Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9 Enhanced Gene Replaceme

Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T–B+NK–). Although the functions of JAK3 have been extensively studied in mouse models, there are important differences in JAK3 mutant phenotypes between mouse and man; therefore, new experimental strategies are required to study lymphopoiesis in JAK3 deficient human cells. Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a compete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9 enhanced gene replacement restores normal T-cell development including the production of mature T-cell populations with a broad T-Cell Receptor (TCR) repertoire. Whole genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe a novel approach for the study of human lymphopoiesis and provide a foundation for gene replacement therapy in humans with immunodeficiencies.