65. Long-Term Effects of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott-Aldrich Syndrome: Persistence of Functional Correction of T Cells and Lack of Malignant Trasformation
Authors: Francesco Marangoni, Loïc Dupré, Samantha Scaramuzza, Cristina Panaroni, Sara Trifari, Raisa Jofra Hernández, Adrian J. Thrasher, Anne Galy, Alessandro Aiuti, Luigi Naldini, Maria Grazia Roncarolo
Journal: Molecular Therapy (2006)

Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Transplantation of genetically corrected autologous HSC could represent an alternative treatment, potentially applicable to all patients. In a murine model of WAS (WAS−/−), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupré, Marangoni, et al. Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS−/− mice.Transduction of WAS−/− HSC was performed with two lentiviral vectors encoding human WASP under the control of either the human PGK promoter (pW) or the full-length (1.6Kb) human WAS autologous promoter (wW). Transduced HSC were transplanted into non-lethally irradiated WAS−/− mice. Mice were sacrificed either 7 months or 16 months after gene therapy. WASP expression was detected in approximately 50% and 40% of splenic T cells from mice treated with pW and wW, respectively. These expression levels were sufficient to fully correct TCR-driven proliferation and IL-2 production. Interestingly, the percentage of WASP-expressing cells was higher in FSChi and in CD44+ T cells, as compared to other T cell subsets. This finding suggests a selective advantage for gene corrected cells within activated and memory T cells. Additionally, WASP expression was detected in T cells, B cells and granulocytes isolated from peripheral blood, as well as in bone marrow CD45+ cells.The safety of the gene therapy treatment was evaluated by hemogram and histopathologic analysis of thymus, spleen, lymph nodes and bone marrow from gene therapy treated mice. In parallel, untransplanted age-matched WAS−/− and wild-type mice were tested as controls. Normal organ architecture and histology together with the absence of leukemias or lymphomas could be demonstrated in the gene therapy treated mice.In conclusion, we provide evidence of engraftment of WASP- expressing cells and correction of T cell defects without toxicity, up to 16 months after HSC gene therapy. Experiments aimed at investigating whether WAS gene therapy can correct the defects of platelets, B cells and dendritic cells, and restore normal in vivo immune response to pathogens are ongoing. Results from these studies will contribute to the design of a clinical trial for Wiskott- Aldrich Syndrome. Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Transplantation of genetically corrected autologous HSC could represent an alternative treatment, potentially applicable to all patients. In a murine model of WAS (WAS−/−), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupré, Marangoni, et al. Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS−/− mice. Transduction of WAS−/− HSC was performed with two lentiviral vectors encoding human WASP under the control of either the human PGK promoter (pW) or the full-length (1.6Kb) human WAS autologous promoter (wW). Transduced HSC were transplanted into non-lethally irradiated WAS−/− mice. Mice were sacrificed either 7 months or 16 months after gene therapy. WASP expression was detected in approximately 50% and 40% of splenic T cells from mice treated with pW and wW, respectively. These expression levels were sufficient to fully correct TCR-driven proliferation and IL-2 production. Interestingly, the percentage of WASP-expressing cells was higher in FSChi and in CD44+ T cells, as compared to other T cell subsets. This finding suggests a selective advantage for gene corrected cells within activated and memory T cells. Additionally, WASP expression was detected in T cells, B cells and granulocytes isolated from peripheral blood, as well as in bone marrow CD45+ cells. The safety of the gene therapy treatment was evaluated by hemogram and histopathologic analysis of thymus, spleen, lymph nodes and bone marrow from gene therapy treated mice. In parallel, untransplanted age-matched WAS−/− and wild-type mice were tested as controls. Normal organ architecture and histology together with the absence of leukemias or lymphomas could be demonstrated in the gene therapy treated mice. In conclusion, we provide evidence of engraftment of WASP- expressing cells and correction of T cell defects without toxicity, up to 16 months after HSC gene therapy. Experiments aimed at investigating whether WAS gene therapy can correct the defects of platelets, B cells and dendritic cells, and restore normal in vivo immune response to pathogens are ongoing. Results from these studies will contribute to the design of a clinical trial for Wiskott- Aldrich Syndrome.