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，困扰着约10万美国人（3）， ~6000加利福尼亚人，以及全球数十万人（4）。它通常会缩短寿命， 即使有最佳的医疗护理也要几十年。SCD是由β-珠蛋白基因的单一突变引起的; 编辑可以直接纠正突变。我们开发了一种自体干细胞产品， CRISPR/Cas9核酸酶刺激造血干细胞中镰状突变的修复。可再现地编辑 根据临床经验，产生足以达到治疗效果的矫正水平。我们 我建议完成这种疗法的临床前开发，从而导致IND申请， 在严重SCD成人中进行的临床试验。我们于2018年10月30日举行了IND前会议， 申请CIRM资助以下活动，以支持与FDA讨论的IND提交： 1.证明在GMP条件下以临床规模生产最终细胞产品的能力。 我们将在cGMP下生产至少3个符合所有放行标准的临床规模批次，用于普乐沙福动员 HSPC。我们还将完成制剂稳定性研究。这些报告将完成 IND的CMC部分。 2.完成严格的遗传毒性评估。一种支持人类的鼠异种移植模型， 造血细胞将用于对在以下条件下生产的最终细胞产品进行毒理学研究： cGMP条件。在长期植入后，将评估小鼠的恶性人类肿瘤的证据。 使用组织病理学、流式细胞术和基因组读数对细胞进行分析。（IND前问题8）。 3.完成额外的临床前研究，以确定产品的安全性和效力。详尽 使用最终生产试剂询问脱靶基因组修饰将解决 潜在遗传毒性。我们还将评估HBB靶位点的大缺失和易位， 编辑程序诱导的HBB突变的性质和潜在影响，以及 可能反映早期肿瘤进展的克隆扩增（IND前问题5、11）。 4.起草并提交IND及最终临床试验设计。我们将制定最终的临床方案， 表格、数据和安全性管理计划，以支持IND进行I期安全性和 严重SCD成人的可行性。我们将向要求的监管机构（例如IRB、IBC、NIH）提交这些文件 RAC）审查，等待IND的最终批准。 这种疗法有可能通过产生一种治愈性治疗来改变SCD的护理， 适用于任何SCD患者，比异基因造血干细胞移植（HSCT）更安全， 从而使得有可能在SCD的并发症造成不可逆的损害之前预防它们。

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 plerixafor-mobilized 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.