生产人类CRISPR工程的CAR-T细胞
概要
在这里，我们提出了一个协议，在原人类T细胞的基因编辑使用CRISPR卡斯技术来修改CAR-T细胞。
Abstract
使用心绞细胞抗原受体T细胞（CAR-T细胞）的采用细胞疗法在血液恶性肿瘤患者中已显示出显著的临床疗效，目前正在对各种实体肿瘤进行调查。CAR-T细胞通过从患者的血液中取出T细胞并设计它们来表达一种合成免疫受体来产生，该受体将T细胞重定向以识别和消除目标肿瘤细胞。CAR-T细胞的基因编辑有可能提高当前CAR-T细胞疗法的安全性，并进一步提高CAR-T细胞的功效。在这里，我们描述了人类CRISPR工程CD19定向CAR-T细胞的激活、扩展和定性方法。这包括CAR扁豆载体的转导，以及使用单导RNA（sgRNA）和Cas9内分泌酶瞄准T细胞感兴趣的基因。本协议中描述的方法可以普遍应用于除本研究所用基因以外的其他CAR结构和靶向基因。此外，本协议还讨论了gRNA设计、铅GRNA选择和靶向基因淘汰验证的策略，以可重复实现临床级人类T细胞的高效率、多重CRISPR-Cas9工程。
Introduction
奇美利安抗原受体（CAR）-T细胞疗法彻底改变了采用细胞疗法和癌症免疫疗法领域。CAR-T细胞是一种表达合成免疫受体的工程T细胞，它结合了抗原特异性单链抗体片段和来自TCRzeta链的信号域，以及对于T细胞激活和共同刺激1、2、3、4所必需且足以进行成本激励的域.CAR-T细胞的制造首先提取患者自己的T细胞，然后对CAR模块进行前体内病毒转导，并扩大CAR-T细胞产品，磁珠作为人工抗原呈现细胞5。扩大的CAR-T细胞被重新注入到患者，在那里他们可以移植，消除目标肿瘤细胞，甚至持续多年后输液6，7，8。虽然CAR-T细胞治疗在B细胞恶性肿瘤中取得了显著的反应率，但固体肿瘤的临床成功受到多种因素的挑战，包括不良T细胞渗透9、免疫抑制肿瘤微环境10、抗原覆盖和特异性，以及CAR-T细胞功能障碍11、12.目前CAR-T细胞治疗的另一个限制包括使用自体T细胞。经过多轮化疗和高肿瘤负担后，CAR-T细胞的质量可能较健康捐赠者的异基因CAR-T产品差，此外还有与制造自体CAR-T细胞相关的时间和费用。CRISPR/Cas9对CAR-T细胞产品的基因编辑代表了克服目前CAR-T细胞13、14、15、16、17的局限性的新策略。
CRISPR/Cas9是一个两个组分系统，可用于哺乳动物细胞18，19的靶向基因组编辑。CRISPR相关内分泌酶Cas9可以通过与目标DNA序列20的碱基配对，诱导由小RNA引导的特定部位双链断裂。在没有修复模板的情况下，通过容易出错的非同位素端连接 （NHEJ） 通路修复双链断裂，导致帧移位突变或通过插入和删除突变 （INDELs） （INDELs）19、20、21过早停止 codons。效率、易用性、成本效益和多路复用基因组编辑能力使CRISPR/Cas9成为提高自体和异基因CAR-T细胞的功效和安全性的强大工具。此方法还可用于编辑 TCR 定向 T 单元格，方法是将 CAR 结构替换为 TCR。此外，基因CAR-T细胞，有有限的潜力，导致移植与宿主疾病也可以通过基因编辑TCR，b2m和HLA细胞位点产生。
在此协议中，我们展示了如何将 T 细胞的 CRISPR 工程与 CAR-Transgene 的病毒载体介导传递相结合，以生成基因组编辑的 CAR-T 细胞产品，提高功效和安全性。图 1显示了整个过程的示意图。利用这种方法，我们已经在原人类CAR-T细胞中展示了高效基因敲除。 图 2A 详细描述了编辑和制造 T 单元的每个步骤的时间表。还讨论了指导RNA设计和淘汰验证的策略，以便将这种方法应用于各种目标基因。
Protocol
Representative Results
Discussion
在这里，我们描述了使用CRISPR Cas9技术和制造产品来进一步测试功能和功效的基因编辑CAR-T细胞的方法。上述协议已优化，用于在原发性人类T细胞中执行CRIPSR基因编辑，并结合具有幻想抗原受体的工程T细胞。此协议允许高淘汰效率，以最小的供体到捐赠者的变异性。使用CRISPR进行改造，通过消除抑制T细胞功能的受体并制造异基因CAR-T细胞，可以提高CAR-T细胞的功效和安全性。
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開示
The authors have nothing to disclose.
Acknowledgements
我们感谢宾夕法尼亚大学提供正常供体T细胞和流细胞学核心的人类免疫学核心。
Materials
| 4D-Nucleofactor Core Unit | Lonza | AAF-1002B | |
| 4D-Nucleofactor X-Unit | Lonza | AAF-1002X | |
| Accuprime Pfx Supermix | ThermoFisher | 12344040 | |
| Beckman Optima XPN ultracentrifuge | Beckman Coulter | ||
| Brilliant Violet 605 anti-human CD3 Antibody | Biolegend | 317322 | Clone OKT3 |
| BV711 Anti-human PD1 | Biolegend | Clone EH12.2H7 | |
| Cas9-Electroporation enhancers | IDT | 1075915 | |
| CD3/CD28 Dynabeads | ThermoFisher | 40203D | |
| CD4+ T cell isolation Kit | StemCell technologies | 15062 | |
| CD8+ T cell isolation Kit | StemCell technologies | 15063 | |
| Corning 0.45 micron vacuum filter/bottle | Corning | 430768 | |
| Corning T150 cell culture flask | Millipore Sigma | CLS430825 | |
| DMSO | Millipore Sigma | D2650 | |
| DNAeasy Blood and Tissue Kit | Qiagen | 69504 | |
| DynaMag Magnet | ThermoFisher | 12321D | |
| Glutamax supplement | ThermoFisher | 35050061 | |
| HEK293T cells | ATCC | CRL-3216 | |
| HEPES (1 M) | ThermoFisher | 15630080 | |
| huIL-15 | PeproTech | 200-15 | |
| huIL-7 | PeproTech | 200-07 | |
| Lipofectamine 2000 | ThermoFisher | 11668019 | |
| Nucleospin Gel and PCR cleanup | Takara | 740609.25 | |
| Opti-MEM | ThermoFisher | 31985062 | |
| P3 Primary cell 4D-nucleofactor X Kit L | Lonza | V4XP-3024 | |
| Penicilin-Streptomycin-Glutamine | ThermoFisher | 10378016 | |
| pTRPE expression Plasmid | in house | ||
| Rabbit Anti-Mouse FMC63 scFv Monoclonal Antibody, (R19M), PE | CytoArt | 200105 | |
| RPMI1640 | ThermoFisher | 12633012 | |
| sgRNA | IDT | ||
| Spy Fi Cas9 | Aldevron | 9214 | |
| Ultracentrifuge tubes | Beckman Coulter | 326823 | |
| Viral packaging mix | in house | ||
| X-Vivo-15 Media | Lonza | BE02-060F |
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