Production of Human CRISPR-Engineered CAR-T Cells
Production of Human CRISPR-Engineered CAR-T Cells
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DOI：
10.3791/62299
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发表时间：
2021-03-01
影响因子：
1.2
通讯作者：
June, Carl H.
中科院分区：
文献类型：
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作者：
Agarwal, Sangya;Wellhausen, Nils;June, Carl H.
使用嵌合抗原受体T细胞（CAR-T细胞）的免疫细胞疗法已在血液恶性肿瘤患者中显示出显著的临床疗效，目前正在研究各种实体瘤。CAR-T细胞是通过从患者血液中去除T细胞并对其进行工程改造以表达合成免疫受体来产生的，该受体可重定向T细胞以识别和消除靶肿瘤细胞。CAR-T细胞的基因编辑有可能提高目前CAR-T细胞疗法的安全性，并进一步提高CAR-T细胞的疗效。在这里，我们描述了用于人CRISPR工程化的CD 19指导的CAR-T细胞的活化、扩增和表征的方法。这包括CAR慢病毒载体的转导和使用单向导RNA（sgRNA）和Cas9内切核酸酶靶向T细胞中的目标基因。本方案中描述的方法可普遍应用于本研究所用以外的其他CAR构建体和靶基因。此外，该方案讨论了gRNA设计、前导gRNA选择和靶基因敲除验证的策略，以可重复地实现临床级人类T细胞的高效、多重CRISPR-Cas9工程化。
Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.