In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors
Year: 2026
Type: preprint
Abstract: 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.
Authors: Paula Barbao, Alba Rodriguez-Garcia, Ariel Galindo-Albarrán, Marta Giménez-Alejandre, Pablo Clavero, Teresa Lobo-Jarne, Marta Botas, Guillem Colell, Joan Castellsagué, Guim Cascalló, Irene Andreu-Saumell, Marta Soria-Castellano, Salut Colell, Berta Marzal, Beatriz Martín-Mur, Anna Esteve-Codina, Alvaro Urbano-Ispizua, Aleix Prat, Luca Gattinoni, Marco Mendoza-Parra, Sonia Guedan
Institutions: Consorci Institut D'Investigacions Biomediques August Pi I Sunyer, Fundació de Recerca Clínic Barcelona-Institut d’Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, Centre National de la Recherche Scientifique, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Génomique Métabolique du Genoscope, Genoscope, Centro Nacional de Análisis Genómico, Hospital Clínic de Barcelona, University Hospital Regensburg, Leibniz Institute for Immunotherapy, University of Regensburg
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
Funders: Breast Cancer Research Foundation, European Federation of Pharmaceutical Industries and Associations, European Commission, Generalitat de Catalunya, Ministerio de Ciencia e Innovación, Universitat de Barcelona, Fundación Fero, Instituto de Salud Carlos III, HORIZON EUROPE European Innovation Council
Awards: Heterogeneidad intra-tumoral e impacto clínico en cáncer de mama precoz HER2-positivo y triple-negativo, G2332273855, Healthcare Alliance for Resourceful Medicines Offensive against Neoplasms in HematologY - Sofia ref.: 116026, G3690828678, RESISTANCE UNDER COMBINATORIAL TREATMENT IN ER+ AND ER- BREAST CANCER., G7378298282, G7425137858, G8273605960, G8377150363, FINE-TUNING T CELL NETWORKS OF EXHAUSTION BY SYNTHETIC SENSORS
OpenAlex ID: W7160451083