Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
MaxCyte Inc., Gaithersburg, MD 20878, USA.
Sci Transl Med. 2017 Jan 11;9(372). doi: 10.1126/scitranslmed.aah3480.
Gene repair of CD34 hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34 HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro. Transplant of gene-repaired X-CGD HSPCs into NOD (nonobese diabetic) SCID (severe combined immunodeficient) γc mice resulted in efficient engraftment and production of functional mature human myeloid and lymphoid cells for up to 5 months. Whole-exome sequencing detected no indels outside of the CYBB gene after gene correction. CRISPR-mediated gene editing of HSPCs may be applicable to other CGD mutations and other monogenic disorders of the hematopoietic system.
CD34 造血干细胞和祖细胞 (HSPCs) 的基因修复可能避免与基因治疗相关的问题，例如载体相关的突变和转基因表达失调。我们使用 CRISPR（成簇规律间隔短回文重复）/Cas9（CRISPR 相关蛋白 9）来修复 X 连锁慢性肉芽肿病（X-CGD）患者的 CD34 HSPCs 中 CYBB 基因的突变。来自 X-CGD 患者的 HSPCs 中超过 20%的序列确认修复恢复了这些前体细胞分化的髓样细胞中 NADPH（烟酰胺腺嘌呤二核苷酸磷酸）氧化酶和超氧自由基产生的功能。将基因修复的 X-CGD HSPCs 移植到 NOD（非肥胖糖尿病）SCID（严重联合免疫缺陷）γc 小鼠中，可有效植入并产生功能性成熟的人髓样和淋巴样细胞，长达 5 个月。全外显子组测序在基因校正后未发现 CYBB 基因以外的插入缺失。HSPCs 的 CRISPR 介导的基因编辑可能适用于其他 CGD 突变和其他造血系统的单基因疾病。
