Method for preparing general type CAR-T cells by using CRISPR/Cas9+AAV

A general-purpose, cell-based technology, applied in the field of preparation of general-purpose CAR-T cells, can solve problems such as limited expansion capacity, inability to obtain cells, and inability to adopt quality, so as to improve safety and controllability, promote application, and reduce Effect of treatment time cost and financial cost

Inactive Publication Date: 2019-04-12
广州白云山拜迪生物医药有限公司
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the current CAR-T technology generally has the following problems: (1) It takes a longer time to observe whether the viral vector will bring about harmful insertional mutations: the CAR sequence is the core component of CAR-T cells, and the current manufacture of CAR-T is mainly The method adopted is to use lentivirus as a carrier to randomly integrate the CAR sequence into the T cell genome, thereby realizing the internalization of the exogenous CAR sequence; however, due to the randomness of the integration site of the lentivirus, there is a certain potential risk of random integration , resulting in canceration of transduced T cells, random expression of irrelevant genes or gradual non-expression of anti

Method used

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  • Method for preparing general type CAR-T cells by using CRISPR/Cas9+AAV
  • Method for preparing general type CAR-T cells by using CRISPR/Cas9+AAV
  • Method for preparing general type CAR-T cells by using CRISPR/Cas9+AAV

Examples

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Embodiment 1

[0027] Example 1 Preparation of Universal CD19 CAR-T

[0028] (1) Preparation process of general-purpose CD19 CAR-T:

[0029] Separation and purification of CD3+ T cells → static culture → activation → CRISPR / Cas gene editing → adeno-associated virus infection of T cells → magnetic bead sorting to remove TCRαβ-positive cells → aliquot storage.

[0030] (2) Selection of functional region gene sequences: KOZAC, scFv CD19 fmc63 (19), CD8A, CD28, CD137 (4-1BB), CD247 (CD3-zeta ) functional region coding sequence.

[0031] (3) Whole-gene synthesis of the CAR gene sequence, including: BstXⅠ restriction site + TRAC left homology arm + KOZAC + CD19 monoclonal antibody fmc63 (19) single-chain variable region + hinge + transmembrane region CD8a + co-stimulatory Molecule CD137 (4-1BB) + intracellular signal transduction region CD247 (CD3-zeta) + stop codon + TRAC right homology arm + BamHI restriction site.

[0032] (4) Insert the vector pAAV-MCS through the restriction site to comple...

Embodiment 2

[0045] Embodiment 2 Quality Inspection

[0046] (1) Mismatch enzyme method (Surveyor method) detects CRISPR / Cas9 activity or mutation efficiency, and evaluates the success rate of gene editing.

[0047] DNA extraction and PCR amplification: Extract gene-edited T cell DNA and perform PCR amplification, using PD-1, TRAC, and B2M gRNA as primers respectively,

[0048]

[0049] Amplification conditions: pre-denaturation at 94°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 63°C for 20 seconds, extension at 72°C for 1 minute, amplification for 3 to 5 cycles, and final extension at 72°C for 7 minutes. The PCR product was stored at 4°C for later use.

[0050] DNA hybridization: The unedited T cell DNA amplification product is mixed with the amplification product of the sample to be tested in equal volumes, denatured at 95°C for 5 minutes, and then gradually lowered to 25°C at a rate of 0.1°C every 4 seconds.

[0051] SURVEYOR digestion: 600ng of homoduplex or ...

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Abstract

The invention relates to a method for preparing general type CAR-T cells by using CRISPR/Cas9+AAV, and belongs to the field of immunocytotherapy. The method utilizes CRISPR/Cas9+AAV to knock out an immunological checkpoint PD-1, a human leukocyte antigen (HLA) and TCR; multi-gene knockout is employed to prepare the general type CAR-T cells; and a CRISPR/Cas9 gene editing technology is used to perform precise gene knockout, and is used in combination with adeno-associated virus (AAV) to replace lentiviruses for precise gene knock-in and to avoid the potential risk of random integration of the lentiviruses. The method of the invention can realize multi-target gene knock-in and knock-out, construct the general type CAR-T cells for allogeneic treatment, and solve the problems existing in traditional CAR-T cell therapy such as risk of random insertion mutation of lentiviruses, high production cost and long preparation period for autologous CAR-T design and culture, and insufficient cell number and expansion ability.

Description

technical field [0001] The invention relates to a method for preparing universal CAR-T cells by using CRISPR / Cas9+AAV, which belongs to the field of immune cell therapy. Background technique [0002] Chimeric antigen receptor T cells (chimeric antigen receptor T cells, CAR-T) are T cells that have been genetically engineered to obtain single-chain variable regions (scFv) of monoclonal antibodies to target antigens. CAR-T specifically recognizes antigens on the surface of tumor cells through the principle of antigen-antibody binding, so it is not restricted by the major histocompatibility complex (MHC), and it can also avoid immune escape caused by down-regulation or loss of tumor cell MHC. Moreover, by adding co-stimulatory molecular signals during chimeric gene design, the tumor-killing activity of CAR-T cells can be enhanced. After more than 20 years of development, the design of CAR-T has become more and more perfect, and there are more and more methods. [0003] Accord...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0636C12N15/86C12N2750/14143
Inventor 邱壮伟丘力功符美娟李润明倪世明郝宇
Owner 广州白云山拜迪生物医药有限公司
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