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Method for efficiently knocking chimeric antigen receptor (CAR) genes into T cell specific genome loci through clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas9, and application of method

A genome and gene technology, applied in the field of cell therapy, can solve the problems of CAR-T cell exhaustion, more difficult, cancerous and other problems

Active Publication Date: 2019-12-27
西安桑尼赛尔生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In late 2017, two CAR-T drugs were approved by the US FDA, however, this ACT therapy is still in its early stages
Its disadvantages are: a. Random insertion into the cell genome can easily lead to exhaustion of CAR-T cells and potential cancer risk; b. After infecting T cells, the residual virus has strong immunogenicity
In primary T cells, gene knock-in of large fragments is even more difficult. Studies have shown that the efficiency of gene knock-in with the above-mentioned different forms of repair templates does not exceed 5%, and both plasmid DNA and double-stranded DNA have different degree of cytotoxicity, not suitable for gene knock-in repair templates in T cells

Method used

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  • Method for efficiently knocking chimeric antigen receptor (CAR) genes into T cell specific genome loci through clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas9, and application of method
  • Method for efficiently knocking chimeric antigen receptor (CAR) genes into T cell specific genome loci through clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas9, and application of method
  • Method for efficiently knocking chimeric antigen receptor (CAR) genes into T cell specific genome loci through clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas9, and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1: PBMC extraction

[0099] Recruit healthy volunteers (inconvenience to disclose information), without symptoms of cold and fever. Professional medical personnel take 100ml of blood from the median vein of the human elbow and put it into the BD anticoagulant blood vessel. After blood collection, the blood was mixed with an equal amount of PBS buffer (containing 2% fetal bovine serum). Take the PBMC separation tube Sepmate-50, carefully add 15ml of Ficoll buffer, then add the blood PBS mixture, carefully add about 30ml to each tube. After centrifuging at 1200g for 10 minutes, quickly pour the supernatant into a new 50ml tube, centrifuge at 200g for 8 minutes, discard the supernatant, add 10ml PBS buffer to resuspend the pellet, discard the supernatant, add 10ml PBS buffer to resuspend, centrifuge and discard After clearing, resuspend all pellets in 10ml of supernatant PBS buffer. To count the resuspended cells, take 10 μl of the suspension and add 10 μl of...

Embodiment 2

[0102] Example 2: T cell activation

[0103] Take the above PBMC cells 1×10 7 One, resuspended in 6ml VIVO-15 medium, activated with anti-CD3 / anti-CD28 antibody magnetic beads. Take anti-CD3 / anti-CD28 antibody magnetic beads (Life Technology) 6×10 6 Each was resuspended with PBS buffer (containing 2 mM EDTA and 1% fetal bovine serum), added to a magnetic pole and allowed to stand for 2 minutes, then carefully discarded the supernatant. Repeat the above process 4 times. Take the magnetic beads after washing, add the magnetic beads to PBMC cells, mix well, and culture them at 37 degrees for 3 days. After 3 days, the magnetic beads were taken out, and the T cells were first resuspended several times with a pipette gun. Place the cell suspension in the magnetic pole, and after standing for two minutes, discard the magnetic beads on the tube wall. Count again, and the counting results are shown in Table 2.

[0104] Table 2

[0105] T cell density 1.9×10 6 pcs / ml...

Embodiment 3

[0106] Example 3: Adeno-associated virus vector construction

[0107] In the present invention, the CAR gene is knocked into the TRAC gene locus using the method of CRISPR-Cas9 technology combined with adeno-associated virus vector transduction. Firstly, CRISPR-Cas9 is used to make a cut at the TRAC gene site, and then the CAR gene template delivered by adeno-associated virus is used to repair the gap by homologous recombination, and finally the CAR gene is knocked into the target site. Schematic such as figure 2 .

[0108] (1) The adeno-associated virus vector comprises two parts:

[0109] a. Left and right homology arms. The left and right homology arms are used to identify the target DNA and carry out recombination and exchange. In order to ensure the correct expression of the knock-in gene, the present invention "logs in" the knock-in gene to the 5' end region of exon 1 of the TRAC gene. According to the length of the base sequence, the following pairs of homology arm...

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Abstract

The invention relates to a method for efficiently knocking chimeric antigen receptor (CAR) genes into T cell specific genome loci through clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas9, and application of the method, in particular to a method for accurately knocking the CAR genes into target genome DNAs of T cells. The method comprises the steps: (a) the target genome DNAs are shorn through gene editing substances, wherein the target genome DNAs are located behind a gene promoter region and between initiation codons and exon 3-ends where the initiation codons are located; and (b) the CAR genes are cloned to a repair template vector and then guided into the T cells, the shorn target genome DNAs are repaired by the cells in a homologous recombination mode, thus theCAR genes are embedded, and an adeno-associated virus vector is adopted as the repair template vector. The method can be applied to preparation of the various CAR-T cells.

Description

technical field [0001] The invention relates to the field of cell therapy, in particular to a method for efficiently knocking in a chimeric antigen receptor gene to a specific genomic site of a T cell by CRISPR-Cas9 and its application. Background technique [0002] CAR-T therapy [0003] Traditional tumor treatment drugs include chemotherapy drugs and targeted drugs. Although they have improved the survival period of cancer patients to a certain extent, they have also brought serious side effects and greatly reduced the quality of life of patients. Even more unfortunately, most patients will still relapse after receiving these traditional treatments, and once relapse, there is a high probability that there is no cure. [0004] In recent years, with the development of immunotherapy, the emergence of immune checkpoint inhibitor drugs (such as CTLA-4, PD-1 / PD-L1 antibody) has completely changed the way of tumor treatment. However, the effective rate of such drugs in differe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/864C12N15/113A61K35/17A61P35/00A61P35/02A61P31/18A61P37/02
CPCA61K35/17A61P31/18A61P35/00A61P35/02A61P37/02C07K14/7051C07K2319/02C07K2319/33C12N15/113C12N15/86C12N2310/10C12N2750/14143C12N2310/20
Inventor 彭作翰李玏
Owner 西安桑尼赛尔生物医药有限公司
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