Curing Sickle Cell Disease with CRISPR-Cas9 genome editing

镰状细胞病(SCD)是一种毁灭性的隐性遗传疾病，困扰着大约10万美国人。 大约6000加州人，以及全世界数十万人(4)。它通常会缩短寿命 几十年，即使有最好的医疗服务。SCD是由？珠蛋白基因的单一突变引起的。 编辑可以直接纠正突变。我们已经开发出一种自体干细胞产品，它使用一种 CRISPR/Cas9核酸酶刺激血液干细胞镰状突变的修复。可重复编辑 产生的矫正水平，根据临床经验，将足以达到疗效。我们 建议完成这种疗法的临床前开发，导致早期IND应用 成人重度SCD的分期临床试验。我们于2018年10月30日举行了IND前会议，并 请求CIRM为以下活动提供资金，以支持与FDA讨论的IND提交： 1.在临床规模上展示在GMP条件下制造最终细胞产品的能力。 我们将在cGMP下产生至少3个临床规模的批次，满足所有释放标准，在plerixafor动员中 HSPC。我们还将完成一项药品稳定性研究。这些报告将完成 工商局CMC部分。 2.完成严格的遗传毒性评估。一种支持人类的小鼠异种移植模型 造血细胞将用于对在以下条件下制造的最终细胞产品进行毒理学研究 CGMP条件。在长期植入后，老鼠将被评估为人类恶性肿瘤的证据。 应用组织病理学、流式细胞术和基因组读数。(预考问题8)。 3.完成额外的临床前研究，以确定产品的安全性和有效性。详尽无遗的 询问最终制造试剂的脱靶基因组修饰将解决 潜在的遗传毒性。我们还将评估HBB目标位点上的大量缺失和易位， 编辑程序引起的HBb突变的性质和潜在影响，以及 可反映早期肿瘤进展的克隆性扩张(IND前问题5，11)。 4.起草并提交具有最终临床试验设计的IND。我们将制定一份最终的临床方案，同意 表格、数据和安全管理计划，以支持IND进行安全性和安全性的I期临床试验 在患有严重SCD的成年人中是可行的。我们将提交这些文件以进行必要的监管(例如IRB、IBC、NIH RAC)审查，等待IND的最终批准。 这种疗法有可能改变SCD的护理，因为它产生了一种根治疗法，即 适用于任何SCD患者，比异基因造血干细胞移植(HSCT)更安全， 从而使在SCD造成不可逆转的损害之前预防其并发症成为可能。

Sickle Cell Disease (SCD) is a devastating recessive genetic disorder, afflicting ~100,000 Americans (3), ~6000 Californians, and hundreds of thousands more worldwide (4). It typically shortens lifespan by decades even with optimal medical care. SCD is caused by a single mutation in the ß-globin gene; gene editing can directly correct the mutation. We have developed an autologous stem cell product that uses a CRISPR/Cas9 nuclease to stimulate repair of the sickle mutation in blood stem cells. Editing reproducibly yields levels of correction that, based on clinical experience, will be sufficient for a curative effect. We propose to complete preclinical development of this therapy, leading to an IND application for an early phase clinical trial in adults with severe SCD. We held a pre-IND meeting on October 30, 2018, and request CIRM funding for the following activities to support IND submission as discussed with the FDA: 1. Demonstrate the capacity to manufacture final cell product under GMP conditions at clinical scale. We will generate at least 3 clinical-scale lots under cGMP that meet all release criteria, in plerixaformobilized HSPCs. We will also complete a drug product stability study. These reports will complete the CMC section of the IND. 2. Complete a rigorous genotoxicity assessment. A murine xenograft model that supports human hematopoietic cells will be used to perform a toxicology study on final cell product manufactured under cGMP conditions. After long-term engraftment, mice will be assessed for evidence of malignant human cells using histopathology, flow cytometry, and genomic readouts. (pre-IND Question 8). 3. Complete additional preclinical studies to establish product safety and potency. An exhaustive interrogation for off-target genomic modifications with the final manufacturing reagents will address potential genotoxicity. We will also assess large deletions and translocations at the HBB on-target site, the nature and potential impact of HBB mutations induced by the editing procedure, and evidence of clonal expansion that could reflect early-stage neoplastic progression (pre-IND Questions 5,11). 4. Draft and file an IND with a final clinical trial design. We will generate a final clinical protocol, consent form, and data and safety management plan to support the IND for a Phase I clinical trial of safety and feasibility in adults with severe SCD. We will submit these for required regulatory (e.g. IRB, IBC, NIH RAC) review, pending final approval of IND. This therapy has the potential to transform the care of SCD by producing a curative treatment that is applicable to any SCD patient and safer than allogeneic hematopoietic stem cell transplantation (HSCT), thus making it possible to prevent complications of SCD before they have done irreversible damage.