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CD22-taregted replication-defective recombinant lentivirus CAR-T transgenic vector as well as construction method and application thereof

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

Active Publication Date: 2016-09-21
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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  • CD22-taregted replication-defective recombinant lentivirus CAR-T transgenic vector as well as construction method and application thereof
  • CD22-taregted replication-defective recombinant lentivirus CAR-T transgenic vector as well as construction method and application thereof
  • CD22-taregted replication-defective recombinant lentivirus CAR-T transgenic vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction of recombinant lentiviral vector

[0069] 1. Material

[0070] 1. Lentiviral backbone plasmid pLenti-3G ​​basic, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein particles pEnv-G, HEK293T / 17 cells, homologous recombinase from Shiao (Shanghai) Biomedical Technology Co., Ltd. provide;

[0071] 2. Primers: Design primers needed to amplify DNA fragments and target sites according to the principle of primer design. 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] CD8leader-F: 5’-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3’ (SEQ IDNO.28)

[0075] CD8leader-R: 5’-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3’ (SEQ ID NO.29)

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

[0077] VL-R: 5'-GCGTTTAATTTCCACTTTGGTG-3' (SEQ ID NO.31)

[0078] CLA-VH-F: 5’-AGTGGAAATTAAACGC GG...

Embodiment 2

[0160] Example 2 Concentration and detection of recombinant lentiviral vector

[0161] 1. Purification of recombinant lentiviral vector by ultracentrifugation;

[0162] (1) Dispense the collected supernatant into 50ml centrifuge tubes, centrifuge at 500g for 10min at room temperature to remove cells and large debris;

[0163] (2) Filter the supernatant with a 0.22μm-0.8μm filter;

[0164] (3) Take 6 Hitachi 40PA ultracentrifuge tubes, spray 70% ethanol on the surface for disinfection, and place them on the ultra-clean table and irradiate them with ultraviolet light for 30 minutes. It can also be sterilized by high temperature, humidity and heat;

[0165] (4) Aliquot 32ml of the cell supernatant sample processed in step 2 into a centrifuge tube;

[0166] (5) Cover the metal cover, balance the centrifuge tube together with the metal cover, adjust with 1XPBS to make the weight deviation within 0.02g;

[0167] (6) Place the balanced centrifuge tube symmetrically in the ultracentrifugation ro...

Embodiment 3

[0245] Example 3 Functional detection of recombinant lentiviral vectors lvCAR22-CLA, lvCAR22-CLB, and lvCAR22-OLC.

[0246] 1. Cell-level expression detection of CAR gene:

[0247] (1) After the recombinant lentiviral vectors lvCAR22-CLA, lvCAR22-CLB, and lvCAR22-OLC infect PBMC cells, collect the cells and use RT-PCR to detect the CAR mRNA transcription level to verify the expression of CAR gene. If the CAR mRNA transcription level increases, It indicates that the transcription level expression of CAR gene is successful;

[0248] (2) After the recombinant lentiviral vectors lvCAR22-CLA, lvCAR22-CLB, and lvCAR22-OLC infect PBMC cells, collect the cells and use western blot to detect the expression level of CAR protein to verify the expression of CAR gene. If the expression level of CAR protein increases, then It shows that the translation level of CAR gene is successfully expressed;

[0249] (3) Infect the cells with lvCAR22-CLA, lvCAR22-CLB, lvCAR22-OLC and the control virus MOCK wi...

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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 specifically relates to a vector, in particular to a CAR-T transgene vector targeting CD22 replication-deficient recombinant lentivirus. In addition, the present invention also relates to the construction method and application of the vector. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-related antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). This biological process is very complex and is still under study. In the 1990s, multiple 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 usually divided into two categories, non-specific immunity and specific immunity. Non-specific immunotherapy mainly incl...

Claims

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Application Information

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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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