IPEX (immunedysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a severe congenital autoimmune disorder in males resulting from hemizygous inheritance of a mutant FOXP3 allele. FOXP3 encodes a transcription factor that governs the development, maintenance, and function of regulatory T cells (Treg). We have developed a cell therapy strategy for treatment of IPEX using a gene-editing approach in which CRISPR/Cas9 RNPs are co-delivered with an AAV6 donor template designed to integrate into the FOXP3 locus an expression cassette containing the MND promoter driving expression of a functional FOXP3 cDNA and a surface LNGFR tag linked by a 2A ribosomal skip peptide. This approach enforces heterologous FOXP3 expression in IPEX CD4 effector T cells (Teff), while simultaneously eliminating expression of the endogenous FOXP3 allele. The resultant high level and stable expression of functional FOXP3 converts Teff to Treg-like cells with immunosuppressive activity. Using an optimized protocol, we obtained efficient HDR rates across multiple healthy donors. Edited cells were consistently enriched to >95% purity by a magnetic LNGFR antibody selection and expanded 50-fold in a week. Expression of FOXP3 cDNA in edited cells was sufficient to enforce Treg-like phenotypes including the up-regulation of Treg-associated markers (CD25, CTLA-4, and ICOS), and down-regulation of CD127 and inflammatory cytokines (IL2, IFNgamma, TNFalpha). Importantly, we demonstrate sustained in vivo suppressive activity of these edited Treg-like cells (edTreg) in a xeno-GvHD mouse model. edTreg (as well as expanded natural Treg) limited effector T cell expansion and tissue infiltration and significantly protected mice from xeno-GvHD induced by co-transferred autologous effector T cells. Along with preliminary data showing successful editing in CD4 T cells from IPEX patients, our data provide key pre-clinical proof-of-concept and safety data supporting use of edTreg in a clinical trial for IPEX and, potentially, for use in other autoimmune diseases.

Disclosures

Torgerson:Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership; Generation Bio: Equity Ownership.

Author notes

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Asterisk with author names denotes non-ASH members.