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

Effects of TET2 on T Cell Proliferation by Electroporation

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T7EⅠ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。

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 T7EⅠenzyme 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.