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Method of knocking out T cell immunity detecting point pathway genes and application

A gene knockout and cellular immunity technology, applied in the field of genetic engineering, can solve the problems of expensive antibody drugs, imbalanced immune tolerance, and no immune response at immune checkpoints, and achieves high safety and a simple and easy method. , effective effect

Inactive Publication Date: 2018-12-07
SUZHOU MAXIMUM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The problems existing in the immunodetection and blockade treatment of tumors with existing clinical blocking antibodies: (1) The effect of antibodies is only a temporary blocking effect; (2) There are many kinds of inhibitory receptors, how to use multiple antibodies to block at the same time (3) It is not easy to develop effective antibodies; (4) Only for extracellular targets; (5) Antibody drugs are expensive; (6) The overall remission rate of inhibitors is relatively low; (7) ) Long-term application of immune checkpoint inhibitors can unbalance immune tolerance and lead to no checkpoint immune response at immune checkpoints, etc.

Method used

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  • Method of knocking out T cell immunity detecting point pathway genes and application
  • Method of knocking out T cell immunity detecting point pathway genes and application
  • Method of knocking out T cell immunity detecting point pathway genes and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 sgRNA design and construction

[0066] (1) sgRNA primer pair design

[0067] Select the target DNA region of the T cell immune checkpoint pathway gene. The T cell immune checkpoint pathway genes are: cytotoxic T lymphocyte-associated protein (T-lymphocyte antigen-4, CTLA-4), programmed death receptor-1 (programmed death-1, PD-1), lymphocyte activation gene-3 (Lymphocyte activation gene-3, LAG-3), T-cell immunoglobulin and mucin-3 (T-cell immunoglobulin and mucin-3, TIM- 3), according to the selected target sequence, a pair of sgRNA was synthesized for each target gene, the sequence is as follows:

[0068]

[0069]

[0070] (2) Denature and anneal each pair of sgRNAs in the above table

[0071] The annealing system is:

[0072]

[0073] Run in the PCR machine according to the following landing PCR program: 95°C, 5min; 95-85°C at-2°C / s; 85-25°C at-0.1°C / s; 4°C, save.

[0074] (3) Linearization of pGL3-U6-sgRNA plasmid

[0075] Enzyme digestion syst...

Embodiment 2

[0085] Example 2 Functional verification of sgRNA

[0086] (1) Cell culture and transfection

[0087] 1) HEK293T cells were inoculated and cultured in DMEM high-glucose medium (HyClone, SH30022.01B), which contained 10% FBS, penicillin (penicillin, 100 U / ml) and streptomycin (streptomycin, 100 μg / ml);

[0088] 2) Inoculate HEK293T cells in a 12-well plate before transfection, and when the cells grow to 70%-80% density of the 12-well plate, replace with antibiotic-free medium to prepare for transfection;

[0089] 3) According to Lipofectamine TM 2000 Transfection Reagent (Invitrogen, 11668-019) operating manual, mix 0.5 μg pGL3-U6-hPD1sg1 and 1.5 μg pST1374-h13Lb-cpf1 plasmid, co-transfect into cells in each well, change the medium after 6 hours, Add blasticidin (Blasticidin, invitrigen, ant-bl-1) and puromycin (Puromycin, invitrigen, ant-pr-1) drug sieve, and collect the cells after 48 hours.

[0090] (2) T7EN1 enzyme digestion detection

[0091] 1) Extract DNA from the c...

Embodiment 3

[0098] Example 3 Preparation of PBMC cells

[0099] Use an anticoagulant tube to collect peripheral blood from healthy people, and shake it while collecting to fully mix the peripheral blood with the anticoagulant; slowly add the anticoagulant blood to a 50ml centrifuge tube filled with an equal volume of lymphocyte separation medium (Ficoll), 450g, Slowly ascend and descend slowly for 25 minutes, and the centrifugation cannot be stopped in the middle. After the centrifugation, carefully absorb the buffy coat cells above the lymphocyte separation solution, transfer to a new 50ml centrifuge tube, add PBS, 300g, and slowly ascend and descend for 10 minutes. , discard the supernatant, and keep the cell pellet at the bottom of the centrifuge tube; add PBS again, 160g, centrifuge slowly for 15min, discard the supernatant; finally add PBS, 300g, centrifuge slowly for 10min, discard the supernatant, and obtain PBMC cell.

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Abstract

The invention provides a method of knocking out T cell immunity detecting point pathway genes by using CRISPR-Cpf1 / sgRNA rapidly, simply, efficiently and specifically. The method effectively solves the defects occurring when a CRISPR-Cas9 system knocks out PD-1, CTLA-4, LAG-3 and TIM3 genes. The method comprises the following specific steps: identifying a target DNA region of T cell immunity detection point pathway genes, designing an sgRNA primer pair according to the selected target DNA region, and specifically and directionally knocking out the T cell immunity detection point pathway genesby using the CRISPR-Cpf1 system. The method is simple and practicable, and has relatively good effectiveness on knock-out of T cells through an immunity detection point and relatively high safety.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering; in particular, it relates to the transformation of T lymphocytes; more specifically, it relates to the preparation of immune checkpoint blocking T cells by using the CRISPR-Cpf1 system, the prepared T cells and applications thereof. technical background [0002] The morbidity and mortality of malignant tumors remain high, and they are the number one killer threatening human health. Finding effective tumor treatment methods and completely conquering tumors are important research topics in the world's medical community. In recent years, with the development of genetic engineering technology, the deepening of understanding of immune regulation mechanism, and the support of more experimental data of anti-tumor immunomodulatory drugs, immune checkpoint therapeutic drugs have become a new means of fighting tumors. [0003] Immune checkpoint blockade has become a research hotspot in cellular...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0636C12N15/85C12N2510/00
Inventor 刘慧莹许先进
Owner SUZHOU MAXIMUM BIO TECH CO LTD
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