A replication-defective recombinant lentiviral car-t transgene vector targeting CD22 and its construction method and application

A technology of recombinant lentivirus and transgenic vector, applied in the field of medical biology

Active Publication Date: 2019-04-30
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 CD22 and its construction method and application
  • A replication-defective recombinant lentiviral car-t transgene vector targeting CD22 and its construction method and application
  • A replication-defective recombinant lentiviral car-t transgene vector targeting CD22 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 provided 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] VL-F: 5'-CACGCCGCCAGGCCGGATATTCAGCTGACCCAGAGC-3' (SEQ ID NO.30)

[0077] VL-R: 5'-GCGTT...

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, and centrifuge at 500g room temperature for 10min to remove cells and large debris;

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

[0165] (3) Take 6 Hitachi 40PA ultracentrifuge tubes, spray the surface with 70% ethanol for disinfection, put them on the ultra-clean table and irradiate them with ultraviolet light for 30 minutes to sterilize. 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 trimmed centrifuge tubes symmetrically in the...

Embodiment 3

[0250] Example 3 Functional detection of recombinant lentiviral vectors lvCAR22-CLA, lvCAR22-CLB, and lvCAR22-OLC.

[0251] 1. Cell-level expression detection of CAR gene:

[0252] (1) After infecting PBMC cells with recombinant lentiviral vectors lvCAR22-CLA, lvCAR22-CLB, and lvCAR22-OLC, collect cells and use RT-PCR to detect CAR mRNA transcription levels to verify CAR gene expression. If CAR mRNA transcription levels increase, This indicates that the transcription level of the CAR gene is successfully expressed;

[0253] (2) After infecting PBMC cells with recombinant lentiviral vectors lvCAR22-CLA, lvCAR22-CLB, and lvCAR22-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;

[0254] (3) Cells were infected with lvCAR22-CLA, lvCAR22-CLB, lvCAR22-OLC and the control virus MOC...

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Abstract

The invention discloses a CD22-taregted replication-defective recombinant lentivirus CAR-T transgenic vector. The vector comprises a prokaryotic replicon pUC Ori sequence for plasmid replication, an ampicillin-containing resistance gene AmpR sequence for mass amplification of a target strain, a virus replicon SV400ri sequence for enhancing replication within eukaryotic cells, a lentivirus packaging cis element for lentivirus packaging, ZsGreen1 green fluorescence protein for expression of green fluorescence of the eukaryotic cells, an IRES ribosome binding sequence for common transcriptional expression of the protein, a human EF1[alpha] promoter for eukaryotic transcription of a chimeric antigen receptor gene, a chimeric antigen receptor for constituting the second or the third generation of CAR integrating identification, transferring and promoting, and an eWPRE element for enhancing a transgenic expression efficiency. In addition, the invention also discloses a construction method and an application of the vector. The vector disclosed by the invention can significantly improve secretion of cell factors and an in vitro killing effect of CAR-T cells; and the vector is obvious in effect on the clinical treatment of precursor B-cell acute lymphocytic leukemia.

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 CD22. 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, multiple scientific research groups have discovered tumor antigens (tμmor antigens), which can be recognized by T lymphocytes in a major histocompatibility complex (MHC)-dependent manner. [0003] Tumor immunotherapy is generally divided into two categories, nonspecific immunity and specific immunity. Non-specific immunotherapy ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867A61K35/17A61P35/02
CPCA61K35/17C12N15/86C12N2740/15043C12N2800/107
Inventor 祁伟俞磊林高武
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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