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Method for preparing cells over-expressing exogenous genes

A technology of exogenous genes and cells, applied in the field of preparing cells overexpressing exogenous genes, can solve the problems of low transfection efficiency, high death rate of immune effector cells, excessive cell death, etc.

Active Publication Date: 2020-05-08
SHANGHAI CELL THERAPY GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its existing problems are also relatively obvious: it is easy to cause excessive cell death under instantaneous high voltage, and the transfection efficiency is low, which is specifically related to the cell type and electroporation conditions (including voltage, waveform, pulse time and composition of electroporation buffer)
Especially for immune effector cells, such as PBMC and TIL cells, electrotransfer is relatively more difficult. Although there are relatively mature electrotransfer instruments and supporting buffer systems on the market, the problem of high mortality after electrotransfer of immune effector cells is still relatively difficult. protrude

Method used

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  • Method for preparing cells over-expressing exogenous genes
  • Method for preparing cells over-expressing exogenous genes
  • Method for preparing cells over-expressing exogenous genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Isolation and culture of liver cancer tissue-derived TIL cells

[0058] Collect freshly resected HCC specimens and process them immediately under sterile conditions. The specific method is as follows: remove the normal tissue and necrotic area around the liver cancer specimen, and remove the 1-2mm in size from different areas of the specimen 3 Place a small tissue block in each well of a 24-well plate. Add 2 mL of complete medium (GT-T551 medium containing 10% FBS) and 3000 IU / mL IL-2 to each well. Place the 24-well plate at 37 °C, 5% CO 2 cultured in an incubator. On the 5th to 6th day after the initiation of culture, a half-volume medium change was performed for all wells. Afterwards, according to the growth of TILs, a half-volume medium change was performed every 1-2 days. Once the wells were overgrown with TILs and all adherent cells had been removed, the TILs in each overgrown well were collected.

Embodiment 2

[0059] Embodiment 2: Construction of recombinant expression vector

[0060] The coding sequence of EGFP was artificially synthesized and loaded between the EcoRI and SalI restriction sites of vectors pNB328 and pS328 based on the PB transposon system (see CN201510638974.7 for the structure and sequence of pNB328, the entire contents of which are cited herein Incorporated in this article; compared with pNB328, pS328 lacks the PB transposon sequence; the EGFP coding sequence is shown in SEQ ID NO:9 in CN201510638974.7), and the constructed recombinant expression vectors were named pNB328-EGFP and pS328-EGFP respectively .

[0061] The coding sequence of PB transposase is artificially synthesized, and it is loaded between the EcoRI and SalI restriction sites of pS328 based on the PB transposon system. The coding sequence of PB transposase is as SEQ ID NO: 5 in CN201510638974.7 As shown, the constructed recombinant expression vector was named pS328-PB.

Embodiment 3

[0062] Example 3: Preparation of TIL expressing EGFP by electroporation of pNB328-EGFP (TIL-single transfection plasmid)

[0063] Follow the steps below to electroporate TIL-single transfection expressing EGFP:

[0064] 1) Add AIM-V medium to 3 wells in a 12-well plate in advance, numbered a, b, c, 2 mL per well, and then transfer to a cell culture incubator at 37°C 5% CO 2 Preheat for 1 hour;

[0065] 2) According to the table below, the ratio of the electrotransfer solution for a single dosage per well:

[0066] 100 μL Nucleocuvette TM Strip (μL)

Nucleofector TM volume of solution

82 Electroporation Supplementary Solution 18

[0067] 3) Get the TIL obtained in Example 1 into 3 EP tubes, and add 5×10 6 centrifuge at 1200rpm for 5min, discard the supernatant, then resuspend the cells with 500μL normal saline, and repeat the centrifugation step to wash the cell pellet;

[0068] 4) Add 6 μg of plasmid pNB328-EGFP to the electrotransfer sol...

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Abstract

The invention relates to a method for preparing cells overexpressing exogenous genes. According to the invention, a STING signal channel inhibitor, especially carbonyl cyanide 3-chlorophenylhydrazone,is utilized to culture electroporated cells, especially electroporated immunologic effector cells, so the survival rate and total number of cells after electroporation can be obviously improved.

Description

technical field [0001] The present invention relates to the cultivation of electroporated cells, in particular to a method for preparing cells overexpressing exogenous genes. Background technique [0002] Adoptive cell therapy (ACT) using immune effector cells expressing chimeric antigen receptors (CAR), such as CAR-T cells, for the treatment of tumors has the potential to permanently change the status quo of cancer treatment. Such treatments rely on efficient, stable and safe gene transfer platforms. The introduction of synthetic genes encoding chimeric antigen receptors into immune effector cells, such as T cells, is the first step towards tumor therapy. Gene transfer techniques mainly include viral methods and non-viral methods. Viral methods mainly include using retroviral vectors or lentiviral vectors to express CAR genes, introducing CAR genes into immune effector cells through packaged virus particles, and integrating into the cell genome through the integration sys...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
CPCC12N5/0636C12N15/85C12N2510/02C12N2500/30
Inventor 金华君徐飞黄晨刘祥箴钱其军
Owner SHANGHAI CELL THERAPY GRP CO LTD
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