Method for detecting copy number of virus vector in CAR-T cell genome
A virus vector and copy number technology, applied in the field of biomedicine, can solve the problems of incomplete elimination of integration risks and achieve the effect of effectiveness and safety guarantee
Pending Publication Date: 2020-08-07
深圳科诺医学检验实验室
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Problems solved by technology
Carrier gene integration may bring harm to cells such as the activation of some original cancer cell genes or the inactivation of cancer suppressor genes, resulting in the risk of secondary infection of malignant tumors, although the current gene integration design of vectors has greatly reduced the risk of gene integration. The risk of secondary infection, but the risk of this integration has not been completely eliminated, so the virus copy number in the genomic vector that has been integrated into the cell still needs to be tested
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Embodiment 1
[0037] 1. The main instruments and equipment include: ultra-clean workbench, micro-drop digital PCR instrument series, desktop centrifuge, vortex suspension device, Nanodrop ultra-micro spectrophotometer;
[0038] 2. The main reagents used include: Cell Genome Extraction Kit (QIAGEN), HPLC purified PCR primers and probes (GeneScript), DNA fragments ((GeneScript), ddPCR Supermix for Probes (No. dUTP), Droplet Generation Oil, TE Buffer.
[0039] 3. The method for detecting the copy number of the viral vector in the CAR-T cell genome specifically includes the following operations:
[0040] 1) Sample extraction: Take CAR-T cell sample suspension and uninfected T cell suspension in a sterile environment, and use the cell genome extraction kit produced by QIAGEN to extract CAR-T cell sample and uninfected T cell respectively The extracted genome was analyzed for DNA concentration and purity using a Nanodrop instrument;
[0041] 2) Prepare PCR solution: PCR reaction volume is 20 μL...
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The invention discloses a method for detecting the copy number of a virus vector in a CAR-T cell genome. A DNA sequence which simultaneously contains a target gene (lentiviral gene) sequence and a reference gene sequence of a CAR-T cell and has a copy number ratio of 1 is designed, and is used for determining the amplification efficiency ratio of primers, meanwhile, a specific primer and a probe which can be used for detecting target genes of most CAR-T cells are designed, a digital PCR detection mode is selected and used in cooperation, detection errors can be reduced, and the copy number ofvirus vectors in the CAR-T cell genome can be accurately reflected, so the effectiveness and safety of tumor treatment through CAR-T cells are guaranteed.
Description
technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for detecting the copy number of viral vectors in the genome of CAR-T cells. Background technique [0002] Chimeric antigen receptor T cell (CAR-T, Chimeric antigen receptor T cell) therapy refers to the modification of T cells through gene editing technology to enhance the effect of T cells on killing tumor cells, so as to achieve the purpose of treating tumors. The safety of drugs is the top priority. Although CAR-T cell therapy has made some progress, it still belongs to an undeveloped field. During its development, many new technologies continue to enter this field, and There are not enough data yet to assess its potential risk. [0003] The difficulty of CAR-T cell therapy is not the early cell therapy, but how to deal with the side effects of cell therapy and the control of product quality in the later stage. CAR-T cells are different from traditional drugs, ...
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IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2563/159C12Q2563/107
Inventor 陈洁莹汤赞胡樾曾桂芳梁晓刘沐芸
Owner 深圳科诺医学检验实验室