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

标题 确定用于治疗血友病A的基因编辑策略的新型安全港 项目摘要 血友病A是一种遗传性血液病，由功能性凝血因子因子（因子A）的缺失引起。 第八（f）段。预防性给予重组凝血因子VIII可减轻失血， 血液中的FVIII蛋白量。然而，目前的治疗是侵入性的， 昂贵，每年花费高达30万美元。此外，高达30%的重度血友病A患者 产生针对FVIII的灭活抗体，使得当前治疗无效。作为一种单基因疾病， 血友病A是具有相对大的治疗窗的基因治疗的有希望的候选者。然而， 开发血友病A基因治疗方案的主要障碍是实现足够的 以安全可控的方式从FVIII转基因表达。此外，基因治疗方案， 血友病A忽视了免疫敏感患者人群。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，该方法将安全地控制整合并靶向高- 在一个实施方案中，所述方法包括将表达fVIII转基因（HPFVIII）转移至基因组中的特定位置。通过利用修复 同源重组机制，CRISPR-Cas9系统将用于编辑基因组 造血干细胞和祖细胞。两个活性位点，1）RhD位点和2）von Willebrand位点 将评价因子（vWF）基因座，每个血友病A亚群各一个 中和抗体和含有中和抗体的那些）。由于RhD基因座被破坏， 一般人群的相当一部分，并发现表型无症状，该位置 对于外源基因的添加是最佳的。这些研究将证实， RhD基因座作为一个安全港，扩展了本研究的应用范围， 没有中和抗体的血友病A。另一方面，vWF基因座是一个有吸引力的基因座， 整合HPFVIII用于在免疫敏感患者中矫正血友病A。这是由于 vWF局限于血小板α颗粒。因此，HPFVIII，如果由相同的监管机构监管 像vWF这样的元素，可以安全地从血小板颗粒中的免疫系统中隔离出来， 生理上必要的。这些研究将证实，血小板中HPFVIII的隔离是 可用于治疗产生抑制剂的血友病A患者。以这种方式，两个新基因 将评价整个血友病A人群治疗的编辑方案。

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.