330. Lentiviral Vector Mediated Hematopoietic Stem Cell Gene Therapy Combined with Non-Lethal Conditioning Restores T Cell Function in the Murine Model of Wiskott-Aldrich Syndrome

Abstract

Top of pageAbstract Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. The present therapeutic strategy is based on transplantation of bone marrow from HLA-identical sibling donors. Because of the limited availability of such donors, the development of alternative approaches, such as hematopoietic stem cells (HSC) gene therapy is highly desirable. We previously demonstrated that lentiviral vectors encoding the Wiskott-Aldrich syndrome protein (WASP) can efficiently correct the functional defects of patients' T cells [Dupr|[eacute]| L, et al. Mol Ther. 2004, 10:903|[ndash]|15]. In the present study we investigated the efficacy of HSC gene therapy in a WAS knockout murine model (WKO) [Zhang J, et al. J Exp Med. 1999, 190:1329|[ndash]|42]. We tested three lentiviral vectors encoding for human WASP under the control of the following promoters: human PGK promoter (pW), full-length (1.6Kb) human WASP autologous promoter (1.6wW) or minimal (0.5 Kb) human WASP autologous promoter (0.5wW). Lineage depleted WKO female bone marrow cells were transduced with the different vectors and transplanted into non-lethally irradiated WKO male mice. Four months after gene therapy, chimerism in T cells, B cells and bone marrow was more than 80% donor. Human WASP expression was detected in T cells, B cells and platelets. Correction of IL-2 production and proliferation by splenic T cells was observed in the majority of the treated mice. T cell correction was achieved when a threshold number of viral integrations in the T cells was reached. Notably, the 1.6wW vector allowed functional T cell correction with the lowest lentiviral vector copy number (1 proviral integration per donor T cell). HSC gene therapy was well tolerated, and no malignancy occurred in more than 80 transplanted WKO mice, as indicated by blood analysis and autopsy. In conclusion, we demonstrate that HSC gene therapy combined with non-lethal conditioning allows robust engraftment and differentiation of transduced cells without toxicity. Correction of T cell proliferation and IL-2 production was highly reproducible. Future studies will determine whether a non myeloablative conditioning regimen based on busulfan leads to successful HSC gene therapy and whether, in this setting, genetically corrected T cells have a selective advantage over non corrected T cells.