Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome
Kristina Pavlovic, María Carmona, Giulia I. Corsi, Noelia Maldonado‐Pérez, Francisco J. Molina-Estévez, Esther Peralbo-Santaella, Marina Cortijo-Gutiérrez, Pedro Justicia-Lirio, María Tristán-Manzano, Víctor Ronco-Díaz, Antonio Ballesteros-Ribelles, Alejandro Millán-López, Paula Heredia-Velázquez, Carla Fuster‐García, Toni Cathomen, Stefan E Seemann, Jan Gorodkin, Francisco Martı́n, Concha Herrera, Karim Benabdellah
Abstract
Introduction Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro . This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.