CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells

Abstract

The prototypical genetic autoimmune disease is immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a severe pediatric disease with limited treatment options. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which plays a critical role in immune regulation. As a monogenic disease, IPEX is an ideal candidate for a therapeutic approach in which autologous hematopoietic stem and progenitor (HSPC) cells or T cells are gene edited ex vivo and reinfused. Here, we describe a CRISPR-based gene correction permitting regulated expression of FOXP3 protein. We demonstrate that gene editing preserves HSPC differentiation potential, and that edited regulatory and effector T cells maintain their in vitro phenotype and function. Additionally, we show that this strategy is suitable for IPEX patient cells with diverse mutations. These results demonstrate the feasibility of gene correction, which will be instrumental for the development of therapeutic approaches for other genetic autoimmune diseases.

Human‐engineered Treg‐like cells suppress FOXP3‐deficient T cells but preserve adaptive immune responses in vivo

“…This finding was despite the fact that patient GD0037 had been exposed to long-term immunosuppression and his T cells have residual expression of mutant FOXP3, while T cells from patient GD0064 were harvested at disease onset and had no FOXP3 expression. 12 These results further prove that LV-mediated Treg-like 'conversion' can be achieved in cells completely lacking FOXP3 expression, as well as T cells from patients being treated with ongoing immune suppression.…”

“…In agreement with our previous findings, CD4 LVFOXP3 cells generated from two new IPEX patients, GD0037 and GD0064, had a Treg‐like phenotype that was similar to the CD4 LVFOXP3 cells generated from healthy donors. This finding was despite the fact that patient GD0037 had been exposed to long‐term immunosuppression and his T cells have residual expression of mutant FOXP3, while T cells from patient GD0064 were harvested at disease onset and had no FOXP3 expression 12 . These results further prove that LV‐mediated Treg‐like ‘conversion’ can be achieved in cells completely lacking FOXP3 expression, as well as T cells from patients being treated with ongoing immune suppression.…”

“…An attempt that was not feasible at this time. To repopulate the mouse with human cells completely lacking FOXP3 expression, cells would need sequential editing, first CRISPR/Cas9 KO of FOXP3, followed by the knock‐in of the AAV6 construct‐expressing NGFR, allowing purification and in vivo injection of pure FOXP3 KO cells 12 . This set of experiments requires large numbers of HSPCs (autologous to the T cells from which CD4 LVFOXP3 cells are generated) which are difficult to obtain in a research setting but would be feasible in a preclinical development context.…”

IPEX as a Consequence of Alternatively Spliced FOXP3

“…Essential for this strategy is the targeting of splice-shifting elements that has shown promising potential in other isoform-related diseases (90,91). Alternatively, also gene editing that enhances FOXP3fl expression may provide a new approach to regulate human T-cell responses (76,92).…”

Gene Therapy for Primary Immunodeficiency

“… 140 Another recent study using gene editing tools to place FOXP3 cDNA under control of its native promoter reported partial correction of FOXP3 expression and suppressive function restored to within the lower range of healthy control cells, indicating this methodology could provide an alternative approach for IPEX patients. 141 …”