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Publications

Journal Article Ppublish 1 citation

PubMed: 39905729 DOI: 10.1016/j.ymthe.2025.01.045

Gene editing of CD3 epsilon to redirect regulatory T cells for adoptive T cell transfer.

Mol Ther 33(3):997-1013

Authors: Weijie Du, Fatih Noyan, Oliver McCallion, Vanessa Drosdek, Jonas Kath, Viktor Glaser, Carla Fuster-Garcia, Mingxing Yang, Maik Stein, Clemens Franke, Yaolin Pu, Olaf Weber, Julia K Polansky, Toni Cathomen, Elmar Jaeckel, Joanna Hester, Fadi Issa, Hans-Dieter Volk, Michael Schmueck-Henneresse, Petra Reinke, Dimitrios L Wagner

Affiliations

  • Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), 13353 Berlin, Germany; BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany.
  • Department of Gastroenterology, Infectious Diseases and Hepatology & Endocrinology, Hannover Medical School, Hannover, Germany.
  • Transplantation Research and Immunology Group, Nuffield Department of Surgical Science, University of Oxford, Oxford OX3 9DU, UK.
  • Institute for Transfusion Medicine and Gene Therapy, Medical Center, University of Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, Medical Center, University of Freiburg, 79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.
  • BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany.
  • Institute of Molecular Medicine and Experimental Immunology (IMMEI), University of Bonn, Biomedical Center II, Venusberg Campus 1, 53127 Bonn, Germany.
  • BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany; German Rheumatism Research Centre, Deutsches Rheuma-Forschungszentrum, ein Leibniz Institut, Berlin, Germany.
  • Department of Gastroenterology, Infectious Diseases and Hepatology & Endocrinology, Hannover Medical School, Hannover, Germany; Department of Liver Transplantation, Multi Organ Transplant Program, University Health Network, University of Toronto, Toronto, ON, Canada.
  • Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), 13353 Berlin, Germany; BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany; Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.
  • Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), 13353 Berlin, Germany; Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.
  • Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), 13353 Berlin, Germany; BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health at Charité - Universitätsmedizin Berlin, 13353 Berlin, Germany; Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany; Institute of Transfusion Medicine, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany; Center for Cell and Gene Therapy, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Abstract

Adoptive transfer of antigen-specific regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. However, their low frequency in peripheral blood poses challenges for both manufacturing and clinical application. Chimeric antigen receptors have been used to redirect the specificity of Tregs, using retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral CRISPR-Cas12a gene editing to redirect Tregs, using human leukocyte antigen (HLA)-A2-specific constructs for proof-of-concept studies in transplantation models. Knock-in of an antigen-binding domain into the N terminus of CD3 epsilon (CD3ε) gene generates Tregs expressing a chimeric CD3ε-T cell receptor fusion construct (TRuC) protein that integrates into the endogenous TCR/CD3 complex. These CD3ε-TRuC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TRuC-redirected Tregs over CD3ε knockout Tregs via repetitive CD3/CD28 stimulation in a good manufacturing practice-compatible expansion system. CD3ε-TRuC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3ε-TRuC Tregs demonstrate superior protection of allogeneic HLA-A2 skin grafts from rejection compared with polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.

MeSH

Cells, Genes, T-Lymphocytes, Regulatory, Gene Editing, Hla-A2 Antigen, Receptors, Chimeric Antigen, T-Lymphocytes, Organ Transplantation, Transplantation, Genetic Therapy

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