MULTIPLEX GENE EDITING IN CAR T CELLS

• Published in: Cytotherapy (Oxford, England) England), 2024-06, Vol.26 (6), p.S173-S173
• Main Authors: Jafarzadeh, L., Boudreau, G., Rulleau, C., Delisle, J.
• Format: Article
• Language: English
• Subjects: CAR T
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.340

Chimeric Antigen Receptor (CAR) T-cell therapy for cancer is limited by several mechanisms that impact T-cell function. The pan T-cell receptor CD5 is an inhibitory co-receptor that can be targeted through CRISPR-Cas9 gene editing techniques to improve T-cell function. We found that CD5 deletion alone had little impact on T-cell functionality and aimed to assess whether the co-deletion of other immune checkpoints alongside CD5 was feasible in CAR T cells. Focusing on the inhibitory receptor TIM-3, we assessed the impact of multiple gene editing in CAR T cells. Human CD3+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 antibodies. After a three-day activation, T cells were transduced with a lentivirus carrying a second-generation CAR construct. Two days post-transduction, CRISPR-Cas9-mediated electroporation induced Tim3 and/or CD5 knockout in CAR T cells. CD5/Tim3 knockout CAR T cells underwent additional activation, and after one week, their phenotype and functionality were assessed using flow cytometry. Our study confirms the efficacy of single and double knockout strategies targeting CD5 and TIM-3 in CAR T cells. Initial investigations revealed comparable effectiveness of editing both genes to single deletions in CD3/CD28-stimulated T cells. This pattern extended to anti-CD19 and anti-BCMA CAR T cells, where edited CAR T cells mirrored single deletions' phenotype and functionality. Despite a slight reduction in expansion post-gene knockout, both single and double knockout cells exhibited similar differentiation, primarily favoring effector memory T cells, and consistently expressed PD-1. Functionally, edited CAR T cells displayed high levels of IFN-g and TNF-a expression, with no discernible differences among single or double CD5 and TIM-3 knockout CAR T cells in comparison with unedited CAR T cells. Importantly, all CAR T cells maintained robust cytotoxicity, confirming specific antigen-dependent cytotoxicity through specific target killing. In conclusion, our findings affirm the integrity of our production procedure, demonstrating that single or double deletions do not compromise CAR T cell phenotype or function. Future studies will involve serial stimulation of unedited, single, and double knockout CAR T cells to investigate CAR T-cell exhaustion/dysfunction and the expansion of antigen-specific CAR T cells with single and double editions.