cGMP Manufacture Of FVIII-Expressing Placental Cells For Hemophilia A

项目摘要 目前血友病A的治疗包括频繁输注FVIII以维持止血。更换 治疗是终身的，昂贵的，对世界上约75%的血友病患者不可用，并且>30%的患者发展为 因子VIII（FVIII）的抑制剂。因此，迫切需要开发负担得起的HA疗法， 持久的益处或永久的治愈。直接输注AAV载体用于体内FVIII基因递送的临床试验 已经在成人中产生了有希望的结果，但在成人中存在预先存在的AAV中和抗体（NAb）。 当AAV被全身递送时，高达50%的群体和抑制靶细胞转导。作为 因此，使用腺相关病毒的临床试验目前只能招募不含或低滴度既存腺相关病毒NAb的受试者。 血友病A的产前治疗（PNTx）将绕过成人患者中存在的免疫屏障， 并且可以诱导对FVIII的终身耐受，从而消除FVIII抑制剂形成的风险， 血友病A治疗/管理中的恐惧问题。虽然直接注射病毒载体到 产前接受者可能会成功，在此之前需要解决许多安全问题 方法可以进入诊所。一种基于细胞和基因治疗的方法，其中细胞在体外转导， 能够在生产中实施多重保障措施，例如，拷贝数和整合位点 分析，这是不可能与直接载体注射。这种做法还将消除 脱靶基因修饰的可能性。使用PNTx的大型动物模型，我们表明， 治疗包括用慢病毒载体（LV）转导的人胎盘细胞（PLC），所述慢病毒载体编码 一种生物工程化的、密码子优化的FVIII转基因，命名为mcoET 3，导致血浆FVIII活性水平 超过对照非移植动物的20%以上，并且这些水平维持了 出生后至少1年。根据这些结果，与FDA举行了IND前会议，讨论 通往临床试验的道路。与会者一致认为，虽然考虑产品的安全性至关重要， 病人，也必须保护母亲，特别是关于可能暴露于基因- 修饰的细胞和/或基因产物。为了直接解决本FDA指南，我们在此提出：目标1： 在体外生产、表征和验证基因修饰的基于PLC的血友病A治疗药物 目的2：确认mcoET 3 - 3的治疗潜力和安全性。 在PNTx的体内、小鼠和大型动物模型中表达人PLC，确定是否存在 PNTx后产品通过胎盘转移到母亲，如果是，定义免疫 这种转移的后果。在这个项目完成后，我们希望已经证明了 PNTx方法的无毒性，并开发出安全有效的基于PLC的血友病A 可以用于PNTx I期临床试验的治疗剂。

PROJECT SUMMARY Current treatments for hemophilia A consist of frequent FVIII infusions to maintain hemostasis. Replacement therapy is lifelong, expensive, unavailable for ~75% of the world's hemophiliacs, and >30% of patients develop inhibitors to Factor VIII (FVIII). There is thus a critical need to develop affordable HA therapies with longer- lasting benefit or permanent cure. Clinical trials directly infusing AAV vectors for in vivo FVIII gene delivery have yielded promising results in adults, but pre-existing neutralizing antibodies (NAbs) to AAV are present in up to 50% of the population and inhibit target cell transduction when AAV is delivered systemically. As a result, clinical trials using AAV can currently only enroll subjects with no- or low-titer pre-existing AAV NAbs. Prenatal treatment (PNTx) for hemophilia A would circumvent the immune barriers present in adult patients, and could induce lifelong tolerance to FVIII, and thereby eliminate the risk of FVIII inhibitor formation, the most feared problem in treatment/management of hemophilia A. Although direct injection of viral vectors into prenatal recipients would likely be successful, numerous safety concerns need to be addressed before this approach can enter the clinic. A cell and gene therapy-based approach, in which cells are transduced in vitro, would enable multiple safeguards to be implemented in production, e.g., copy number and integration site analyses, that are not possible with direct vector injection. Such an approach would also eliminate the possibility of off-target gene-modification. Using a large animal model of PNTx, we showed that an off-the-shelf therapy comprised of human placental cells (PLCs) transduced with a lentiviral vector (LV) encoding a bioengineered, codon-optimized FVIII transgene, designated mcoET3, resulted in plasma FVIII activity levels that exceeded that of control non-transplanted animals by over 20%, and that these levels were maintained for at least 1 year after birth. Based on these results, a Pre-IND meeting with the FDA was held to discuss the path to a clinical trial. It was agreed that while it was vital to consider safety of the product to safeguard the patient, it was also imperative to protect the mother, particularly with respect to possible exposure to the gene- modified cells and/or gene product. To directly address this FDA guidance, we herein propose to: Aim 1: Manufacture, characterize, and authenticate, in vitro, a gene-modified PLC-based hemophilia A therapeutic product under cGMP conditions; and Aim 2: Confirm the therapeutic potential and safety of mcoET3- expressing human PLCs in vivo, in mice and in a large animal model of PNTx, determine whether there is transfer of the product across the placenta to the mother following PNTx, and if so, define the immune consequences of such transfer. At the completion of this project, we expect to have demonstrated the innocuity of the PNTx approach, and to have developed a safe and effective PLC-based hemophilia A therapeutic that could be used in a PNTx Phase-I clinical trial.