Preparation And Verification Of Universal CD19 CAR-T Cells Using CRISPR/Cas9 Technology

Chimeric antigen receptor(CAR)T cell therapy is currently the most promising method for the treatment of cancer globally,and its surprising efficacy in relapse and refractory acute B-cell lymphocytic leukemia(B-ALL)was inspiring.CD19 antigen is the most popular target for the study of B-line blood tumor in CAR-T cell therapy.Nowadays,the response of autologous CD19CAR-T cells to patients tends to be stable.In order to make it more accessible to more patients,to develop universal CD19 CAR-T cells therapy is the key breakthrough.In this strategy,it's necessary to eliminate T cell receptors(TCRs)on allogeneic CAR-T cells to avoid graft-versus-host disease(GVHD)and major histocompatibility complex(MHC),so that can minimize CAR-T cell's immunogenicity to the full extent.In relating study of universal CD19 CAR-T cells,it was found that the multiple gene knockout efficiency,safety and function change is the nonnegligible problems.Human MHC molecules contain HLA class I molecules and HLA class II molecules.Most researchers believe that TCR and HLA class I molecules are the keys to immune rejection in CAR-T cells treatment.And the presence of HLA class II molecules in this process wouldn't cause the immune rejection.Although in which case the existence of HLA class II molecules won't be immunologic rejection theoretically,however there is by far no sufficient data to support this view.So further study of HLA molecules deletion CAR-T cells holds great significance.In this study,we used a gene-editing technology,CRISPR/Cas9,which is relatively technically mature and simple to use.It achieves multiple genes knockout for CAR-T cells targeting CD19,and obtained TCR null,HLA I null,and HLA II null CD19 CAR-T cells.In addition,via in vitro cytotoxic and ELISA Cytokine testing to verify its function.First,the programs for electroporation of PBMCs was rapidly screened by electroporation of LV-GFP plasmids.The next step,we designed several g RNAs that can target TCR,B2M(HLA class I co-subunit)and CIITA(HLA class II transcriptional regulator)gene respectively.The optimal sequences were screened separately and a knockout plasmid(PX330 A-1X3-sg TRAC-1-sg B2M-3-sg CIITA2)that can be used to simultaneously knock out TCR,HLA class I molecules and HLA class II molecules was contrusted by us.Then,the PBMCs infected by the CD19 CAR lentivirus which previously designed and maintained by our laboratory were used to electroporate the knockout plasmids.And HLA I^null HLA II^null TCR^null CD19 CAR-T cells were obtained by flow sorting.In this process,a series of electroporation multigene knockout plasmid transfection efficiency optimization experiments were performed.Finally,to test whether multiple genes knockout have an influence on CAR-T cell function,we conducted experiments on both the ability of cells to kill tumors and cytokine secretion after killing.We set different ratio of the obtained cells to CD19 antigen-positive cells Raji or negative cells K562 in vitro cytotoxic testing,and,analysis of the results.Also,the IFN-?cytokine secretion level of the supernatant which remained was detected.The above series of experimental results show that the g RNA sequences used in this study could cut genome targeting sites(T7E I digestion and genome sequencing identification)successfully,and no off-target effects(sequencing of the predicted off-target site region)was be found.The HLA I^null HLA II^null TCR^null CD19 CAR-T cells obtained finally in this study.They can successfully kill Raji cells and release cytokine IFN-?in vitro.However,apparent killing effect and release of cytokine IFN-?on K562 cells was not been found.In summary,we successfully established a rapid and economical method to preparing universal CD19 CAR-T cells and obtained universal CD19 CAR-T cells that can be used for in vitro studies preliminary.The above research results not merely open up a new road for universal CD19 CAR-T cell drug development,but also provide a theoretical basis and practical basis for the development of universal CAR-T cells.