Hematopoietic stem cell gene editing rescues B cell development in X-linked agammaglobulinemia.

BACKGROUND: X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton's Tyrosine Kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. Here we report on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA.

METHODS: To rescue BTK expression, we employed a CRISPR/Cas9 system that creates a DNA double strand break in an early exon of the BTK locus and an AAV6 virus that carries the donor template for homology directed repair. We evaluated the efficacy of the gene editing approach in HSPCs from XLA patients that were cultured in vitro under B cell differentiation conditions or that were transplanted in immunodeficient mice to study B cell output in vivo.

RESULTS: A (feeder-free) B cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B cell maturation observed in XLA patients. Using this system, we could show the rescue of B cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B cell lineage compartment in the bone marrow and immunoglobulin production in the periphery.

CONCLUSIONS: Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, our work demonstrates the potential of this strategy as a future definitive therapy for XLA.