利用电转的方法对T细胞TET2基因敲除并探讨TET2对T细胞增殖的影响

doi:10.13560/j.cnki.biotech.bull.1985.2019-0891

摘要/Abstract

摘要： 近年来,利用重组病毒对T细胞进行基因编辑用于免疫治疗,受到了广泛重视。然而,重组病毒因存在随机整合,制备耗时长且昂贵的缺点制约了其应用。与此同时,电转染技术的应用能够快速将外源DNA带入细胞内,有助于提高T细胞基因编辑效率。TET（Ten-eleven translocation）家族蛋白可以催化5-甲基胞嘧啶（5mC）转化为5-羟甲基胞嘧啶（5hmC）,5hmC作为细胞中的DNA去甲基化酶,在细胞基因组表观遗传学中起着重要调控作用。研究表明TET2 基因的缺失能够促进CAR-T 细胞的快速繁殖,产生强力的 CAR-T 细胞。该研究利用CRISPR/Cas9基因编辑技术对TET2基因进行敲除。首先对sgRNA进行体外转录,与大肠杆菌诱导表达的Cas9蛋白孵育形成Cas9：gRNA核糖核蛋白复合物（RNP）,并在体外酶切验证sgRNA的活性。接着利用电转染技术将Cas9：gRNA核糖核蛋白复合物（RNP）带入细胞内,并检测T细胞基因编辑效率。最后利用流式细胞分析技术检测T细胞的增殖情况。基因测序与T7E Ⅰ 酶切结果表明,T细胞中的TET2基因被成功敲除,T细胞活力和功能并未受到影响。流式细胞技术,CCK-8以及台盼蓝细胞活率检测结果显示缺失Tet2蛋白后,T细胞的增殖速率明显快于野生型T细胞。该研究为非病毒载体替代传统的慢病毒载体构建携带嵌合抗原T细胞奠定了基础,具有制备周期短,安全性高的特点。同时TET2缺失促进了CAR-T细胞的增殖,使其能够引发有效的抗肿瘤反应,为CAR-T细胞免疫治疗提供了新的思路。

关键词: Cas9核糖核蛋白（Cas9 RNP）, TET2, 电转染, 基因敲除, T细胞增殖

Abstract: Gene editing of T cells using recombinant viruses for immunotherapy has attracted widespread attentions. However,the application of recombinant viruses was constrained due to the disadvantages of random integration,long-time preparation and high cost. However,the application of electroporation technique can quickly bring exogenous DNA into cells,which is conducive to improving gene editing efficiency of T cells. The TET（Ten-eleven translocation）family proteins,such as DNA demethylase,can catalyze the conversion of 5-methyl cytosine（5mC）to 5-hydroxymethyl cytosine（5hmC）and play an important regulatory role in cell genome epigenetics. The researches demonstrated that the CAR-T cells lacking TET2 gene proliferated more rapidly and functioned more powerfully. In this study,the CRISPR/Cas9 technology was used to knock out the TET2 gene. First,sgRNA was transcribed in vitro and incubated with the Cas9 protein expressed by Escherichia coli expression system to form Cas9：sgRNA ribonucleoprotein complex（RNP）,and the activity of sgRNA was verified by in vitro enzyme digestion. Then,RNP was introduced into cells by electroporation,and the gene editing efficiency of T cells was tested. Finally,the proliferation of T cells was detected by flow cytometry. The results of gene sequencing and T7E Ⅰ enzyme digestion showed that the TET2 gene in the T cells was successfully knocked out,while the activity and function of the T cells were not affected. The results of flow cytometry analysis,CCK-8 and trypan blue cell viability test showed that the proliferation of T cells lacking TET2 gene was significantly faster than wild-type T cells. This study provides a basis for the application of non-viral vector replacing traditional recombinant virus used in the preparation of chimeric-antigen-carrying T cells,and it characterizes with short preparation cycle and high safety. Meanwhile,TET2 gene deficiency promotes the proliferation of CAR T cells,and enables them to initiate effective anti-tumor responses,which provides a new insight into CAR T cell immunotherapy.

Key words: Cas9 ribonucleoprotein, TET2, electroporation, gene knockout, T cell proliferation

杨雷, 叶洲杰, 李兆龙, 沈阳坤, 傅雅娟. 利用电转的方法对T细胞TET2基因敲除并探讨TET2对T细胞增殖的影响[J]. 生物技术通报, 2020, 36(1): 229-237.

YANG Lei, YE Zhou-jie, LI Zhao-long, SHEN Yang-kun, FU Ya-juan. Effects of TET2 on T Cell Proliferation by Electroporation[J]. Biotechnology Bulletin, 2020, 36(1): 229-237.

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