CRISPR-Cas9 Mediated Gene Editing In Primary Human T Cells

Objective:Since being studied and developed from 2012,CRISPR-Cas9 has been applied in various fields,including genome editing in various organisms and gene regulation.Meanwhile,the adoptive immunotherapy has also shown promising results in treating cancer patients.Extensive efforts are being made to develop more effective T cells through gene editing,represented by chimeric antigen receptor(CAR)T cells,in order to enhance their response and durability.The immune checkpoint molecules,such as CTLA-4 and PD-1,are found to be inhibitory receptors on immune cells,which bind to their ligands,lead to T cell failure,and become obstacles to cell therapy.Thus,in this article we sought to permanently silence these checkpoints in primary T cells by taking use of CRISPR-Cas9 and a serial of sgRNAs targeting them.By combining with CD 133 CAR,we also tried to explore what effects would be made to the functions of T cells when checkpoints inhibition and CAR were used together.Methods:We designed and produced the sgRNA vectors targeting CTLA-4,TIM-3,2B4,LAG-3 and so on,which were co-transfected with Cas9 into HeLa cell lines.T7EN1 test and T-A clones sequencing were performed in the targeting area,in order to obtain the most efficiency ones.The sgRNAs with the highest efficiency of targeting PD-1 and TIM-3 were cloned into one sgRNA-expression vector with multiple U6 promoters in tandem.We transfected the vectors into primary human T cells with Cas9 plasmid by electroporation.The efficiency of multiple genes editing is confirmed by T7EN1 test and T-A clones sequencing.Meanwhile,the Cas9 and sgRNA vectors were also eletroporated into primary human T cells with CD 133 CAR vectors,which was built based on Piggybac transposon/transposase system.The T cells were further activated and expanded with drug selection,which turned to PD-1 knockout;CD 133 CAR-T cells.The expression of CAR and knockout effciency of PD-1 were confirmed by flow cytometry,T7EN1 and T-A clone test.And the cytotoxic assay against the glioma cell lines U251-CD133,the cytokine release assay and stimulation-induced-proliferation assay were performed in vitro,in order to check the effects of PD-1 disruption in CD133 CAR-T cells.After that,In vivo model using intracranial glioma implantation in immunodeficiency mice was built to check the effects of PD-1 disruption in CD 133 CAR-T cells in further.Tracing the insertion of Cas9 by monitor the GFP tagged Cas9,T7EN1 assay on the potential off-target sites of PD-1 and tracing the human CAR-T cells in immunodeficiency mice without specific antigens,those three experiments were performed to test the safety of PD-1 KO;CD133 CAR-T cells.Results:We selected those sgRNAs with the effciency of more than 50%in HeLa to target the checkpoints including CTLA-4,TIM-3,2B4,LAG-3 and so on.When multiple-U6-sgRNA vector P2T2 was electroporated into T cells,the targeting effciency on PD-1 and TIM-3 is 55%and 60%,respectively,which is higher than those using individual sgRNA vectors,with the efficiency of 35%and 40%,respectively.When Cas9 mediated PD-1 disruption was combined with CD 133 CAR,the efficiency of PD-1 disruption and CAR expression were both higher than 85%.In vitro cytotoxic assay has shown that PD-1 disruption could enhance the killing effect on glioma cell lines,which is about 20%when the rate of effector to target is 8:1.Similarly,PD-1 disruption could enhance the tumor suppresion effect of CD 133 CAR-T cells in intracranial glioma implantation mice model and extend overall survival time for about 2 weeks.In the aspect of bio-safety,we didn't find the insertion of Cas9,nor obvious editing in off-target sites.The persistence assay in vivo also showed that the rate of CAR-T cells in blood quickly decreased from about 0.1%in day 7 to about 0.05%in day 28.Conclusion:Our work suggests that the use of the CRISPR-Cas9 system allowed us to efficiently perform gene editing on T cells and endogeneously block the checkpoint genes including PD-1 and TIM-3.Combining the Cas9 mediated PD-1 disruption with CD133 CAR can improve the anti-glioma function of T cells,which may benefit the cellular immunotherapy.