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

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 (CARs) have been used to redirect the specificity of Tregs, employing 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 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 which 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 KO Tregs via repetitive CD3/CD28-stimulation in a GMP-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 to polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.