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Method for preparing T cells with knockout of double genes TCR and HLA

A double-gene, cell-based technology, applied to cells modified by introducing foreign genetic material, recombinant DNA technology, using vectors to introduce foreign genetic material, etc., can solve the problem of limiting the use of peripheral blood mononuclear cells, the number, activity and proliferation of T cells Limited capacity, loss of treatment options, etc., to achieve the effect of facilitating tumor treatment

Active Publication Date: 2018-01-26
上海兴瑞一达生物科技有限公司
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CAR-T also has some shortcomings in the treatment of tumors. On the one hand, it will cause side effects such as cytokine storms. On the other hand, the immune system of patients after chemotherapy and radiotherapy is destroyed, and the number, activity and The proliferation ability is extremely limited, which limits the use of autologous peripheral blood mononuclear cells, which is the best treatment option for patients with advanced cancer
Therefore, people turn their attention to a kind of chimeric antigen receptor cells that can be reinfused. On the one hand, healthy donors have a strong immune system that can effectively kill tumor cells. On the other hand, their blood is easy to collect for the isolation of T cells. However, allogeneic reinfusion has a strong graft-versus-host reaction

Method used

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  • Method for preparing T cells with knockout of double genes TCR and HLA
  • Method for preparing T cells with knockout of double genes TCR and HLA
  • Method for preparing T cells with knockout of double genes TCR and HLA

Examples

Experimental program
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Effect test

Embodiment example 1C

[0031] Implementation case 1 CRISPR / Cas9-TCR and CRISPR / Cas9-HLA vector construction

[0032] 1. Recombinant expression vector

[0033] The vectors used in the present invention are pX330A and pX330S (the map is as follows figure 1, shown in 2), the pX330A expression gene vector contains an original replicon, a cytomegalovirus promoter sequence (CMV), and restriction endonuclease sites (BbsI, Bsa I, etc.), resistance screening gene (anti ampicillin); pX330S is similar in structure to pX330A, and the differences are mainly in the different enzyme cutting sites and the different resistance selection genes (spectinomycin). The artificially synthesized sgRNA gene is connected with the linearized backbone carrier DNA under the action of T4 ligase to form a recombinant vector.

[0034] 2. Synthesis of TRAC-sgRNA, TRBC2-sgRNA and B2M-sgRNA

[0035] The sgRNA of TRAC, TRBC2 and B2M is located in the exon of the gene (SEQ ID NO.1~3), and the target sequence on the gene is unique. Ad...

Embodiment example 2T

[0040] Implementation Case 2 Acquisition of TCR and HLA Double Gene Knockout T Cells

[0041] Fresh peripheral blood mononuclear cells were separated by density gradient, activated by CD3 and CD28 monoclonal antibodies, cultured with IL-2 (final concentration 300IU / ml), activated into T cells, and then the recombinant vector CRISPR / Cas9 prepared in Example 1 -HLA and CRISPR / Cas9-TCR plasmids were co-transfected into T cells, and TCR and HLA double gene knockout was performed. Transfection has been called:

[0042] Add the plasmids (CRISPR / Cas9-TCR, CRISPR / Cas9-HLA) into the electroporation cuvette respectively, each electroporation plasmid is 200ng, add cells (1×10 7 ), invert the electric shock cup to make it evenly mixed, put the electric shock cup into the electric shock tank, pulse electric shock once (2.0KV, 25uFD), then transfer the cells to the well containing 0.5ml DMEM medium, shake the well gently After 24h-48h of electroporation, the gene was transiently expressed...

Embodiment example 3T

[0045] Implementation case 3 TCR and HLA double gene knockout T cells combined with CAR to treat tumors

[0046] The above isolated TCR and HLA double gene knockout T cells were infected by lentivirus with the CAR for the treatment of different types of tumors. The specific process is: Leader-scFv-CD8-CD137-CD3ζ-T2A-HSV-TK nucleotide Synthesis, the gene fragment of the synthetic Leader-scFv-CD8-CD137-CD3ζ-T2A-HSV-TK is inserted into the NotI-AsiSI site of the pLent-C-GFP vector, after the sequencing is correct, a plasmid is constructed, and the plasmid Transfect 293T cells, package into a lentivirus carrying the encoding gene, add 1 mL of the lentivirus carrying the Leader-scFv-CD8-CD137-CD3ζ-T2A-HSV-TK encoding gene to culture TCR and HLA double gene knockout T cells (1×10 7 ) in a petri dish, mix well, and replace with fresh culture medium after 24 hours.

[0047] Further, after the infection is completed, the CAR-expressing TCR and HLA double-gene knockout T cells are exp...

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Abstract

The invention discloses a method for preparing T cells with knockout of double genes TCR and HLA. The method comprises the following steps: co-transfecting T cells by plasmids of CRISPR / Cas9-HLA and CRISPR / CAs9-TCR, and carrying out knockout of double genes TCR and HLA to obtain the T cells with knockout of double genes TCR and HLA. The method can be used for allotransplantation without causing immunological rejection.

Description

technical field [0001] The invention relates to the field of biological genes, more specifically, a method for preparing T cells with double gene knockout of TCR and HLA. Background technique [0002] Tumors have always been a major disease that plagues the world. At present, there are at least a hundred kinds of tumors, which seriously endanger human health. At present, the treatment methods mainly include surgery, chemotherapy, radiotherapy, monoclonal antibodies, traditional Chinese medicine, etc., but the therapeutic effect is limited and the side effects are obvious. In recent years, tumor cell immunotherapy has become the most active and promising treatment method. Immunotherapy includes immunotherapy drugs and cellular immunotherapy. At present, chimeric antigen receptor cell CAR-T therapy has brought hope to tumor treatment. The basic principle is to isolate and cultivate T cells from the tumor patient's own body, and use genetic technology to study and transform th...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
Inventor 刘明录刘敏王立新冯建海张传鹏强邦明金海锋万磊韩庆梅王亮
Owner 上海兴瑞一达生物科技有限公司
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