Abstract
Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.
Highlights
In the more frontier point of view, cell-intrinsic disruption of immune checkpoints by gene targeting in T-cells is likely to display a better safety profile than the systemic administration of blocking antibody[13]
No difference of the activation marker CD28, CD27, CD69 and HLA-DR on CD3+ T cells was detected between the sgRNAhPD-1 T cells and control T cells (p > 0.01) (Fig. 5d). These results indicated that the CD4 or CD8 subsets constitution or the memory and activation status of the T cells is stable with sgRNA:Cas9-mediated programmed death-1 (PD-1) disruption
The effective activation of the tumor reactive T cells and the suppression of checkpoint inhibitor has long been the key problem of immunotherapy
Summary
In the more frontier point of view, cell-intrinsic disruption of immune checkpoints by gene targeting in T-cells is likely to display a better safety profile than the systemic administration of blocking antibody[13]. We show that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA-Cas[9] DNA is technically feasible and efficient, and this genome disruption in primary human lymphocytes sustained over prolonged in vitro culture in the presence of antigen stimulation This electroporation mediated intrinsic PD-1 gene disruption will not affect the proliferation capacity of primary T cells whereas enhance cellular immune responses and cytotoxicity on tumor cell lines. These results underscore the therapeutic potential of a non-viral mediated CRISPR-Cas[9] genome editing method for the disruption of immune inhibitory checkpoints, and may realize its clinical application in adoptive T-cell transfer therapy of cancer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
