Identification of novel safe harbors to be used in a gene editing strategy for the treatment of hemophilia A

标题 用于血友病A基因编辑策略的新的安全港的鉴定 项目摘要 血友病A是一种遗传性血液疾病，由功能性凝血因子、凝血因子 VIII(FVIII)。预防性应用重组FVIII可减少失血量，且出血量相对较小 血液中循环中的FVIII蛋白的数量。然而，目前的治疗是侵入性的， 价格昂贵，每年花费超过30万美元。此外，高达30%的严重血友病A患者 产生针对FVIII的灭活抗体，使目前的治疗无效。作为一种单基因疾病， 血友病A是一种有希望的基因治疗候选者，具有相对较大的治疗窗口。然而，一个 开发血友病A的基因治疗方案的主要障碍是取得了足够的 以一种安全可控的方式表达FVIII转基因。此外，基因治疗方案可用于 血友病A忽略了免疫敏感的患者群体。为了克服这些缺点， 我们打算利用一种非病毒基因组编辑方法，这种方法将安全地控制整合并针对高... 表达FVIII转基因(HPFVIII)到基因组中的特定位置。通过利用修复 同源重组的机制，将利用CRISPR-Cas9系统编辑基因组 造血干细胞和造血祖细胞。两个活跃的基因，RhD基因和von Willebrand基因 因子(VWF)基因座将被评估，每个血友病A亚群一个(那些没有 中和抗体和包含中和抗体的抗体)。由于RhD基因座在 一般人群的很大一部分被发现是无症状的，这个地方 在基因组中添加外源基因是最理想的。这些研究将证实利用 RhD基因座作为一个安全港扩展了这项研究的应用范围，超越了对患有 无中和抗体的血友病A。另一方面，vWF轨迹是一个吸引人的轨迹 整合HPFVIII纠正免疫敏感患者的血友病A。这是由于 VWF对血小板α颗粒的限制作用。因此，如果HPFVIII受到相同的监管机构的监管 像vWF这样的成分将安全地从免疫系统中隔离在血小板颗粒中，直到 从生理上来说是必要的。这些研究将证实HPFVIII在血小板中的隔离是 可用于治疗产生抑制物的血友病A患者。通过这种方式，两个新基因 将对治疗整个血友病A人群的编辑方案进行评估。

TITLE Identification of novel safe harbors to be used in a gene editing strategy for the treatment of hemophilia A Project Summary Hemophilia A is a hereditary blood disorder caused by the loss of the functional coagulation factor, factor VIII (fVIII). Prophylactic administration of recombinant fVIII can alleviate blood loss with relatively small amounts of circulating fVIII protein in the bloodstream. However, the current treatment is invasive and expensive, costing upwards of $300,000 per year. In addition, up to 30% of severe hemophilia A patients develop inactivating antibodies to fVIII rendering the current therapy ineffective. As a monogenic disorder, hemophilia A is a promising candidate for gene therapy with a relatively large therapeutic window. Yet, a major barrier to developing gene therapy protocols for hemophilia A has been achieving sufficient expression from fVIII transgenes in a safe controllable manner. In addition, gene therapy protocols for hemophilia A overlook the immunosensitive patient population. In order to combat these shortcomings, we intend to utilize a non-viral genome editing method that will safely control integration and target a high- expression fVIII transgene (HPFVIII) to a specific location in the genome. By exploiting the repair mechanism of homologous recombination, the CRISPR-Cas9 system will be utilized to edit the genome of hematopoietic stem and progenitor cells. Two active loci, the 1) RhD locus and the 2) von Willebrand Factor (vWF) locus, will be evaluated, one for each hemophilia A subpopulation (those without neutralizing antibodies and those containing neutralizing antibodies). Since the RhD locus is disrupted in a substantial portion of the general population and found to be phenotypically asymptomatic, this location in the genome is optimal for addition of an exogenous gene. These studies will confirm the utilization of the RhD locus as a safe harbor extending the application of this study beyond treating individuals with hemophilia A without neutralizing antibodies. The vWF locus, on the other hand, is an attractive locus for the integration of HPFVIII for the correction of hemophilia A in immunosensitive patients. This is due to the confinement of vWF to the α granules of platelets. Thus HPFVIII, if regulated by the same regulatory elements as vWF, would be safely sequestered from the immune system in the granules of platelets until physiologically necessary. These studies will confirm that the sequestration of HPFVIII in platelets is feasible for the treatment of hemophilia A patients that produce inhibitors. In this manner, two novel gene editing protocols for the treatment of the entire hemophilia A population will be evaluated.