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Recombinant retrovirus for primary T cell infection, infection method and application thereof

A retrovirus and cell infection technology, applied in the field of recombinant retroviruses for primary T cell infection, can solve the problems of low feasibility, no means of delivery, hindered development, etc., and achieve a simple activation method, good infection effect, and streamlined operation. Effect

Active Publication Date: 2020-12-29
汉恒生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used vectors include viral vectors and non-viral vectors (Non-Viral Vectors). Although non-viral vectors have advantages over viral vectors in terms of immunogenicity, they not only have to overcome intracellular and extracellular barriers, but also overcome many barriers from The challenge of improving transfection efficiency, secondly, because there is no universal delivery method, the current feasibility is very low, and viral vectors have absolute advantages in achieving gene transduction and expression efficiency, and are currently the mainstream vectors for gene therapy
[0004] Due to the biological characteristics of T cells, the efficiency of virus infection to achieve foreign gene expression is very low. Based on continuous research, more and more research and development work requires gene editing of primary T cells. At present, there is no international consensus. Mature products can efficiently infect primary T cells and ensure simple operation, which severely limits and hinders the development of related research work

Method used

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  • Recombinant retrovirus for primary T cell infection, infection method and application thereof
  • Recombinant retrovirus for primary T cell infection, infection method and application thereof
  • Recombinant retrovirus for primary T cell infection, infection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: preparation kit

[0049] (1) Preparation of HB-Tmax2.0-M / Hplate24

[0050] a) Dilute the anti-CD3 and anti-CD28 antibodies to 8ug / ml with pH 7.4 PBS buffer, add 500ul diluted antibody solution to each well of a 24-well plate with no surface treatment, and coat at 4°C for 14h;

[0051] b) Discard the antibody coating solution, block the 24-well plate with 1% BSA in PBS buffer, and block at room temperature for 45 minutes;

[0052] c) Discard the blocking solution, dry it at 37°C for 3 hours;

[0053] d) Put it into an aluminum foil bag, add a desiccant, seal it and label it, and store it at 4°C.

[0054] (2) Assembly:

[0055] Assemble the recombinant retrovirus (HB-RV3) and HB-Tmax2.0-M / Hplate24 described in the present invention into a cassette. The virus needs to be stored at -80°C, and the remaining components are stored at 2- 8°C.

[0056] The HB-RV3 was obtained by using HB-RV mutations.

Embodiment 2

[0057] Example 2: Infection of mouse primary T cells

[0058] a) Isolation of mouse primary T cells ( Untouched TM Mouse CD4Cells Kit, 11416D, thermo), resuspended cells with complete medium containing 200U / ml rmIL-2 (R&D, 402-ML-020), 2*10 5 Every hole of each cell is paved with HB-Tmax2.0-Mplate24 (prepared in Example 1), the volume of the culture medium is maintained at 2ml, and 5% CO is put into 2 Incubator, activate at 37°C for 48h.

[0059] b) To observe the increase in the volume of activated cells, suck off 1.5ml of the medium and leave about 500ul of the medium and resuspend the cells by blowing, counting, and the number becomes 60% of the original, that is, 1.2*10 per well 5 Activated cells, add HB-RV3 virus, according to MOI25, that is, add 30ul of HB-RV3 virus per well, the virus titer is 1*10 8 TU / ml, add the virus and mix by pipetting.

[0060] c) Centrifuge the plate at 2000g for 2h at room temperature.

[0061] d) Return to 5% CO after centrifugation 2...

Embodiment 3

[0064] Example 3: Infection of human primary T cells

[0065] a) Isolation of human primary T cells ( Untouched TM Human CD4Cells Kit, 11352D, thermo), resuspended cells with complete medium containing 200U / ml rhIL-2 (R&D), 2*10 5 Spread HB-Tmax2.0-Hplate24 for each cell, keep the volume of the culture medium at 2ml, put in 5% CO 2 Incubator, activate at 37°C for 48h.

[0066] b) Observe the increase in the volume of activated cells, suck off 1.5ml of the medium, leave about 500ul of medium, and blow the cells to resuspend, count, and the number becomes 70% of the original, that is, 1.4*10 per well 5 For activated cells, add HB-RV3 virus, according to the MOI50, add the virus and mix well by pipetting.

[0067] c) Centrifuge the plate at 2000g for 2h at room temperature.

[0068] d) After centrifugation, put it back into the 5% CO2 incubator, infect at 37°C for 12 hours, discard 70% of the supernatant after 12 hours, add a complete medium containing 200U / ml rhIL-2 to ma...

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Abstract

The invention provides a recombinant retrovirus for primary T cell infection, an infection method and an application of the recombinant retrovirus. The retrovirus sequentially comprises a long-chain repetitive sequence coding area, a packaging signal coding area, a promoter coding area and a puro gene coding area from a 5' end to a 3' end, the sequence of nucleotide codes of a (786-788) bit of the packaging signal coding area is ATA, the sequence of nucleotide codes of an (829-831) bit of the packaging signal coding area is GCC, and the sequence of nucleotide codes of a (70-72) bit of the promoter coding area is TGC. By the primary T cell infection method, the recombinant retrovirus for infection can efficiently infect human and mouse primary T cells and is high in infection efficiency.

Description

technical field [0001] The invention relates to a recombinant retrovirus for primary T cell infection, an infection method and application thereof, especially for transfecting T cells. Background technique [0002] Cancer Immunotherapy is one of the most promising methods for treating malignant tumors. Tumor immunotherapy is the application of immunological principles and methods. It can stimulate and enhance the body's anti-tumor immune response by improving the immunogenicity of tumor cells and enhancing the killing ability of effector cells. It can also infuse the host with immune cells and effector molecules. Cooperate with the body's immune system to kill tumors and inhibit tumor growth. The current chimeric antigen receptor T-cell immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-T) and T-cell receptor (TCR) chimeric T cells (TCR-T) as adoptive cell reinfusion therapy (Adoptive Cellular Therapy (ACT) technology, two of the latest immune cell technolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/861A61K48/00A61P35/00A61P35/04
CPCA61K48/005C12N7/00C12N15/86C12N2740/10021C12N2740/10043
Inventor 陈意雄曹元元蔡晓龙邹杰
Owner 汉恒生物科技(上海)有限公司
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