-3次静脉注射FVIII 在病人的一生中。不幸的是，反复出现的静脉通路，低依从性，突破性 尽管进行了FVIII预防，但仍可能发生出血和关节损伤。此外，大约30%的HA会形成 抑制其恢复止血能力的FVIII替代产品的同种抗体。因此，治疗 这些患者的出血涉及昂贵的“旁路”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， 通过几次严格的FVIII输注（通常在预防性治疗中），使60%的HA与抑制性抗体结合 使用旁路剂），尽管FVIII抑制剂的治疗仍然是HA患者的关键问题。 由于其单基因性质，HA是基因替代疗法的理想候选者，具有潜在的治疗作用。 纠正HA。有前景的方法包括通过免疫组织化学将人FVIII靶向表达至肝脏。 静脉输注裸DNA，产生一种新的腺相关病毒（AAV）载体， 免疫原性较低的外壳蛋白，以及掺入小活性形式的FVIII的载体（其符合 4.4 kb的AAV包装容量）。然而，这些策略排除了具有1）抑制性抗体的个体 对FVIII（约30%PWHA），2）预先存在的对AAV的抗体（约40%人）和3）慢性肝损伤。 为了解决这个具有相当临床意义的问题，我们提出了一个首次在人体I期临床试验 采用造血干细胞（HSC）基因治疗策略，该策略利用慢病毒基因转移载体 在巨核细胞特异性ITGA 2B基因启动子的转录控制下编码人FVIII 靶向巨核细胞中FVIII基因的表达，引起异位合成、储存和 精确地在血管损伤部位从活化血小板的α颗粒调节释放因子VIII。 该提议得到了临床前研究的支持，这些研究表明，血小板FVIII安全有效地改善了 在HA的鼠和犬模型中止血，不产生FVIII抑制性抗体， 即使在小鼠中存在预先存在的FVIII抑制性抗体的情况下。总之，拟议的审判 对于目前患有FVIII抑制性抗体的PWHA患者， 采用因子旁路剂、耐受性治疗和其它遗传治疗的策略是不够的。 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