Multiplex HDR for Disease and Correction Modeling of SCID by CRISPR Genome Editing in Human HSPCs

Severe combined immunodeficiency (SCID) is a group of monogenic primary immunodeficiencies caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patients own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID patients. Using CRISPR-Cas9/rAAV6 gene-editing, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs, thus eliminating the need for rare patient samples, that model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology directed repair (HDR). Firstly, we developed a SCID disease model via knock-out of both alleles of genes critical to the development of lymphocytes; and secondly, we established a knock-in/knock-out (KI-KO) strategy to develop a proof-of-concept gene correction. Lastly, we performed successful gene correction of RAG2-SCID patient-derived CD34+ HSPCs, highlighted by their successful development into CD3+ T cells with diverse TCR repertoires. By using CRISPR-Cas9, multiplexed HDR, and HD-derived CD34+ HSPCs, we outline an approach for the study of human lymphopoiesis and present a way for researchers to determine the optimal configuration for CRISPR-Cas9 gene correction for other SCIDs and additional recessive blood disorders.

重症联合免疫缺陷（Severe combined immunodeficiency, SCID）是一类由参与淋巴细胞成熟与功能调控的基因突变所引发的单基因原发性免疫缺陷病。对患者自体造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）开展CRISPR-Cas9体外基因编辑，可作为当前SCID患者治疗金标准——异基因造血干细胞移植的替代疗法。本研究借助CRISPR-Cas9/重组腺相关病毒6型（rAAV6）基因编辑技术，在健康供者（HD）来源的CD34+造血干祖细胞中构建目标基因型，从而无需依赖稀缺的患者样本，并通过多重同源定向修复（homology directed repair, HDR）同时构建SCID疾病模型与模拟基因编辑疗法对SCID的治疗效果。具体而言，我们首先通过敲除淋巴细胞发育关键基因的双等位基因，建立了SCID疾病模型；其次，我们开发了敲入/敲除（KI-KO）策略以完成概念验证性基因校正研究；最后，我们成功对RAG2缺陷型SCID患者来源的CD34+造血干祖细胞实施基因校正，其可顺利分化为具有多样化T细胞受体库的CD3+ T细胞，该结果验证了该方案的可行性。本研究通过整合CRISPR-Cas9、多重同源定向修复技术与健康供者来源的CD34+造血干祖细胞，提出了一种研究人类淋巴细胞生成的实验路径，并为研究者确定针对其他SCID及其他隐性血液疾病的CRISPR-Cas9基因校正最优配置提供了参考思路。