Fumagalli, M.; An, D.; Simula, L.; Combe, C.; Aziez, L.; Simoni, Y.; Alves-Guerra, M.-C.; Valentini, A.; Marchais, M.; Vermare, A.; Bercovici, N.; Donnadieu, E.; Pendino, F.

CAR-T cell therapies are revolutionizing the treatment of refractory and relapsed haematological malignancies, but many patients do not exhibit long-term responses, and these therapies are less effective against solid tumours. Poor persistence of CAR-T cells in patients is associated with therapeutic failure, highlighting the need to identify genes that promote in vivo expansion. Here, we developed an accessible in vivo competitive screening method to identify genes whose inactivation provides a selective advantage to CAR-T cells. Inactivation of 50 genes in a heterogeneous population of T cells expressing an EGFR-targeting CAR revealed that disruption of REGNASE-1, SOCS1, PTPN2, and P16INK4A conferred a selective advantage to CAR-T cells in human lung tumour-bearing mice. Coherently, inactivation of these genes resulted in improved tumour eradication by CAR-T cells. Interestingly disruption of other genes, described to improve CAR-T cell function against other tumours, had a negative impact in this orthotropic lung tumour model. Further evaluation of late effects, within a subcutaneous model, highlighted SOCS1 gene inactivation as the most promising strategy for in vivo CAR-T cell persistence. These results support the importance of evaluating CAR-T cell editing strategies in specific tumours models and the screening method proposed provides a versatile pre-clinical tool for assessing persistence and function in context-specific settings.