Ghazaleh Zarrinrad, Lisa-Marie Burkhardt, Claudia Beltran-Mestres, Silvina Romero-Suárez, Dimitrios L. Wagner, Stephan Schlickeiser, Maik Stein, Désirée J. Wendering, Iván Juky Carrera Diaz de la Cebosa, Lukas Ehlen, Frederik Hamm, Gavin Kurgan, Pawel Durek, Frederik Heinrich, Anamika Giri, Yaolin Pu, Kristy Ou, Henrike Hoffmann, Sandra Muench, Insa Stuewe, Sven Dolling, Oliver McCallion, Jaspal Kaeda, Jonas Kath, Sarah Schulenberg, Niklas Wiese, Abdolreza Nazari, Lena Peter, Samira Picht, Andrea Sánchez-Peña, Olalekan H. Usman, Christian M. Brommel, Rolf Turk, Garrett R. Rettig, Morgan Sturgeon, Thomas L. Osborne, Ashley M. Jacobi, Rebecca Friedrich, Masako Monika Kaufmann, Julia Klermund, Simon Fink, Markus F. Templin, Andy Roemhild, Daniel R. Kaiser, Oliver Klein, Toni Cathomen, Mir‐Farzin Mashreghi, Fadi Issa, Julia K. Polansky, Hans-Dieter Volk, Michael Schmueck-Henneresse, Petra Reinke, Leila Amini
Unwanted alloimmune responses are a central driver of solid organ transplant rejection and currently managed with life-long immunosuppression, which imposes substantial risks and burdens on patients. Adoptive transfer of regulatory T cells (Tregs) offers a strategy to restore immunological balance and reduce long-term adverse effects of generalized immunosuppression. However, although Tregs can potently inhibit incipient immune activation, they struggle to suppress established memory effector T cells, necessitating the continued use of immunosuppression. Calcineurin inhibitors such as Tacrolimus effectively control both, newly activated and pre-existing effector T cells, but unfortunately, they also impair Treg function. Therefore, we hypothesized that gene-editing of Tregs inducing tacrolimus resistance (FKBP12 KO ) would enable combined therapy that curbs effector T cell responses without compromising Treg efficacy. Here, we developed FKBP12 KO -Tregs using a ribonucleoprotein-based CRISPR-Cas9 approach and characterized them extensively in vitro . FKBP12 KO -Tregs retained phenotype, high viability, and suppressive function comparable to unedited Treg WT , and they remained functionally impervious to Tacrolimus, while preserving sensitivity to alternative CNIs. We additionally established a good-manufacturing practice process for FKBP12 KO -Tregs. Comprehensive in vitro phenotypic, functional, and molecular characterization, together with the established manufacturing, provide the rationale for a proof-of-concept clinical trial assessing the feasibility and safety of co-administration of FKBP12 KO -Tregs with Tacrolimus in living-donor kidney transplant recipients.