An in vivo CRISPR screen unveils promising target genes to improve CAR-T cell efficacy in a solid tumor model

  • Mol Ther. 2026 Jul 1;34(7):3976-4000. doi: 10.1016/j.ymthe.2026.04.009.
Mattia Fumagalli  1 ,  Dongjie An  2 ,  Luca Simula  2 ,  Camille Combe  2 ,  Lisa Aziez  3 ,  Yannick Simoni  3 ,  Marie-Clotilde Alves-Guerra  3 ,  Andrea Valentini  4 ,  Maude Marchais  3 ,  Anaïs Vermare  2 ,  Josquin Moraly  5 ,  Simona Manni  6 ,  Maria Cecilia Quadraccia  6 ,  Concetta Quintarelli  7 ,  Biagio De Angelis  6 ,  Nadège Bercovici  2 ,  Emmanuel Donnadieu  2 ,  Frédéric Pendino  8
Affiliations
  • 1. Université Paris Cité, Institut Cochin, INSERM, CNRS, 75014 Paris, France; Equipe Labélisée Ligue Nationale Contre le Cancer, Paris, France; Thèse Financée par la Ligue Nationale Contre le Cancer, Paris, France. Electronic address: [email protected].
  • 2. Université Paris Cité, Institut Cochin, INSERM, CNRS, 75014 Paris, France; Equipe Labélisée Ligue Nationale Contre le Cancer, Paris, France.
  • 3. Université Paris Cité, Institut Cochin, INSERM, CNRS, 75014 Paris, France.
  • 4. Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
  • 5. Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
  • 6. Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, 00165 Rome, Italy.
  • 7. Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, 00165 Rome, Italy; Department of Clinical Medicine and Surgery, Federico II University of Naples, 80131 Naples, Italy.
  • 8. Université Paris Cité, Institut Cochin, INSERM, CNRS, 75014 Paris, France; Equipe Labélisée Ligue Nationale Contre le Cancer, Paris, France. Electronic address: [email protected].
PMID: 41935953 DOI: 10.1016/j.ymthe.2026.04.009
Abstract

CAR-T cell therapies are revolutionizing the treatment of refractory or relapsed hematological malignancies, but many patients do not achieve durable responses, and these therapies remain ineffective against solid tumors. Therapeutic failure is closely associated with a poor persistence of CAR-T cells in patients, highlighting the need to identify strategies promoting in vivo expansion. Although numerous gene-editing strategies have been proposed, comparative studies to identify the most effective ones are still lacking. Here, using a focused CRISPR-knockout library targeting 50 selected gene candidates, we developed a competitive screening that revealed ZC3H12A, SOCS1, PTPN2, and CDKN2A as the most robust targets to improve persistence of EGFR CAR-T cells in human lung tumor-bearing mice. Surprisingly, disruption of Other genes previously reported to improve CAR-T cell efficacy in Other preclinical models-MED12, PRDM1, and BATF-had a detrimental effect in this context. These results suggest that some gene-editing strategies can yield beneficial, neutral, or even deleterious effects on CAR-T cell persistence, depending on specific conditions. Altogether, these findings highlight the importance of performing context-specific evaluations of genetic modifications to accelerate the clinical translation of the most promising editing strategies for optimizing CAR-T cell therapies.

Keywords
CAR-T cell therapy; CRISPR screening; cancer immunotherapy; context specificity; gene editing; metastases; mitochondrial metabolism; solid tumors.
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