Lentivirus used for preparing CART cells and having characteristics of efficient transfection capacity and biological activity

A wild-type virus and cell technology, applied in the field of lentivirus, can solve the problems of complex immunotherapy process, high cost and high cost

Active Publication Date: 2015-09-16
SHANGHAI JI KAI GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether you make it yourself or buy a commercial product, the cost is very high
[0009] 2. The operation is cumbersome
However, the current common lentivirus infection efficiency of T lymphocytes is not high, and the infection efficiency is closely related to the activation scheme of T lymphocytes
The current solutions to improve the efficiency of lentivirus infection include superinfection of T lymphocytes, centrifugation during infection, and addition of pro-infection reagents such as Retronectin, but such operations have a greater impact on the state of cells and will affect the subsequent proliferation and expansion of cells. Multiplication requires special instrument support, and the operation is cumbersome
[0013] The existing immunotherapy process is complex and expensive, so those skilled in the art are committed to developing technical solutions that can simplify the treatment process and reduce treatment costs

Method used

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  • Lentivirus used for preparing CART cells and having characteristics of efficient transfection capacity and biological activity
  • Lentivirus used for preparing CART cells and having characteristics of efficient transfection capacity and biological activity
  • Lentivirus used for preparing CART cells and having characteristics of efficient transfection capacity and biological activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1: Construction of recombinant lentiviral vector

[0159] The schematic diagrams of the recombinant lentiviral vectors αCD3-VSVG and αCD28-VSVG have been described above.

[0160] The construction of the recombinant lentiviral vector is based on the viral packaging helper plasmid H2 (purchased from Addgene, namely pVSV-G) carrying the VSVG gene.

[0161] Construction of αCD3-VSVG recombinant lentiviral vector:

[0162] a) Obtaining the αCD3 gene fragment:

[0163] Whole gene synthesis signal peptide-6His-αCD3-flexible peptide fragment (the nucleotide sequence information of the signal peptide is SEQ ID NO.:8, the nucleotide sequence information of 6His is SEQ ID NO.:9, the nucleotide sequence information of αCD3 The nucleotide sequence information of the flexible peptide is SEQ ID NO.: 3, and the nucleotide sequence information of the flexible peptide is SEQ ID NO.: 10). Using the whole gene synthesis plasmid as a template, the αCD3 gene primer was designed, a...

Embodiment 2

[0209] Example 2: Recombinant lentivirus packaging and preliminary verification

[0210] 1. Supply HEK-293T cells (purchased from ATCC) according to the 24h passage cycle, change the medium before transfection, and use an electric pipette to replace 5ml of DMEM medium containing 2% FBS for each plate of cells.

[0211] 2. Add HBW buffer, plasmid H1 (purchased from Addgene, 12 μg / plate, plate refers to 10cm cell culture dish), recombinant H2 plasmid (10 μg / plate), vector plasmid (lentiviral vector containing exogenous genes) in sequence , purchased from Addgene, 24 μg / plate), CaCl2 (50 μl / plate), and finally 2×HBS (500 μl / plate) was added dropwise while shaking on a vortex shaker, and the transfection system was 1 ml / plate. The vector plasmid is a vector expressing EGFP initiated by EIF1α, and the recombinant H2 is a mixture of αCD3-VSVG, αCD28-VSVG and wild-type H2. Among them, αCD3-VSVG and αCD28-VSVG are added at a ratio of 1:1, and wild-type H2 is matched in different rati...

Embodiment 3

[0216] Example 3: Recombinant lentivirus infection of T lymphocytes

[0217] Infection experiments were performed according to conventional methods known to those skilled in the art. A brief description of the infection steps is as follows:

[0218] 1. Peripheral blood mononuclear lymphocytes (PBMC) were obtained, and >1x107 cells were obtained through the blood apheresis system.

[0219] 2. Use complete medium (TexMACS medium + 5% autologous serum) to adjust part of the cell suspension to make the final concentration 0.7 × 106 / ml, and add interleukin 2 (IL-2), penicillin (penicillin) and streptavidin streptomycin and bovine serum albumin (BSA), so that the final concentrations were 600IU / ml, 100U / ml, 100μg / ml and 0.2%.

[0220] 3. Gently mix the resuspended cells, take out 0.4ml of cell suspension (total cell volume 0.28×106) and transfer to a 1.5mL centrifuge tube.

[0221] 4. Add the required virus, and calculate the amount of virus added according to the MOI of 10.

[...

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Abstract

The present invention provides lentivirus used for preparing CAR-T cells and having characteristics of efficient transfection capacity and biological activity. According to the present invention, the lentivirus can be used as the vector of the exogenous gene in CAR-T lymphocyte therapy and can infect T lymphocytes at the high efficiency to transform the exogenous gene into the T lymphocytes so as to carry out genome modification on the T lymphocytes while activate the T lymphocytes during the process, such that the tumor immunotherapy process is substantially simplified.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, in particular, the invention relates to a lentivirus with high-efficiency transfection ability and biological activity for preparing CART cells. Background technique [0002] Cancer has always been a huge problem plaguing human beings. According to the official website of the World Health Organization, the number of newly discovered tumor patients in the world will increase from 10 million to 15 million every year. The "2013 China Cancer Registration Annual Report" released in early 2014 shows that there are about 3.12 million new cancer cases in my country every year, with an average of 8,550 people per day, which means that 6 people are diagnosed with malignant tumors every minute. It is a big killer that threatens health. [0003] Over the years, the main methods of cancer treatment are surgery, chemotherapy and radiotherapy, and the effective rate of clinical treatment for tumor patients ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N15/861C12N5/10C12R1/93
Inventor 曹跃琼王庆亮胥建源李军朱向莹
Owner SHANGHAI JI KAI GENE TECH CO LTD
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