A method for engineering high-fidelity regulatory T cells

SUMMARY Regulatory T cells (Tregs) dominantly suppress effector T cells, serving as a living therapeutic agent for immunological diseases such as type I diabetes and multiple sclerosis. However, Tregs exhibit a plastic fate at inflammatory conditions characterized by the loss of lineage identity and the acquisition of effector function, which compromises their efficacy. Furthermore, human Tregs cannot be reliably sorted at high purity by surface markers, because conventional T cells transiently upregulate Foxp3 expression upon activation. The presence of contaminating effector T cells would exacerbate inflammation. Technically, retroviral or lentiviral systems, which are widely employed for engineering Tregs, are susceptible to integration site-related effects known as position effects, resulting in substantial variations in gene expression levels. These factors pose challenges for modifying Tregs with enhanced performance for research and therapeutic applications. Therefore, expressing antigen-specific receptors or booster genes in Treg samples presents significant uncertainties. To address this issue, we hypothesize that tethering the Treg master regulator Foxp3 to genes of interest will generate Tregs with high lineage fidelity and robust regulatory function. In preliminary experiments, we began testing this hypothesis. We creatively combined CRISPR/Cas9 genome editing with bacteriophage integration system to insert genes of interest into the 3’ untranslated region (3’-UTR) of the endogenous Foxp3 gene. We leveraged CRISPR/Cas9 genome editing to first insert a landing pad attP site at Foxp3 3’-UTR and subsequently performed site-specific insertion of genes of interest via the attP-attB integration system. We validated this strat- egy in experimental mice and generated knock-in strains bearing Tregs with three representative features, demonstrating the feasibility of our approach. Our method surpasses ectopic Foxp3 expression via retroviral or lentiviral systems in preventing Treg fate loss. The latter cannot convert contaminating conventional T cells into fully functional Tregs, and Foxp3 expression is significantly influenced by the insertion sites. Based on preliminary results, we propose to rigorously test our hypothesis. We will establish a versatile platform to engineer Tregs by expressing genes of interest under the control of endogenous Foxp3 gene via its 5’-UTR or 3’-UTR. This will be first tested in murine Tregs through genome editing in germline. Subsequently, we will extend our approach to human primary Tregs and develop protocols for inserting genes into Foxp3 5’- UTR or 3’-UTR via sequential CRISPR/Cas9- and integrase-mediated insertions. Successful completion of our study will establish an innovative method to engineer Tregs with high lineage fidelity and robust expression of modifying genes, thereby minimizing the consequences of transdifferentiation of Tregs into disease-causing ef- fector T cells or of empowering contaminating conventional T cells. Tregs engineered through this approach will serve as a reliable source for both basic research and translational applications.