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Method for preparing CAR-T cell by CRISPR/Cas9

A cell and DNA sequence technology, applied in the field of biology, to achieve the effect of high editing efficiency, avoiding potential safety hazards and high safety

Inactive Publication Date: 2015-09-09
NANJING KAEDI BIOTECH INC
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Problems solved by technology

[0011] However, CAR-T cells currently used in clinical trials in the world generally use viral vectors to introduce CAR molecules into T cells. From the perspective of clinical treatment safety, there is a greater risk

Method used

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  • Method for preparing CAR-T cell by CRISPR/Cas9

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Embodiment 1

[0020] Embodiment 1 (preparation embodiment)

[0021] Production of leukemia CAR-T cells using CRISRP / Cas9 system:

[0022] 1) T cell isolation and culture

[0023] a. Separation of fresh peripheral blood mononuclear cells (PBMC, peripheral blood mononuclear cell) by density gradient centrifugation;

[0024] b. Use paramagnetic beads (Dynabeads ClinExVivo CD3 / CD28, Invitrogen, Camarillo, CA, USA) coupled with anti-CD3 and anti-CD28 antibodies to enrich CD3+ cells. The ratio of magnetic beads to cells is 3:1; cells are diluted to TNC (total nucleated cell) at a concentration of 20-30x106 / mL, co-incubate with magnetic beads in a petri dish for 2 hours at room temperature;

[0025] c. Use Magnetic particles concentrator (MPC) (Invitrogen) to enrich CD3+ cells; cells containing CD3+ are resuspended in culture medium (OpTmizer TM CTS TMT-Cell Expansion SFM, Life Technologies), the final concentration was 1×106cells / mL. Incubate for 2 days at 37°C in a 5% CO2 incubator.

[00...

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Abstract

The invention relates to a method for preparing a CAR-T cell by CRISPR / Cas9. The method comprises integrating a CAR molecule into a first intron of a human 19th chromosome AAVS1 site by a CRISPR / Cas9 technology, and integrating antibody molecules of various tumor surface antigens into a human T cell genome AAVS1 site by CRISPR / Cas9. The method realizes accurate integration of the CAR molecule into a human T cell genome specific safe port site, does not influence human normal gene functions, and prevents a series of clinical risks such as viral vector safety hidden trouble and genetic toxicity and immunogenicity caused by insertion of an exogenous gene into a genome.

Description

technical field [0001] The invention relates to a method for preparing CAR-T cells by using CRISPR / Cas9, which belongs to the field of biology. Background technique [0002] The immune system is the defense system of the human body. On the one hand, it plays the role of removing bacteria, viruses, and foreign substances. On the other hand, it eliminates aging cells and mutated cells in the body. Some mutated cells will become cancer cells. The result of the interaction between the body's immune system and cancer cells determines the ultimate evolution of cancer. For healthy people, their immune system is strong enough to eliminate cancer cells caused by mutations in the body in time. However, for patients with cancer cells, it is common for the immune system to be low and unable to effectively identify and kill cancer cells; To escape the recognition and killing of immune cells, the immunotherapy of cancer is to improve the immunogenicity of cancer with the help of molecul...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/02
Inventor 熊波
Owner NANJING KAEDI BIOTECH INC
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