UCLA

X-Linked Agammaglobulinemia (XLA) is a primary immunodeficiency characterized by a lack of mature B lymphocytes and antibody production. Patients with XLA have loss of function mutations in the Bruton’s Tyrosine Kinase (BTK) gene. The standard of care for XLA is immunoglobulin supplementation, which has a profound effect on patient wellbeing and life expectancies. However, immunoglobulin supplementation requires frequent, expensive injections throughout a patient’s life and patients remain susceptible to certain recurring illnesses. The only permanent cure for XLA is an allogeneic hematopoietic stem cell (HSC) transplant, though it is rarely performed due to the associated risks. Gene therapy-based methods to replace or repair the BTK gene in autologous HSCs provide an alternative with the benefits of a permanent cure for XLA while circumventing much of the risk. While previous efforts to deliver a functional copy of the BTK gene using viral vector mediated gene transfer have shown promise, these vectors carry a risk of insertional oncogenesis which may not be tolerated for treatment of XLA. Instead, this dissertation lays a foundation for targeted integration of a functional copy of the BTK gene into HSCs using Cas9 endonuclease mediated gene editing to drastically reduce those risks. Initial work identified and optimized a target site for integration into both cell lines and primary cells. However, integration of the BTK sequence alone generated insufficient transgene expression. We identified three modifications that improved integration and expression of the construct: mutation of the protospacer adjacent motif, re-addition of the BTK terminal intron, and addition of the woodchuck hepatitis virus posttranscriptional regulatory elemental. Together, these modifications generated expression of transgenic BTK nearing wildtype levels in cell lines with seamless DNA integration. Finally, an unbiased comparison of three different methods of targeted integration (homology directed repair, homology independent targeted integration, and precise integration into target chromosome) was performed to identify the optimal donor design for treatment of XLA. This work has produced a novel treatment for XLA that is ready to progress into in vivo models and towards the clinic. The new, effective donor modifications may be invaluable for similar treatments in development for other genetic disorders.