Correction of Hemophilia B using Factor IX-expressing T cells

 描述（由申请人提供）：我的论文项目的目标是开发一种新的治疗方法，将实现血友病和其他潜在遗传疾病的终身治疗。 A型血友病和B型血友病分别是由凝血因子VIII（FVIII）和IX（FIX）功能障碍或缺乏引起的X连锁疾病。在美国有超过20，000名男性患有血友病，全世界有数十万人。血友病目前通过蛋白质替代疗法治疗，但这是极其昂贵的（每个患者每年> 100，000美元），并且高达25%的严重血友病患者产生中和蛋白质替代的抗FVIII或-IX抗体。 开发一种安全有效的血友病基因治疗方法是血友病的长期目标。尽管在临床上取得了一些成功，但基因治疗尚未成为一种广泛的治疗方法。遗传性疾病的基因和细胞治疗的失败和问题主要是由于治疗的不稳定性、载体的免疫原性和载体介导的肿瘤发生的发展。为了克服这些严重的局限性，我建议利用T细胞作为血友病基因治疗的细胞载体。我假设慢病毒 载体可用于将FIX基因导入人T细胞，并且将T细胞转移回血友病患者将导致FIX的安全和持续表达以及疾病的纠正。 我的假设是基于嵌合抗原受体（CAR）T细胞疗法在治疗人类白血病方面取得的惊人临床成功。CAR T细胞是通过从患者中取出T细胞，并用编码识别白血病细胞的CAR的慢病毒载体转导它们来产生的。工程T细胞被转移回患者体内，在那里它们消灭肿瘤细胞。数十名患者接受了CAR T细胞治疗，没有报告与载体相关的不良事件。此外，已经发现LV工程化T细胞在患者体内持续多年。为了验证我的假设，我将：（1）产生慢病毒载体介导的FIX分泌的人T细胞（体外），（2）进行研究以优化FIX分泌的T细胞转移到动物中并介导持久产生循环免疫球蛋白的能力。 （3）评估CRISPR/Cas9系统将FIX转基因插入人T细胞基因组的安全港基因座中的用途。 这将是首次利用T细胞 作为血友病基因治疗的细胞载体。重要的是，由于慢病毒载体修饰的T细胞已用于人类，因此已经有大量的安全性数据。因此，如果该项目的结果确定了该方法在血友病动物模型中的有效性，则将该策略转移到人类血友病的治疗中是可行的。除了与人类健康相关外，F31的应用还将支持我作为科学家的培训。通过这些研究，我将获得人类T细胞治疗的新兴领域，以及基因工程，CRISPR/Cas9技术和血友病的专业知识。我还将获得血液学和免疫学方面的一般专业知识。这次培训对我成为一名独立调查员的目标非常宝贵。

 DESCRIPTION (provided by applicant): The goal of my thesis project is to develop a new therapy that will achieve life-long treatment of hemophilia and potentially other genetic diseases. Hemophilia A and B are X-linked diseases caused by dysfunction or absence of blood coagulation factors VIII (FVIII) and IX (FIX), respectively. More than 20,000 men in the US have hemophilia, and hundreds of thousands more worldwide. Hemophilia is currently treated by protein replacement therapy, but this is extremely costly (>$100,000 per patient per year) and up to 25% of severe hemophiliacs develop anti-FVIII or -IX antibodies that neutralize the protein replacement. The development of a safe and effective gene therapy for hemophilia is a long-standing goal for the disease. Despite some clinic success, gene therapy has not become a widespread treatment. The failures and problems of gene and cell therapy for genetic disease have been largely due to instability of the treatment, immunogenicity of the vector, and the development of vector-mediated oncogenesis. To overcome these serious limitations, I propose to exploit T cells as a cellular vehicle for hemophilia gene therapy. I hypothesize that lentiviral vectors can be used to introduce a FIX gene into human T cells, and that transfer of the T cells back into a hemophiliac will result in safe and sustained expression of FIX and correction of the disease. My hypothesis is based on the stunning clinical success of chimeric antigen receptor (CAR) T cell therapy in treating human leukemia. CAR T cells are generated by removing T cells from a patient, and transducing them with a lentiviral vector encoding a CAR that recognizes leukemic cells. The engineered T cells are transferred back to patients where they wipe out tumor cells. Dozens of patients have been treated with CAR T cells, and no vector-related adverse events have been reported. Moreover, LV-engineered T cells have been found to last for years in patients. To test my hypothesis, I will: (1) Generate lentiviral vector-mediatd FIX- secreting human T cells (in vitro), (2) Carry out studies to optimize the ability of the FIX-secreting T cells to be transferred into animals and mediate long-lasting production of circulating FIX, and (3) Evaluate the use of the CRISPR/Cas9 system to insert a FIX transgene into a safe harbor locus of the genome of human T cells. This will be the first time that T cells are utilized as a cellular vehicle for hemophilia gene therapy. Importantly, because lentiviral vector-modified T cells have been used in humans, there is already substantial safety data. Thus, if the outcome of this project establishes the effectiveness of the approach in animal models of hemophilia, it will be feasible to move this strategy to the treatment of human hemophilia. In addition to its relevance for human health, this F31 application will also support my training as a scientist. Through these studies I will gain expertise in the burgeoning area of human T cell therapy, as well as in genetic engineering, CRISPR/Cas9 technology, and hemophilia. I will also gain general expertise in hematology and immunology. This training will be invaluable towards my goal of becoming an independent investigator.