| CAR-T cell therapy has strong therapeutic effects on the treatment of hematological malignancies.However,the current CAR-T cell therapy is highly personalized.The T cells used for modification in cell therapy are all derived from the patient themselves so that CAR-T cell therapy are expensive,time consuming and uneven in quality,greatly limiting its large-scale clinical application.To genetically engineer healthy donor-derived T cells for an‘off the shelf’ CAR-T cell drugs of all cancer patients not only greatly reduces the cost of treatment but also guarantees the quality.That would greatly expand its applications in clinic.The primary strategy for the preparation of allogeneic universal CAR-T cells is to knock out TCR and HLA-I molecules of the healthy donor-derived T cells by using CRISPR/Cas9 genome editing technology for eliminating immunological rejection between different individuals.However,to obtain a high proportion of TCR-HLA-I-T cells to meet clinical needs,the CRISPR/Cas9 gene knockout efficiency needs to be improved.The delivery efficiency of CRISPR/Cas9 system is the main factor affecting the editing efficiency of CRISPR/Cas9 system.Electroporation is often used to deliver the CRISPR/Cas9 system with several advantages such as simplicity,economy,high transfection efficiency,no residual toxicity,good reproducibility,and no risk of cancer.However,T cell electroporation is highly challenging because T cells are very fragile.There are low cell viability and efficiency using DNA to electroporate T cells.This is the main difficulty in applying electroporation to T cells.Optimizing the conditions of electroporation with T cells and solving the problem of low gene editing efficiency of T cells are of great significance of the large-scale application of CAR-T cell therapy.Objective This study intended to use T lymphocyte leukemia cell lines Jurkat as a model cell to establish an optimized system for plasmid electroporation.To achieve efficient gene editing of Jurkat cells,the CRISPR/Cas9 system was electroporated into Jurkat cells by using this optimized system.The optimized system was further verified on human umbilical cord blood-derived T cells.The CRISPR/Cas9 system was efficiently electroporated into human umbilical cord blood T cells and successfully knocked out the TCR and HLA-I molecules of human umbilical cord blood T cells.The allogeneic universal CD19.CAR-T cells were further prepared,and it was confirmed in vitro that the universal cells constructed by the optimization system did not lose the ability to kill CD19-positive tumor cells.Methods Part I: Establishing an optimized system for plasmid electroporation by using human T lymphoma cell lines Jurkat as model cell The plasmid carrying the green fluorescent protein marker gene and Jurkat cells were used to systematically investigate the effects of electroporation voltage,pulse time,electroporation buffer,plasmid concentration and resting time after electroporation on electrotransfer efficiency and cell viability for an optimized system of plasmid electroporation of Jurkat cells.Part II: Based on the optimized system of plasmid electroporation,an optimized system for electroporation of Jurkat cells with epismoal CRISPR plasmid was established.1.Using different concentrations of episomal CRISPR plasmid to electroporate Jurkat cells,and the optimal plasmid dosage was determined based on cell transfection efficiency and survival rate.2.Using different concentrations of puromycin to screen Jurkat cells for 5 days,and the lowest concentration that killing Jurkat cells completely was selected as the optimal concentration of the final screening.3.Using the episomal CRISPR plasmid targeting the β2M gene to electroporate Jurkat cells,and the Jurkat cells completely deficient in HLA-I molecules were finally obtained under the puromycin resistance screen.Part III: Based on the optimized plasmid electroporation system,an optimized system for RNP electroporation of human umbilical cord blood-derived T cells was established.1.T cells were electroporated with RNP under optimized electroporation conditions to knock out TCR molecules;and the effects of anti-CD3/CD28 magnetic beads and electroporation times on the knockout efficiency of TCR molecules were also investigated to establish an optimized system for RNP electroporation of human umbilical cord blood-derived T cells.2.Editing TCR and HLA-I molecules of T cells with RNP simultaneously,T cells deficient in TCR and HLA-I molecules were obtained.3.Using anti-CD3/CD28 magnetic beads to stimulate CFSE-labeled TCR-T cells and normal T cells,respectively,to observe the proliferation of T cells.4.CFSE-labeled allogeneic PBMCs were mixed with irradiated HLA-I-T cells and irradiated normal T cells,respectively,to observe the proliferation of T cells in allogeneic PBMCs.5.Using the lentiviral system to infect the TCR-HLA-I-T cells to prepare allogeneic universal CD19.CAR-T cells;The CAR-T cells were mixed with CD19+ tumor cells,and the specific killing effects against tumor cells was demonstrated by luciferase assay and cytokine IL-2 and INF-γ secretion experiments.Result Part I: Establishing an optimized system for plasmid electroporation by using human T lymphoma cell lines Jurkat as model cell The optimized system for plasmid electroporating Jurkat cells were: 450 V,20ms,using Opti-MEM as electroporation buffer and resting for 45 min after electroporation.Under this optimized system,the cell transfection efficiency can reach up to 84.3%,and the cell survival rate is 52.2%.Part II: Based on the optimized system of plasmid electroporation,an optimized system for electroporation of Jurkat cells with epismoal CRISPR plasmid was established.1.The optimal concentration of the episomal plasmid electroporated Jurkat cells was 0.4 μg/μl,the transfection efficiency was 33% and the cell viability was 21%.2.The optimal concentration of puromycin for screening Jurkat cells was 0.25 μg/ml,and all negative cells could be killed completely by screening for 5 days. 3.Combining with the resistance screening,the proportion of HLA-I-Jurkat cells gradually increased from 62.23% on day 2 to 96.84% on day 9.Part III: Based on the optimized plasmid electroporation system,an optimized system for RNP electroporation of human umbilical cord blood-derived T cells was established.1.The addition of anti-CD3/CD28 magnetic beads after electroporation significantly improved the knockout efficiency of TCR molecules,and the knockout efficiency increased from 28% to 54.9%.However,there was no significant difference between the 1:1,2:1,and 3:1 magnetic beads/cells ratios.Multiple electroporation could improve the knockout efficiency of TCR molecules,but the more the times of electroporation,the higher the cell death rate.The optimal times of electroporation was 3,and the knockout efficiency of TCR could be increased from 53% to 75%,while the cell survival rate was maintained at 80%.2.Successfully knocked out TCR and HLA-I molecules of T cells and the proportion of TCR-HLA-I-T cells can reach 28.9%;After sorting,TCR-HLA-I-T cells with a purity of 96.5% were obtained.3.Anti-CD3/CD28 magnetic beads could stimulate the proliferation of normal T cells but couldn’t stimulate the proliferation of TCR-T cells.4.Proliferation was observed in T cells in allogeneic PBMC mixed with irradiated T cells,but not with irradiated HLA-I-T cells.5.TCR-HLA-I-T cells were infected with lentivirus,and 50.2% of T cells successfully expressed CAR.The killing experiments confirmed that TCR-HLA-I-CD19.CAR-T had a specific killing effect against CD19+ tumor target cells.The ELISA results showed that TCR-HLA-I-CD19.CAR-T secreted high concentrations of IL-2 and INF-γ upon contact with target cells.Conclusion 1.An optimized system for plasmid electroporation was established by using the human T lymphoma cell line Jurkat as a model cell.The optimized system is not only suitable for efficiently delivering epismoal CRISPR plasmids into Jurkat cells,but also for T cells delivered RNP,and efficient gene editing of T cells can be achieved by using this optimization system.2.Based on the optimized plasmid electroporation system,the optimized episomal CRISPR plasmid electroporation system was further established,and 96.84% of HLA-IJurkat cells were obtained.3.Based on the optimized plasmid electroporation system,an optimized system of RNP electroporation of human umbilical cord blood-derived T cells was established,and allogeneic universal TCR-HLA-I-T cells without immunological rejection were obtained.It was confirmed by TCR-HLA-I-CD19.CAR-T specific killing and cytokine detection experiments that the established RNP electroporated human cord blood-derived T cell optimization system can be applied to universal CAR-T cell therapy. |
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