Hematopoietic stem cell gene therapy for the treatment of X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a rare inborn error of immunity caused by loss-of-function mutations in the gene encoding Bruton's tyrosine kinase (BTK). XLA patients lack mature B cells and have negligible antibody levels, leaving them susceptible to recurrent bacterial and chronic viral infections. Autologous hematopoietic stem cell gene therapy with gene-corrected HSC may serve as a promising treatment of XLA; this therapy would provide a one-time cure and would replace lifelong immunoglobulin replacement therapy. Due to the requirement of strict physiological regulation of gene expression, a site-specific editing strategy was designed to insert a cDNA transgene directly into its endogenous locus. To study the effectiveness of this therapy, murine lineage-negative hematopoietic cells from a murine model of XLA were edited using CRISPR-Cas9/rAAV6 then transplanted into recipient XLA mice. Myeloablated XLA mice that received transplantation of Btk-corrected Lin- cells displayed high levels of engraftment, significant increases in their B cell levels, increased production of various immunoglobulins, improved B cell development in the bone marrow, increased B cell receptor diversity, and the ability to produce antigen-specific antibodies following immunization. Collectively, we have modeled a gene therapy strategy in a disease model of XLA and extensively validated the site-specific genome editing approach.