A novel FOXP3 knockout-humanized mouse model for pre-clinical safety and efficacy evaluation of Treg-like cell products

Forkhead box P3 (FOXP3) is an essential transcription factor for regulatory T cell (Treg) function. Defects in Tregs mediate many immune diseases including the monogenic autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is caused by FOXP3 mutations. Treg cell products are a promising modality to induce allograft tolerance or reduce the use of immunosuppressive drugs to prevent rejection, as well as in the treatment of acquired autoimmune diseases. We have recently opened a phase I clinical trial for IPEX patients using autologous engineered Treg-like cells, CD4(LVFOXP3). To facilitate the pre-clinical studies, a novel humanized-mouse (hu-mouse) model was developed whereby immune-deficient mice were transplanted with human hematopoietic stem progenitor cells (HSPCs) in which the FOXP3 gene was knocked out (FOXP3KO) using CRISPR-Cas9. Mice transplanted with FOXP3KO HSPCs had impaired survival, developed lymphoproliferation 10–12 weeks post-transplant and T cell infiltration of the gut, resembling human IPEX. Strikingly, injection of CD4(LVFOXP3) into the FOXP3KO hu-mice restored in vivo regulatory functions, including control of lymphoproliferation and inhibition of T cell infiltration in the colon. This hu-mouse disease model can be reproducibly established and constitutes an ideal model to assess pre-clinical efficacy of human Treg cell investigational products.