肿瘤细胞治疗研究
英文原题：High-efficiency of genetic modification using CRISPR/Cpf1 system for engineered CAR-T cell therapy.
CAR-T（CAR-T）细胞疗法在多种血液癌症中已显示出有前景的疗效，包括弥漫性大B细胞淋巴瘤以及急性和慢性淋巴细胞白血病等。
CAR-T（CAR-T）细胞疗法在多种血液肿瘤中显示出良好的疗效，包括弥漫性大B细胞淋巴瘤以及急性和慢性淋巴细胞白血病等。通过基因编辑技术为免疫细胞治疗有效生成更强效且更安全的CAR-T细胞至关重要。基于慢病毒、逆转录病毒和转座子的传统方法会将CAR序列随机整合到T细胞基因组中，这可能导致安全性问题。因此，将CAR盒精确敲入特定基因位点如TRAC和PDCD1，可以降低随机整合带来的风险，同时增强修饰后CAR-T细胞的稳定性和功能。当前基于CRISPR/Cas9的基因编辑方法在嵌合抗原受体的敲入效率方面存在局限，而Cpf1作为一种CRISPR-Cas/RNA引导的核酸酶，由于其独特的生化特性，相比Cas9表现出更高的同源定向修复（HDR）率。在此，我们介绍一种结合电穿孔和腺相关病毒（AAV）感染的方法，将CRISPR/Cpf1组分和HDR模板递送到T细胞中，从而以高效率将CAR序列精确整合到特定基因位点。
Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in several kinds of blood cancers, including diffuse large B-cell lymphoma and acute and chronic lymphoblastic leukemia, etc. It is essential to effectively generate more potent and safer CAR-T cells through gene editing technologies for immune cell therapy. Conventional methods based on lentivirus, retrovirus and transposon, randomly integrate CAR sequence into T cell genome, which could lead to safety issues. Therefore, precise knock-in of CAR cassette into specific gene locus like TRAC and PDCD1 can lower the risks caused by random integration, as well as enhance the stability and function of the modified CAR-T cells. Current approaches of CRISPR/Cas9-based gene-editing have limitations in knock-in efficiency of the chimeric antigen receptor, while Cpf1, a CRISPR-Cas/RNA-guided nuclease, shows higher homology-directed repair (HDR) rate compared to Cas9 due to its unique biochemical characteristics. Here, we introduce a method combining electroporation and adeno-associated virus (AAV) infection to deliver CRISPR/Cpf1 components and a HDR template into T cells, thus precisely integrate CAR sequence at a specific gene locus with high efficiency.
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