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A car-t transgene vector based on replication-deficient recombinant lentivirus 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: 2019-06-21
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 car-t transgene vector based on replication-deficient recombinant lentivirus and its construction method and application
  • A car-t transgene vector based on replication-deficient recombinant lentivirus and its construction method and application
  • A car-t transgene vector based on replication-deficient recombinant lentivirus and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of recombinant lentiviral vector

[0072] 1. Materials

[0073] 1. The lentiviral backbone plasmid pLenti-3G ​​basic, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzymes were purchased from Shiao (Shanghai) Biomedical Technology Co., Ltd. supply;

[0074] 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:

[0075] EF1α-F: 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO.26)

[0076] EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO.27)

[0077] CD8leader-F: 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO.28)

[0078] CD8leader-R: 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO.29)

[0079] VH-F: 5'-CACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATC-3' (SEQ ID NO.30)

[0080] VH-R: 5'-TGTGATC...

Embodiment 2

[0164] Example 2 Concentration and detection of recombinant lentiviral vector

[0165] 1. Purification of recombinant lentiviral vector by ultracentrifugation

[0166] (1) Divide the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g room temperature for 10min, and remove cells and large debris;

[0167] (2) Filter the supernatant with a 0.22 μm-0.8 μm filter;

[0168] (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;

[0169] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0170] (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;

[0171] (6) Place the balanced centrifuge tubes symmetrically in the ult...

Embodiment 3

[0246] Example 3 Functional detection of recombinant lentiviral vectors lvCAR19-CLA, lvCAR19-CLB, and lvCAR19-OLC.

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

[0248] (1) After infecting PBMC cells with recombinant lentiviral vectors lvCAR19-CLA, lvCAR19-CLB, and lvCAR19-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 lvCAR19-CLA, lvCAR19-CLB, and lvCAR19-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 lvCAR19-CLA, lvCAR19-CLB, lvCAR19-OLC and the control ...

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Abstract

The invention discloses a CAR-T transgene vector based on replication defective recombinant lentivirus. The CAR-T transgene vector comprises an original nuclear replicon pUCOri sequence, a resistance gene AmpR sequence containing ampicillin, a virus replicon SV40 Ori sequence, a lentivirus packaging cis element, ZsGreen1 green fluorescent protein, an IRES ribosome binding sequence, a human EF1 alpha promoter , a chimeric antigen receptor of second-generation CAR or third-generation CAR and a regulating element, wherein the original nuclear replicon pUCOri sequence is used for plasmid replication; the resistance gene AmpR sequence is used for massively proliferating target strains; the virus replicon SV40 Ori sequence is used for enhancing replication in eukaryocyte; the lentivirus packaging cis element is used for lentivirus packaging; the ZsGreen1 green fluorescent protein is used for expressing green fluorescent for eukaryocyte; the IRES ribosome binding sequence is used for jointly transcribing and expressing protein; the human EF1 alpha promoter is used for conducting eukaryotic transcription on antigen receptor genes; the chimeric antigen receptor is used for forming the second-generation CAR or the third-generation CAR integrating recognition, transfer and start; the regulating element is used for enhancing expression efficiency of transgenes and used after eWPRE-enhanced type woodchuck hepatitis b virus is transcribed. Besides, the invention further discloses a construction method and application of the vector. By means of the CAR-T transgene vector and the construction method and application of the vector, secretion of cell factors and an in vitro killing effect of CAR-T cells can be remarkably improved, and the clinical treatment effect is remarkable.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a vector, in particular to a CAR-T transgene vector based on a replication-deficient recombinant lentivirus. 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 immunotherapy m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/66A61K48/00A61P35/00A61P35/02
CPCA61K48/00C12N15/66C12N15/86C12N2740/15043
Inventor 祁伟俞磊余宙
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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