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A replication-defective recombinant lentiviral car-t transgene vector targeting CD30 and its construction method and application

A technology of recombinant lentivirus and transgenic vector, which is applied in the field of medical biology and achieves remarkable curative effect

Active Publication Date: 2019-04-02
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is worth noting that the above differences are only the conclusions obtained from in vitro experiments, and there is no report comparing the second-generation and third-generation CARs in vivo.

Method used

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  • A replication-defective recombinant lentiviral car-t transgene vector targeting CD30 and its construction method and application
  • A replication-defective recombinant lentiviral car-t transgene vector targeting CD30 and its construction method and application
  • A replication-defective recombinant lentiviral car-t transgene vector targeting CD30 and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction of recombinant lentiviral vector

[0069] 1. Materials

[0070] 1. The lentiviral backbone plasmid pLenti-3G ​​basic, the lentiviral packaging plasmids pPac-GP, pPac-R and the membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzyme were supplied by Shiao (Shanghai) Biomedical Technology Co., Ltd. supply;

[0071] 2. Primers: According to the principles of primer design, the primers required for amplifying DNA fragments and target sites are designed. The primers are synthesized by Shanghai Biological Company, specifically:

[0072] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO.26)

[0073] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.27)

[0074] CD8 leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO.28)

[0075] CD8 leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.29)

[0076] VH-F: 5'-CACGCCGCCAGGCCGCAGGTACAGCTGCAGCAGTCA-3' (SEQ ID NO.30)

[0077] VH-R: 5'-ACTCGA...

Embodiment 2

[0161] Example 2 Concentration and detection of recombinant lentiviral vector

[0162] 1. Purification of recombinant lentiviral vector by ultracentrifugation;

[0163] (1) Divide the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g room temperature for 10min, and remove cells and large debris;

[0164] (2) Filter the supernatant with a 0.22 μm-0.8 μm filter;

[0165] (3) Take 6 Hitachi 40PA ultracentrifuge tubes, spray 70% ethanol on the surface to sterilize them, put them on a clean table and irradiate them with ultraviolet light for 30 minutes to sterilize them. It can also be sterilized by high temperature and moist heat;

[0166] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0167] (5) Cover the metal cover, balance the centrifuge tube together with the metal cover, and adjust with 1XPBS to make the weight deviation within 0.02g;

[0168] (6) Place the balanced centrifuge tubes symmetrically in the ul...

Embodiment 3

[0246] Example 3 Functional detection of recombinant lentiviral vectors lvCAR30-CLA, lvCAR30-CLB, and lvCAR30-OLC.

[0247] 1. Cell-level expression detection of CAR gene:

[0248] (1) After infecting PBMC cells with recombinant lentiviral vectors lvCAR30-CLA, lvCAR30-CLB, and lvCAR30-OLC, collect the cells and detect CAR mRNA transcription levels by RT-PCR to verify the expression of CAR genes. If the CAR mRNA transcription levels increase, This indicates that the transcription level of the CAR gene is successfully expressed;

[0249] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCAR30-CLA, lvCAR30-CLB, and lvCAR30-OLC, collect the cells and detect the expression level of CAR protein by western blot to verify the expression of CAR gene. If the expression level of CAR protein increases, then It shows that the translation level expression of CAR gene is successful;

[0250] (3) Infect the cells with lvCAR30-CLA, lvCAR30-CLB, lvCAR30-OLC and the control ...

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Abstract

The invention discloses a CD 30-targeting replication-defective recombinant lentivirus CAR-T transgenic vector. The CD 30-targeting replication-defective recombinant lentivirus CAR-T transgenic vector comprises a prokaryotic replicor pUC Ori sequence used for plasmid duplication; an ampicillin resistance gene AmpR sequence used for amplification of a large number of target strains; a virus replicor SV40 Ori sequence used for enhancing the replication in eukaryocytes; a lentivirus packaging cis element used for lentivirus packaging; ZsGreen 1 green fluorescent protein used for green fluorescence expression of the eukaryocytes; an IRES ribosome binding sequence used for co-transcription and co-expression of protein; a human EF1 alpha promoter used for eukaryotic transcription of genes of a chimeric antigen acceptor; the chimeric antigen acceptor used for forming second-generation CAR or third-generation CAR integrating recognition, delivery and promoting; and an eWPRE element used for enhancing the expression efficiency of transgenes. In addition, the invention further discloses a construction method and applications of the vector. With the vector, the secretion of the cell factors and the in-vitro lethal effect of the CAR-T cells can be obviously improved, and the effect in treating hodgkin lymphoma or non-hodgkin lymphoma clinically is outstanding.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a carrier, in particular to a CAR-T transgene carrier of a replication-deficient recombinant lentivirus targeting CD30. In addition, the present invention also relates to the construction method and application of the carrier. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). This biological process is complex and is still under investigation. In the 1990s, several scientific research groups have discovered tumor antigens (tμmor antigens), and T lymphocytes can recognize these tumor antigens in a major histocompatibility complex (MHC)-dependent manner. [0003] Tumor immunotherapy is generally divided into two categories, nonspecific immunity and specific immunity. Nonspecific immu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867A61K35/17A61P35/00
CPCA61K35/17C12N15/86C12N2740/15043C12N2800/107
Inventor 祁伟俞磊欧黄思
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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