cGMP Manufacture Of FVIII-Expressing Placental Cells For Hemophilia A

项目总结 目前对血友病A的治疗包括频繁输注FVIII以维持止血。更换 治疗是终生的，昂贵的，世界上75%的血友病患者无法获得治疗，而且30%的患者会患上 因子VIII的抑制物(FVIII)。因此，迫切需要开发负担得起的HA疗法，其具有更长的- 持久的利益或根治。AAV载体体内直接输注FVIII基因的临床试验 在成人中取得了有希望的结果，但先前存在的AAV中和抗体(NAB)存在于 当AAV系统递送时，高达50%的种群并抑制靶细胞转导。作为一名 结果，使用AAV的临床试验目前只能招募没有或低滴度预先存在的AAV NAB的受试者。 血友病A的产前治疗(PNTx)将绕过成人患者存在的免疫屏障， 并可诱导对FVIII的终身耐受，从而消除FVIII抑制剂形成的风险，最 在治疗/管理血友病A方面令人担忧的问题尽管直接将病毒载体注射到 产前接受者很可能会成功，在此之前需要解决许多安全问题 接近即可进入临床。这是一种基于细胞和基因治疗的方法，在这种方法中，细胞在体外进行转导， 将支持在生产中实施多种保障措施，例如拷贝数量和集成站点 分析，这是不可能的直接载体注射。这样的方法还将消除 脱靶基因修饰的可能性。使用PNTx的大型动物模型，我们展示了现成的 由慢病毒载体(LV)转导的人胎盘细胞(PLC)组成的治疗包括 生物工程、密码子优化的FVIII转基因，命名为mcoET3，导致血浆FVIII活性水平 这比对照组的非移植动物高出20%以上，而且这些水平一直保持在 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。会议一致认为，虽然考虑产品的安全性对于保障 对于患者，保护母亲也是当务之急，特别是在可能接触到基因的情况下- 修饰细胞和/或基因产品。为了直接解决FDA的这一指导方针，我们在此建议：目标1： 基因修饰的PLC血友病A治疗剂的研制、鉴定和体外鉴定 目的2：确认mcoET3的治疗潜力和安全性。 在体内、小鼠和PNTx的大型动物模型中表达人PLC，确定是否存在 在PNTx之后通过胎盘将产品转移给母亲，如果是这样的话，定义免疫 这种转移的后果。在这个项目完成时，我们希望已经展示了 无毒的PNTx方法，并已开发出一种安全有效的基于PLC的血友病A 可用于PNTx I期临床试验的治疗性药物。

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.