Universal cart/tcrt cells with chemotherapeutic drug resistance and construction method thereof

A construction method and a technology for chemotherapeutic drugs, which are applied in the fields of genetic engineering and synthetic biology, and can solve the problems of difficult to achieve treatment and inability to generate universal CART cells.

Active Publication Date: 2019-04-23
NANJING BIOHENG BIOTECH CO LTD
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AI Technical Summary

Problems solved by technology

[0018] Knocking out the TCRα and β chains alone cannot generate universal CART cells, because this kind of CART cells still have HLA molecules expressed on the cell surface, so they are recognized and rejected by T cells of the recipient’s immune system, making it difficult to achieve the purpose of treatment

Method used

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  • Universal cart/tcrt cells with chemotherapeutic drug resistance and construction method thereof
  • Universal cart/tcrt cells with chemotherapeutic drug resistance and construction method thereof
  • Universal cart/tcrt cells with chemotherapeutic drug resistance and construction method thereof

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Experimental program
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Embodiment 1

[0096] Primary T cell expansion. Primary human T cells were cultured in RPMI 1640 containing 10% FBS, 100U / ml penicillin, 100μg / ml streptomycin sulfate, 10mM Hepes, and used CD3 / CD28 antibody-coupled magnetic beads at a ratio of 1:3 Perform an activation stimulus. Count the cells, add medium every 2 days, collect T cells at the right time according to the cell size and growth parameters, and perform functional testing or cryopreservation.

[0097] Design and Implementation of CRISPR

[0098] The sgRNA and crRNA targeted sequences within exon 1 of the TCRα constant region, the consensus sequence of exon 1 and 2 of the TCRβ constant region, and the exon 1 sequence of dCK, and were cloned into vectors containing the T7 promoter. These plasmids were linearized with restriction enzymes. sgRNA / crRNA were transcribed using the MEGAscript T7Transcription kit (Life Technologies, Carlsbad, CA), and the RNA was stored at -80°C.

[0099] The sgRNA and crRNA sequences used in the scree...

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Abstract

The invention belongs to the fields of genetic engineering and synthetic biology, and particularly relates to a universal CART / TCRT cell with chemotherapeutic drug resistance and a construction methodof the universal CART / TCRT cell. The universal CART / TCRT cell is an allogeneic T cell with chimeric antigen receptors and T cell receptors, wherein the alpha and beta chains of the T cell receptors and deoxycytidine kinase molecules are knocked out; and the knocking-out technology for the alpha and beta chains of the T cell receptors and the deoxycytidine kinase molecules is a CRISPR technology;for the universal CART / TCRT cell, the targeting property of the tumor specific antigen is reserved, the problems of GvHD and rejection are also eliminated, meanwhile, the sensitivity for the chemotherapeutic medicines Fludarabine and Clofarabine is weakened, and the universal CART / TCRT cell can be used as a universal simple, low-cost and high-activity CART / TCRT cell preparation.

Description

technical field [0001] The invention belongs to the field of genetic engineering and synthetic biology, and specifically relates to a general-purpose CART / TCRT cell with chemotherapeutic drug resistance and a construction method thereof. Background technique [0002] Chimeric antigen receptor T cells (CART) are currently one of the most promising tumor immunotherapies. The basic principle is to extract the patient's own T cells and express specific chimeric antigens through genetic and cell engineering methods. Receptors enable it to recognize and bind to tumor cell surface antigens, thereby targeting and killing tumor cells. CAR-T has achieved remarkable curative effect in the treatment of leukemia and lymphoma. On August 30, 2017, the US Food and Health Safety Committee FDA announced the approval of Novartis CAR-T cell therapy for the treatment of relapsed or refractory children, Juvenile B-cell acute lymphoblastic leukemia (rALL), available under the trade name Kymriah (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/113
CPCC07K14/7051C12N9/1205C12N15/113C12N2310/10C12Y207/01074
Inventor 贺小宏王延宾袁鹏
Owner NANJING BIOHENG BIOTECH CO LTD
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