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mRNA for coding CAR gene, combined mRNA, construction method, CAR-T cell and application

A construction method and encoding technology, applied in the field of genetic engineering, can solve the problems of lack of target antigen, microenvironment, immune escape, etc., and achieve the effects of easy storage and transportation, treatment of malignant tumors, and simplified procedures

Active Publication Date: 2021-03-12
SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the achievements of CAR-T therapy in the treatment of malignant hematological tumors are obvious to all, such as Kymriah for refractory / relapsed acute B-lymphoblastic leukemia and refractory / relapsed non-Hodgkin's Yescarta for lymphoma was launched in the United States last year, but there will be a recurrence rate of at least 50%, and the effect of CAR-T cells on solid tumors is not good, mainly due to the lack of suitable target antigens and the duration of CAR-T cells in the body Short, immune escape, immunosuppressive tumor microenvironment, etc.

Method used

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  • mRNA for coding CAR gene, combined mRNA, construction method, CAR-T cell and application
  • mRNA for coding CAR gene, combined mRNA, construction method, CAR-T cell and application
  • mRNA for coding CAR gene, combined mRNA, construction method, CAR-T cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of CAR-T cells

[0043] PBMC cells were isolated from the peripheral blood of healthy donors, cultured in AIM-V medium added with IL2 (50U / ML), CAR gene mRNA and cytokine mRNA according to seq1, seq2KY, seq3 YE, seq4, seq4+ 5. Cells were transfected with seq4+6, seq4+7+8, and seq4+9 respectively. After the cell culture was completed, the samples were taken and packed into sampling tubes for quality control testing.

[0044] The mRNA encoding the CAR gene, the nucleotide sequence is shown in SEQ ID No.1, referred to as seq1;

[0045] The mRNA encoding the CAR gene, the nucleotide sequence is shown in SEQ ID No.2, referred to as seq2 KY;

[0046] The mRNA encoding the CAR gene, the nucleotide sequence is shown in SEQ ID No.3, referred to as seq3 YE;

[0047] The mRNA encoding the CAR gene, the nucleotide sequence is shown in SEQ ID No.4, referred to as seq4;

[0048] The mRNA combination for preventing and treating tumors includes the mRNA encoding the CAR ...

Embodiment 2

[0069] In vitro killing experiments were performed on CAR-T cells made from peripheral blood. The specific experimental steps are as follows:

[0070] Step 1: Preparation of CAR-T cells

[0071] As in Example 1.

[0072] Step 2: Calcein-AM labeling target cells

[0073] 1) Dilute Calcein-AM to 1 mg / mL with DMSO;

[0074] 2) The human lymphoma cell line Daudi was resuspended into 1×10 6 Density / mL;

[0075] 3) Add 15ul of Calcein-AM, 37°C, 5% CO 2 Incubate for 30 minutes, and mix gently every 10 minutes;

[0076] 4) Centrifuge at 1500rpm, remove the supernatant, resuspend with medium, repeat twice;

[0077] Step Three: Kill

[0078] 1) Resuspend the labeled human lymphoma cell line Daudi at a density of 5000-50000 cells / mL, and add 100ul into a 96-well plate;

[0079] 2) Add 100 μl of CAR-T cells according to an appropriate ratio, so that the number ratios of CAR-T cells and cancer cells are 50:1, 25:1, 12.5:1, 6:1 and 3:1, 3 in each group Parallel; at the same time, t...

Embodiment 3

[0088] After the transfection of the CAR gene, the release of immune factors by T cells under the stimulation of the target cells was detected.

[0089] The CAR-T cells prepared in Example 1 were co-cultured with the human lymphoma cell line Daudi, and the supernatant and cell pellet were collected after 24 hours.

[0090] The human IFNγ detection kit (BD) was used in the supernatant to perform an ELISA test to detect the IFNγ content released by CAR-T cells after target cell stimulation. Cell pellets were detected by flow cytometry for endogenous cellular immune factors, and the antibodies used were allophycocyanin(APC)-Cy7-conjugated mAb to human CD8, PerCP-Cy5.5–conjugated mAb to human CD4, V450-conjugated mAb to human IFNg, PE-Cy7–conjugated antitumor necrosis factor (TNF) mAb, and flfluorescein isothiocyanate (FITC)-conjugated mAb to human IL2 (BD Biosciences). The result is as Figure 3-6 And shown in Table 3, 4, 5.

[0091] Table 3 The content of immune factor IFNγ exc...

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Abstract

The invention provides mRNA for coding a CAR gene, combined mRNA, a construction method, a CAR-T cell and application, which belong to the technical field of gene engineering. The nucleotide sequenceof the mRNA is selected from any one of SEQ ID No.1 to 4; the mRNA for encoding the CAR gene and the mRNA for encoding the cytokine, and the nucleotide sequence of the mRNA for encoding the cytokine is selected from one or more of SEQ ID No.5 to 9. The CAR-T cell constructed by the mRNA for encoding the CAR gene and the combined mRNA can express the CAR gene and has a killing effect on tumors.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an mRNA encoding a CAR gene, a combined mRNA, a construction method, CAR-T cells and applications. Background technique [0002] With the continuous deterioration of global environmental factors, the incidence and mortality of malignant tumors are on the rise in both developed and developing countries, and malignant tumors have become the greatest threat to human life and health. Due to the inherent defects of traditional treatment methods and the rapid development of global high-tech biotechnology, people have been expecting the emergence of new treatments for a long time. As a brand-new treatment concept that has just emerged, biological therapy has its unique advantages including selectively targeting and killing tumor cells, and has no toxic effect on normal cells. Biological therapy in the treatment of cancer can significantly improve the clinical sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/19C12N5/10A61K39/00A61P35/00
CPCC07K14/7051C07K14/52C12N5/0636A61K39/001136A61P35/00C12N2510/00C07K2319/00A61K2039/804
Inventor 张苗苗洪丹胡迅
Owner SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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