Gene-edited Allogeneic Regulatory T Cells as 'Off-the-shelf' Immunomodulatory Therapy for Transplantation

Abstract Text: Regulatory T cells (Tregs) are a pivotal component in maintaining immune homeostasis and have therapeutic potential in autoimmune diseases and transplant rejection. However, the clinical application of Tregs is hindered by several challenges, including donor variability, manufacturing complexities, and the time-intensive nature of Treg expansion. These limitations are particularly pronounced in acute disease phases or scenarios where autologous Tregs are sparse or dysfunctional. Here, we explored the feasibility and potency of unmatched, allogeneic Tregs as an 'off-the-shelf' immunomodulatory cell therapy. In a humanized skin transplant mouse model, allogeneic "third-party" Tregs displayed significantly reduced ability to suppress graft rejection. Gene editing of B2M and CIITA in human Tregs allowed efficient silencing of MHC class I and II, respectively. Targeted insertion of HLA-E cDNA provided partial protection of B2M-edited Tregs against allogeneic NK cell lysis in vitro. Silencing of MHC class II was necessary to shield allogeneic Tregs from killing by expanded allo-specific T cell lines in vitro. The combination of HLA-E-B2M knock-in and CIITA knockout enhanced the suppressive activity of allogeneic, unmatched Tregs in the humanized skin transplant model. With respect to long-term graft survival, allogeneic MHCI/II-edited Tregs had comparable suppressive activity to autologous Tregs. Peripheral blood analysis of humanized mice indicated the presence of circulating MHC-silenced Tregs at day 20 after cell infusion. Further in vivo experiments are ongoing to validate these results. Our results suggest that MHC gene editing can enable more efficacious allogeneic 'off-the-shelf' Tregs.