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

确定用于治疗 A 型血友病的基因编辑策略的新型安全港

• 批准号: 10228561
• 负责人: Jennifer Marie Johnston
• 金额: $ 14.65万
• 依托单位: SAN JOSE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-04 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228561
• 关键词: Adverse effects; Alpha Granule; Antibodies; Antigens; Blood; Blood Circulation; Blood Coagulation Factor; Blood Platelets; CRISPR/Cas technology; Candidate Disease Gene; Cells; Clinical; Clinical Research; DNA; Development; Elements; F8 gene; Factor VIII; General Population; Genes; Genome; Genomics; Hematological Disease; Hematopoietic System; Hematopoietic stem cells; Hemophilia A; Hemorrhage; Immune system; Individual; Inherited; Knock-out; Lentivirus; Lentivirus Vector; Liver; Location; Mendelian disorder; Metabolic Diseases; Methods; Modification; Output; Patients; Phenotype; Physiological; Population; Procedures; Proteins; Protocols documentation; Recombinants; Regulatory Element; Risk; Site; Source; System; Therapeutic; Transgenes; Transplantation; Treatment Protocols; Viral; cell type; cellular targeting; combat; cost; enzyme replacement therapy; functional loss; gene therapy; genome editing; genomic locus; homologous recombination; ineffective therapies; inhibitor/antagonist; lentiviral integration; nervous system disorder; neutralizing antibody; novel; patient population; pre-clinical; promoter; prophylactic; recombinant antihemophilic factor VIII; reduce symptoms; repaired; self-renewal; therapeutic transgene; therapeutically effective; tool; treatment strategy; von Willebrand Factor

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.

标题 确定用于治疗 A 型血友病的基因编辑策略的新型安全港 项目概要 A 型血友病是一种遗传性血液疾病，由功能性凝血因子、凝血因子的丧失引起 VIII (fVIII)。预防性施用重组 fVIII 可以以相对较小的剂量减少失血 血液中循环的 fVIII 蛋白量。然而，目前的治疗方法是侵入性的， 价格昂贵，每年花费高达30万美元。此外，高达30%的严重A型血友病患者 开发针对 fVIII 的灭活抗体，使当前疗法无效。作为一种单基因疾病， A 型血友病是基因治疗的有希望的候选者，具有相对较大的治疗窗口。然而，一个 开发 A 型血友病基因治疗方案的主要障碍是取得了足够的进展 以安全可控的方式从 fVIII 转基因中表达。此外，基因治疗方案 A 型血友病忽视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，该方法将安全地控制整合并针对高 将 fVIII 转基因 (HPFVIII) 表达到基因组中的特定位置。通过利用修复 同源重组机制，将利用CRISPR-Cas9系统来编辑基因组 造血干细胞和祖细胞。两个活跃基因座，1) RhD 基因座和 2) von Willebrand 将评估因子 (vWF) 位点，每个血友病 A 亚群（没有 中和抗体和含有中和抗体的抗体）。由于 RhD 基因座被破坏 一般人群中很大一部分被发现没有表型症状，这个位置 基因组中最适合添加外源基因。这些研究将证实利用 RhD 位点作为安全港，将本研究的应用扩展到治疗患有以下疾病的个体之外 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 HPFVIII 的整合用于纠正免疫敏感患者的 A 型血友病。这是由于 vWF 将其限制在血小板的 α 颗粒中。因此，HPFVIII，如果受到相同监管机构的监管 vWF 等元素将被安全地隔离在血小板颗粒中，与免疫系统隔离，直到 生理上必要的。这些研究将证实 HPFVIII 在血小板中的隔离是 可行用于治疗产生抑制剂的A型血友病患者。以这种方式，两个新基因 将评估用于治疗整个 A 型血友病人群的编辑方案。