Barbao, P.; Rodriguez-Garcia, A.; Galindo-Albarran, A.; Gimenez-Alejandre, M.; Clavero, P.; Lobo-Jarne, T.; Botas, M.; Colell, G.; Castellsague, J.; Cascallo, G.; Andreu-Saumell, I.; Soria-Castellano, M.; Colell, S.; Marzal, B.; Martin-Mur, B.; Esteve-Codina, A.; Urbano-Ispizua, A.; Prat, A.; Gattinoni, L.; Mendoza-Parra, M.; Guedan, S.

CAR-T cell therapy has shown limited efficacy in solid tumors, largely due to T cell dysfunction driven by chronic antigen exposure. To uncover mediators of this dysfunction, we developed an in vivo screening platform using an ovarian xenograft tumor model in which CD28-based CAR-T cells undergo exhaustion leading to tumor escape. Transcriptomic profiling of tumor-infiltrating CAR-T cells at different stages revealed dynamic upregulation of exhaustion-associated genes. We used this data to design a focused CRISPR/Cas9 library and performed an in vivo screen. We identified 14 significantly enriched candidate genes, among which ZC3H12C emerged as the top hit. Single-cell RNA and ATAC-seq confirmed ZC3H12C expression in CAR-T cells undergoing early exhaustion in vivo. ZC3H12C disruption enhanced CAR-T cell persistence and antitumor efficacy while reducing exhaustion, across both CD28- and 4-1BB-based CARs targeting distinct antigens. These results highlight ZC3H12C as a promising target to improve CAR-T therapy in solid tumors.