Development of a pre-clinical model for gene therapy treatment of Wiskott-Aldrich Syndrome

Wiskott-Aldrich Syndrome (WAS) is a rare primary immunodeficiency caused by mutation of Wiskott-Aldrich Syndrome protein (WASp). Defective WASp functionality results in abnormal immunological synapse formation, impaired cellular migration and compromised cellular signaling. As a result, WAS patients present with frequent infections, thrombocytopenia, autoimmunity and increased risk of malignancies. The disease is fatal in the absence of bone marrow transplant and gene therapy is a promising alternative for patients lacking well-matched donors. We investigated selective advantage and promoter activity as variables regulating successful gene therapy treatment. While analyzing selective advantage of WASp+ cells, we identified a novel role of WASp in the function and homeostasis of invariant NKT (iNKT) cells. The absence of WASp resulted in abnormal iNKT development and impaired proliferation and cytokine production. These findings suggest that WASp has a crucial role in regulating homeostasis of iNKT cells. To model gene therapy treatment, we used lentiviral vectors (LVs) to introduce WASp cDNA into WASp-deficient hematopoietic stem cells (HSCs). We compared retroviral MND promoter with the mammalian WS1.6 promoter in vivo . MND LV resulted in endogenous WASp expression in all hematopoietic lineages and selection of WASp+ T, NKT and B cells. MND recipients exhibited normalized T cell proliferation and cytokine production and generated marginal zone (MZ) B cells. We observed a single instance of viral-mediated myeloid clonal expansion with MND LV. The WS1.6-huWASp LV exhibited substantially weaker promoter activity and resulted in partial selection of WASp T cells and minimal selection of WASp+ B cells. The WS1.6 recipients exhibited minimal rescue of MZ B cell formation and increased generation of autoimmune-prone B cells. Our findings identify the benefits and pitfalls of each lentiviral construct and will lead to improved gene therapy approaches for treating Wiskott-Aldrich Syndrome.