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Recombinant gene construction of chimeric antigen receptor (CAR) for treating HIV (human immunodeficiency virus) infection and application of chimeric antigen receptor

A technology of receptors and antigens, applied in the field of recombinant gene construction of chimeric antigen receptors, can solve the problems of low transduction efficiency of retroviral vectors, cell death, loss of CAR molecules, etc., to increase clinical effectiveness and safety , increase broad-spectrum, increase the effect of safety

Active Publication Date: 2017-08-29
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reasons are as follows: 1. The transduction efficiency of retroviral vectors is low. In order to obtain enough reinfused CAR-T cells, excessive in vitro expansion leads to cell death and loss of CAR molecules after reinfusion.
2. There are defects in the design of CAR molecules, in which the CD4 domain may cause transduced CTLs to be infected by HIV or virus-infected cells escape the killing of CAR-T cells by down-regulating the expression of CD4 molecules

Method used

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  • Recombinant gene construction of chimeric antigen receptor (CAR) for treating HIV (human immunodeficiency virus) infection and application of chimeric antigen receptor
  • Recombinant gene construction of chimeric antigen receptor (CAR) for treating HIV (human immunodeficiency virus) infection and application of chimeric antigen receptor
  • Recombinant gene construction of chimeric antigen receptor (CAR) for treating HIV (human immunodeficiency virus) infection and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The present invention provides a chimeric antigen receptor (CAR) recombinant gene for treating HIV infection and its construction method. The specific splicing method is: sequential splicing of signal peptide, gp120 single-chain antibody ScFv capable of recognizing the surface of HIV-infected cells , CD8hinge, leukocyte antigen differentiation group molecule transmembrane region CD28-TM+ICD, 4-1BB and CD3 (leukocyte antigen differentiation group molecule 3) ζ chain, and finally a complete chimeric antigen receptor (CAR) that can treat HIV ) molecule, its amino acid sequence is shown in SEQID NO.4, and its structure is as figure 1 shown; the nucleotide sequence of the gene encoding the chimeric antigen receptor (CAR) is shown in SEQ ID NO.3.

[0029]The amino acid sequence of the single-chain antibody ScFv derived from the chimeric antigen receptor for the treatment of HIV infection is shown in SEQ ID NO.2. The nucleotide sequence of its coding gene is shown in SEQ ID N...

Embodiment 2

[0031] Example 2: CAR Molecular Recombination Construction of PTK-EF-1α-N6 Plasmid Expression Vector

[0032] The CAR was synthesized according to the sequence shown in SEQ ID NO.4, and the gene encoding the full-length CAR was inserted into the target expression vector based on seamless recombination cloning technology (see figure 2 ). After many tests, the preferred plasmid vector is the PTK-EF1α-N6 vector (the nucleotide sequence of which is shown in SEQ ID NO.5) transformed from the PTK881 vector as the backbone, after replacing the CMV promoter with the EF1α promoter. Finally, a recombinant plasmid PTK-EF1α-N6 vector inserted into the CAR gene and capable of expressing CAR was obtained, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0033] The virus packaging steps are as follows:

[0034] 1) Take two centrifuge tubes filled with 16ml DMEM culture medium, add 960μg PEI to one tube, and add 320μg premixed PTK881 vector plasmid to the other tube, vortex, and...

Embodiment 3

[0038] Example 3: Preparation of CD8 capable of expressing chimeric antigen receptors + T cells (CAR-T cells)

[0039] Step 1: Isolation of Patient PBMC Cells

[0040] (1) Collect 60-80ml of human peripheral blood samples, and shake while collecting to fully mix the peripheral blood with the anticoagulant;

[0041] (2) Transfer the peripheral blood into a 50ml centrifuge tube, dilute the peripheral blood 1:1 with DPBS buffer, and mix well. Slowly add the diluted blood sample into the centrifuge tube of 15ml human lymphocyte separation medium at room temperature. The method is as follows: take the blood sample with a 10ml pipette, extend it to 0.5cm above the liquid surface of the separation liquid, the blood sample will naturally slide down and spread on the surface of the separation liquid, then gently add the blood sample, be careful not to break through the liquid surface;

[0042] (3) Trim and centrifuge for 30 minutes, slowly ascend and descend slowly;

[0043] (4) Af...

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Abstract

The invention relates to the technical field of immunotherapy of infectious diseases, in particular to recombinant gene construction of chimeric antigen receptor (CAR) for treating HIV (human immunodeficiency virus) infection and an application of the CAR. A single-chain antibody (ScFv) can identify gp120 of surfaces of HIV infected cells through series connection of antibody light chain and heavy chain variable zones of the gp120 of the surfaces of the HIV infected cells; the ScFv is prepared into the CAR, CAR coding genes are shifted to plasimid vectors, and lentiviral vectors shifted with the CAR coding genes are transduced to CD8+T lymphocytes. The CD8+T lymphocytes are discovered to be remarkable in restraining and killing activities of the HIV infected cells in both in-vitro and in-vivo experiments and can serve as active ingredients to prepare anti-HIV infection drugs, and good application prospect is achieved.

Description

technical field [0001] The invention relates to the technical field of immunotherapy for infectious diseases, in particular to a recombinant gene construction and application of a chimeric antigen receptor for treating HIV infection. Background technique [0002] AIDS is an infectious disease caused by human immunodeficiency virus type 1 (Human Immunodeficiency Virus1, HIV-1) infection, which seriously threatens the safety of human life. people died, and there are still 37 million HIV-infected people in the world. The number of HIV-infected people in my country is increasing year by year, and the total number of patients has exceeded one million. There is currently no effective vaccine and existing drugs cannot completely cure it. [0003] Highly Active Antiretroviral Therapy (HAART) is the first revolution in the history of HIV / AIDS treatment, which greatly reduces the morbidity and mortality of HIV / AIDS, significantly prolongs the life of patients, and even reduces the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C07K19/00C12N15/13C12N15/62C12N15/867C12N5/10A61K35/17A61P31/18
CPCC12N15/86C07K14/7051C07K16/1063C12N2740/15043C07K2317/622C07K2319/02C12N5/0636A61K39/4631A61K2239/31A61K39/464838A61K2239/38A61K39/4611A61P31/18C07K2319/03C07K2319/33C12N2510/00C07K2319/74C07K2317/73C07K2317/76A61K38/00A61K2039/505C07K14/70517C07K14/70521C07K14/70578C07K2319/30
Inventor 张同存胡广顾潮江张尚昆黄政汤杰唐骝
Owner WUHAN BIO RAID BIOTECH CO LTD
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