| BackgroundMalignant tumors are one of the most important diseases that endanger human health and cause great harm to human survival.In recent years,although the treatment of tumors has made great progress in surgery,radiotherapy and chemotherapy,there are still problems such as low cure rate,high recurrence rate and large side effects.A doptive immunotherapy（AIT）is one of the important methods for the biological treatment of malignant tumors.Tumor adoptive immunotherapy of T cells is modified by chimeric antigen receptors,which is a hotspot in recent years.It can reshape the targeting,proliferation and persistence of T cells and effectively increase its specific killing activity[1-2].There are many methods for gene transduction,such as gamma retrovirus（γVV）,adeno-associated virus vector,lentiviral vector,transposon,and mRNA electroporation[3-4].Currently,a common method for T cell gene transduction is lentivirus and retroviral transfection.However,in addition to the limited efficiency of transfection,retrovirus integration and insertional mutations may cause genotoxicity,which may cause some concerns about its clinical application[5-6].The non-viral Piggybac transposon transgenic system used in this study can specifically insert the integrated gene into the TTAA four-base site by the"cleavage-paste"mechanism to achieve long-term expression of the foreign gene[7].Experiments have shown that the PiggyBac（PB）transposon can successfully perform stable genetic modification on human T cells,enhancing the killing ability of T cells to tumors in vitro and in vivo[8].Based on the strategy of PiggyBac transposon to construct CAR-T cells,this study aims to establish a stable genetic modification of T cells,establish an in vitro activation amplification system,achieve superior amplification of CAR-T cells and high expression of CAR in T cells,and retain the memory phenotype of CAR-T cells.ObjectiveA non-viral T cell gene transduction and in vitro activation amplification system was established to provide an experimental basis for CAR-T cell immunotherapy.MethodsCD19CAR cassette（1470bp）was cloned into XbaI and EcoRI sites of the PiggyBac transposon vector PB513B-1 to construct a PiggyBac recombinant vector（pb19CAR）targeting CD19.DNA digestion electrophoresis and gene sequencing confirmed the successful construction of the vector,and the expression of CD19CAR in T cells was detected by Western blot.Dual plasmids（pb19CAR and transposase）were transducted into primary T cells using electroporation（1×20ms830V/840V/850V）and nucleofection（U014/T023）.The transduction efficiency and cell viability were compared and optimized,and compared with traditional lentivirus transfection methods.T cells were activated by plates coated with human CD19（5μg/ml）protein or CD3/CD28mAb（5μg/ml）,and then amplified by IL-2（500U/ml）or mixed cytokines IL-7（10ng/ml）/IL-15（10ng/ml）/IL-21（20ng/ml）CD19CAR expression and phenotype of T cells were compared after activation and amplification.ResultsThe Piggybac transposon vector（pb19CAR）was successfully constructed by DNA gel electrophoresis and gene sequencing after double digestion with XbaI and EcoRI.Western blot showed that the chimeric antigen receptor CD19CAR was successfully expressed in CAR-T cells.By flow cytometry and cell viability counting instrument test,the transduction efficiency and cell viability of electroporation were higher than that of nuclear transfectors（53.2±2.7%vs 33.5±1.7%,75±3.8%vs60±3.0%,both P<0.05）;electroporation,nuclear transduction efficiency was higher than lentiviral transfection（25.9±1.3%）,and cell viability was lower than lentiviral transfection（80±2.8%）.After electroporation,the cells were activated and expanded for 12 days.The expression of CD19CAR on the cell surface by flow cytometry showed that the target antigen CD19 protein group was significantly higher than the CD3/CD28 mAb group（72.5±3.6%and 53.7±2.7%,P<0.05）.Flow cytometry cell phenotypic analysis showed that the mixed cytokine group（IL-7/IL-15/IL-21）had a higher proportion of Tscm/Tscm-like than the IL-2 group（24.7±1.2%vs 12.1±0.6%,P<0.05）,Tcm cells（10.2±0.5%vs 2.9±0.1%,P<0.05）.Conclusion1.This study tested the Celertrix electroporator,Lonza nuclear transilator and traditional lentiviral transfection.In comparison the transduction efficiency,cell viability and cell activation after transfection are better with the Celertrix electroporator.Moreover,the transposition enzyme is indispensable for the transposition and stable expression of the foreign target gene.2.The target antigen CD19 protein activation group CD19CAR expression rate of CAR-T cells was significantly higher than the CD3/CD28 mAb activation group,indicating that the target antigen CD19 protein stimulation can preferentially expand CAR-T cells anti-CD19;3.Mixed cytokines（IL-7/IL-15/IL-21）support CAR-T cell growth with central memory and stem cell memory phenotype.This study provides theexperimental basis for CAR-T cell immunotherapy. |
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