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CAR-NK transgenosis carrier based on replication defective recombinant lentivirus, and construction method and application of CAR-NK transgenosis carrier

A technology of recombinant lentivirus and transgenic vector, applied in the field of medical biology, can solve the problems of large lentivirus vector, affecting the infection efficiency of virus immune cells, low recombinant virus titer, etc., achieving good therapeutic effect and broad application prospects.

Active Publication Date: 2021-02-02
杭州乾合细胞生物科技有限公司
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

For example, the titer of the recombinant virus is not high enough. One of the main reasons for the low titer of the recombinant virus is that the lentiviral vector is too large. A larger vector will directly affect the titer of the virus and the infection efficiency of immune cells.

Method used

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  • CAR-NK transgenosis carrier based on replication defective recombinant lentivirus, and construction method and application of CAR-NK transgenosis carrier
  • CAR-NK transgenosis carrier based on replication defective recombinant lentivirus, and construction method and application of CAR-NK transgenosis carrier
  • CAR-NK transgenosis carrier based on replication defective recombinant lentivirus, and construction method and application of CAR-NK transgenosis carrier

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Embodiment 1

[0034] Construction of recombinant lentiviral vectors

[0035] The Mesothelin single-chain antibody, NKG2D Transmembrane chimeric receptor transmembrane region, 2B4 chimeric receptor co-stimulatory factor, and chimeric receptor activation domain fragments were cloned into the lentiviral backbone plasmid plenti (the size of plenti is 7050bp, and the vector sequence is shown in SEQ ID NO.20, see the attached vector map figure 1 ) to obtain the recombinant lentiviral plasmid plenti-CAR4 (see attached vector map figure 2 ).

[0036] (1) The lentiviral backbone plasmid plenti was double digested with Xba I and BamH I restriction endonucleases, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment V of 7002bp (such as image 3 shown), and the rubber tapping recovery was placed in an Eppendorf tube, and the corresponding fragment was recovered with an agarose gel recovery kit (steps are shown in Table 1), and the purity and concentration of the ...

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Abstract

The invention puts forward a CAR-NK transgenosis carrier based on a replication defective recombinant lentivirus, and a construction method and application of the CAR-NK transgenosis carrier. The CAR-NK transgenosis carrier of the recombinant lentivirus comprises a pronucleus replicon pUC Ori sequence, a sequence containing an ampicillin resistance gene AmpR, a virus replicon SV40Ori sequence, a lentivirus packaging cis element, a RSV promoter, a T2A "self-shearing" peptide sequence, a puromycin resistance gene Puro sequence, a human ubiquitin C promoter, a multiple cloning site MCS sequence,and a WPRE enhanced marmot hepatitis B virus post-transcriptional control element used for enhancing expression efficiency of transgenosis, wherein the lentivirus packaging cis element adopts a thirdgeneration of lentivirus carrier. The above carrier is used for carrying out transfection on NK92 cells, realizes a killing function for tumor cells, can generate a good treatment effect and has a wide application prospect in the CAR-NK treatment field.

Description

【Technical field】 [0001] The invention relates to the technical field of medical biology, in particular to a CAR-NK transgene vector based on a replication-deficient recombinant lentivirus and its construction method and application. 【Background technique】 [0002] In recent years, in the field of tumor treatment, immunotherapy has received more and more attention. Compared with the existing surgery, radiotherapy, chemotherapy, and targeted therapy, it has its own irreplaceable advantages and development potential. Therefore, it is called the cornerstone of tumor treatment. Fifth technology. Adoptive immunotherapy of tumors refers to the infusion of autologous or allogeneic immune effector cells activated in vitro into patients to kill tumor cells in patients. A key issue in adoptive immunotherapy of tumors is to find suitable tumor-killing cells. Since the 1980s, cells including LAK, cytokine-ind μced killers (CIK), TIL and other cells have been used in clinical practice ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12N15/62C12N5/10A61K39/00A61P35/00A61P35/02
CPCC12N15/86C12N15/65C07K16/2821C07K14/7056C07K14/70503C07K14/7051C07K14/70535C12N5/0646A61K39/001168A61P35/00A61P35/02C12N2740/15043C12N2800/107C12N2830/48C12N2830/50C12N2820/10C07K2317/622C07K2319/03C07K2319/33C07K2319/74C12N2510/00A61K2039/5156A61K2039/892A61K2039/804Y02A50/30
Inventor 陈伟刘昊
Owner 杭州乾合细胞生物科技有限公司
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