289. Safe and Effective Gene Therapy for Wiskott – Aldrich Syndrome Using an Insulated Lentiviral Vector

Wiskott–Aldrich Syndrome (WAS) is a severe X-linked immunodeficiency disorder characterized by recurrent infection, thrombocytopenia, and autoimmunity resulting from mutations within the WAS gene. The encoded gene product, WAS protein (WASp), is a scaffold protein important for activation of the actin cytoskeleton downstream of multiple hematopoietic receptors. Development of safe and effective hematopoietic stem cell (HSC) gene therapy for WAS would provide a valuable treatment alternative to allogeneic bone marrow transplantation. Of critical importance for such an approach is the capacity to achieve sufficient WASp expression in relevant lineages to correct the multiple deficits, while avoiding development of autoimmune disease. We previously tested a panel of self-inactivating (SIN) lentiviral (LV) vectors expressing human WAS via alternative internal promoters and observed optimal expression and rescue using the modified γ-retroviral derived LTR, MND. To improve the potential safety and stability of expression, we incorporated a 650bp chimeric insulator from the chicken β-globin locus into this LV. Using in vitro assays the insulated LV exhibited minimal LMO2 transactivation and no evidence for immortalization of cultured HSC. Next, we generated a stable high-titer producer clone expressing this candidate clinical LV, CL20-i650–MND-WAS. Here, we evaluate the efficacy and safety of the insulted vs uninsulated LV (CL20-MND-WAS) in a large cohort of primary and secondary transplanted Was/- mice. Both vectors enabled stable engraftment of WASp expressing cells in all hematopoietic lineages, positive selection of WASp+ cells, and rescued functional and developmental T and B cell defects without evidence for toxicity. Inclusion of the insulator allowed for higher expression levels at lower numbers of viral integration per cell; a feature that was further exaggerated in secondary recipients. Finally, in parallel, we evaluated CL20-i650–MND LV expressing a fluorescent marker using CD34+ autologous HSC transplants in a non-human primate model (Macaca nemestrina) and observed long term stable polyclonal engraftment of LV expressing hematopoietic cells for up to 2 yr. Based on these extensive pre-clinical and toxicity studies we anticipate that CL20-i650–MND-WAS LV will provide sustained rescue of platelet and lymphocyte development and function in vivo (facilitated, in part, via the insulator) without evidence of toxicity. We plan to directly test this hypothesis in an upcoming WAS LV clinical trial.