Cas9/RNP knockout T cell PD-1 and LAG3 genes and preparation method of CAR-T cells

A PD-1, cell technology, applied in receptors/cell surface antigens/cell surface determinants, chemical instruments and methods, reverse transcription RNA viruses, etc., to achieve the effect of releasing inhibition and increasing safety

Inactive Publication Date: 2018-09-14
杭州荣泽生物科技集团有限公司
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Problems solved by technology

Studies have reported that knocking out PD-1 in T cells or CAR-T cells can enhance the anti-tumor function of T cells or CAR-T cells, but there is no information about knocking out PD-1 and LAG-3 in T or CAR-T cells. report

Method used

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  • Cas9/RNP knockout T cell PD-1 and LAG3 genes and preparation method of CAR-T cells
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  • Cas9/RNP knockout T cell PD-1 and LAG3 genes and preparation method of CAR-T cells

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Embodiment Construction

[0035] The present invention provides a method for Cas9 / RNP preparation of PD-1 and LAG3 double gene knockout CD19 CAR-T cells, which comprises CD4+ / CD8+ T cell preparation, PD-1 and LAG3 knockout sgRNA and Cas9 protein preparation, Electroporation transfection of Cas9 / RNP and T cells, transfection of CD19 CAR lentivirus, activation of anti-CD3+CD28 antibody magnetic beads, and CD19 CAR-T cells were obtained by screening and culturing.

[0036] Preparation of CD4+ / CD8+T:

[0037] Use an EDTA anticoagulant tube to collect peripheral blood from healthy people, dilute it with an equal volume of phosphate buffered solution (PBS), add the diluted peripheral blood solution to a 50ml centrifuge tube with an equal volume of lymphocyte separation medium (Ficoll), and centrifuge at 400g for 30 minutes; carefully absorb the lymph The buffy coat cells on the separation solution were transferred to a new 50ml centrifuge tube, and washed 3 times with PBS centrifugation at 300g for 10min t...

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Abstract

The invention relates to a method for preparing PD-1 and LAG3 double knockout CD19 CAR-T cells by Cas9/RNP. The method comprises the following steps: mixing sgRNA and Cas9 protein for knocking out PD-1 and LAG3 genes with an electroporation transformation reagent, and adding the mixture into CD4+/CD8+ cells for electroporation transformation; transfecting CD4+/CD8+ positive cells with CD19 CAR lentivirus; and screening, and culturing and amplifying transfected T cells, so as to obtain the PD-1 and LAG3 double knockout CD19 CAR-T cells. The method, adopting a Cas9/RNP gene editing system, improves the transfection efficiency and the expression efficiency and time of transgenes, simplifies the preparation process of CAR-T cells, and improves the feasibility and increases the load of the system. In addition, the method is free of transfection reagents when the PD-1 and LAG3 double genes are knocked out, and thus the safety is higher.

Description

technical field [0001] The present invention relates to the field of immune cell preparation, in particular, the present invention relates to the preparation of PD-1 and LAG3 knockout CD19 CAR-T cells using Cas9 / RNP. Background technique [0002] After surgery, radiotherapy, and chemotherapy, tumor immunotherapy has become an effective treatment method and has recently received widespread attention. Among them, Chimeric Antigen Receptors-T cell technology (Cheimeric Antigen Receptors-T cell, CAR-T) is a newly emerging adoptive cell therapy (Adoptive cell transfer therapy, ACT), which modifies the patient's T cells in vitro, After activation and proliferation, it is infused back into the patient's body, through the specific receptors expressed by T cells, targeted recognition of tumor cells, and shows strong killing activity and persistence. [0003] Death molecule-1 (Programmed death-1, PD-1, also known as CD279) belongs to the inhibitory co-receptor of T lymphocytes and is...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC07K14/70503C07K14/7051C07K14/70521C07K16/2803C12N15/86C12N2740/15043
Inventor 陈相波雷鸣田朋飞
Owner 杭州荣泽生物科技集团有限公司
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