Effects of TET2 on T Cell Proliferation by Electroporation

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

Abstract

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

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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