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Gene HDAC11 interfered chimeric antigen receptor T-cell and application thereof

A chimeric antigen receptor, HDAC11 technology, applied in the field of biomedicine, can solve the problems of killing tumors, exhaustion, and weak CAR-T cells

Active Publication Date: 2019-02-05
EAST CHINA NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the therapeutic effect of CAR-T therapy in solid tumors is still unsatisfactory. The main reason is that the killing ability is not strong and CAR-T cells are seriously exhausted in the body and cannot sustainably kill tumors.

Method used

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  • Gene HDAC11 interfered chimeric antigen receptor T-cell and application thereof
  • Gene HDAC11 interfered chimeric antigen receptor T-cell and application thereof
  • Gene HDAC11 interfered chimeric antigen receptor T-cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0221] Example 1 Construction of pLL3.7-HDAC11-shRNA interference vector

[0222] 1.1 RNAi target sequence design

[0223] Find the gene sequence number of Homo sapiens histone deacetylase 11 (HDAC11) on the NCBI website: NM_024827.3. According to the gene number, the HDAC11 gene RNAi target sequence was designed on the website (https: / / rnaidesigner.thermofisher.com / rnaiexpress / design.do), and the results are shown in Table 1.

[0224] Table 1. HDAC11 Gene RNAi Target Sequences

[0225]

[0226] 1.2 Design the interference sequence according to the target sequence:

[0227] Based on the screened target sequence, the interference sequence is designed and determined according to the following principles: the 5-terminus starts with G, and the G+C content is set at 30%-50%. According to the requirements of the pLL3.7 vector, (1) add T to the 5' end of the sense strand to rebuild the T at the 1 position of the U6 promoter. (2) Add Loop "TTCAAGAGA" after the interference targ...

Embodiment 2

[0235] Example 2 Packaging of pLL3.7-shRNA-(D / NC)-(EGFP / CAR19) lentivirus

[0236] 2.1. Plasmid transfection

[0237] 1) Place the plasmid, PEI, and Opti-MEM medium at room temperature for 5 minutes;

[0238] 2) Take 436 μl of Opti-MEM into a 1.5ml EP tube, add 64 μg PEI, mix well, and let stand at room temperature for 5 minutes;

[0239] 3) Take 12 μg vector plasmid, 8 μg psPA×2, 4 μg pMD2.G, add Opti-MEM to 500 μl, and let stand at room temperature for 5 minutes;

[0240] 4) Add the prepared PEI-Opti-MEM solution into the Opti-MEM containing the plasmid, and let stand at room temperature for 20 minutes;

[0241] 5) Slowly drop 1ml of the DNA / PEI mixture into the 293T Petri dish laid out the day before, mix gently, and incubate in a 37°C incubator for 6-8h or overnight;

[0242] 6) Replace with fresh medium, put into 37°C incubator and continue to incubate.

[0243] 2.2. Virus collection and concentration

[0244] 1) After 48 hours of plasmid transfection, collect the su...

Embodiment 3

[0258] Example 3 pLL3.7-HDAC11-shRNA-EGFP carrier interference verification test

[0259] Inoculate Jurkat T in a 6-well plate, 2×10 per well 6 cells in a total of 9 wells. Divided into 3 groups: no virus infection negative control group, pLL3.7-shRNA-D-EGFP lentivirus interference group and pLL3.7-shRNA-NC-EGFP negative interference group, with 2 wells in each group. Corresponding volumes of virus were added to the two experimental groups according to MOI=10:1, and 10 μg / ml polybrene was added to promote infection. After 10 hours, the cells were collected, centrifuged at 1000 g for 10 minutes, the medium was discarded, and fresh medium was added.

[0260] Cells were harvested 48 hours after lentivirus infection, and the expression efficiency of GFP was detected by flow cytometry, that is, the positive rate of virus infection. Results The infection efficiency of pLL3.7-shRNA-D-EGFP and pLL3.7-shRNA-NC-EGFP on Jurkat cells was close to 100%.

[0261] Three groups of Jurkat ...

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Abstract

The invention provides a gene HDAC11 interfered chimeric antigen receptor T-cell and application thereof. Particularly, the invention provides an engineering immune cell. The engineering immune cell comprises (a) a chimeric antigen receptor CAR, wherein the chimeric antigen receptor CAR comprises an antigen bonded structural domain, a transmembrane structural domain and an intracellular structuraldomain, and the antigen bonded structural domain is specifically bonded to a surface antigen of a tumor cell; and (b) an inhibiting molecule for lowering or inhibiting expression activity of an HDAC11 protein. The engineering immune cell provided by the invention has a good tumor killing effect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to HDAC11 gene-interfering chimeric antigen receptor T cells and applications thereof. Background technique [0002] Chimeric antigen receptor T cells (chimeric antigen receptor T cells, CAR-T) therapy, as a new technology in tumor immunotherapy, has achieved great success in the field of anti-tumor, and this therapy is likely to become The mainstream direction of cancer treatment. CAR-T therapy is mainly to modify the patient's T cells in vitro through gene editing technology, so that the surface of T cells expresses a single-chain antibody variable region that can specifically recognize tumor surface antigens, and the activation signal of T cells is connected inside. Therefore, CAR-T cells can specifically recognize and kill tumor cells after being reinfused into the patient's body. At present, CAR-T therapy has achieved very good clinical results in some malignant hematological tumor...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/113A61K35/17A61P35/00A61P35/02
CPCA61P35/00A61P35/02A61K35/17C12N15/113C07K14/7051C07K16/30C07K2319/33C12N2310/14C12N2510/00
Inventor 江文正张红梅何聪刘明耀席在喜
Owner EAST CHINA NORMAL UNIVERSITY
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