Evidence for efficacy and safety of lentiviral mediated gene transfer in Wiskott–Aldrich syndrome

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ABSTRACTS / Blood Cells, Molecules, and Diseases 40 (2008) 248–294

size difference was significant in favor of thal spleens over normal ones, both at steady state (19.42 ± 5.31% vs. 4.53 ± 1.56%, p b 0.001) and post G-CSF (31.6 ± 7.63% vs. 10.55 ± 2.27%, p b 0.001), the G-CSF effect on spleen size was stronger in normal than in thal strain (133% vs. 63% increase, respectively). G-CSF caused dramatic alterations in the histology of thal spleens represented by excessive hypercellularity with a greatly expanded red pulp dominating over the white one and intense trilineage hemopoiesis. HBBth-3mice mobilized less effectively than C57 mice (LSK+ cells 1.40 ± 0.87% vs. 3.35 ± 2.40%, respectively, p = 0.005) due to an increased trapping of HSCs and progenitors in the enlarged spleen (LSK+ 10,27 ± 2.85% vs. 6.06 ± 2.53%, p = 0.001). Splenectomy restored the mobilization efficacy in thal mice at comparable levels to normal strain whereas the liver after splenectomy developed compensatory extramedullary hemopoiesis which was greatly increased after G-CSF. Extrapolation of our data to the human situation predicts that non-splenectomized thalassemic patients may mobilize less efficiently than donors or patients without splenomegaly. This will probably require in non-splenectomized thalassemic patients multiple mobilization cycles in order to reach an adequate stem cell number for genetic correction. The excessive splenic hemopoiesis along with the cells’ activation by G-CSF may lead to splenic infarcts and therefore, strategies to reduce extramedullary hemopoiesis as well as the overall cell production (i.e., pretreatment with Hydroxyurea, hypertransfusion), should be explored in a clinical setting. doi:10.1016/j.bcmd.2007.10.071

level of WAS expression and functional correction after lentiviral transduction. Furthermore, CD34+ cells, isolated from mobilized peripheral blood or bone marrow of healthy donors, and transduced using WASP or GFP-encoding lentiviral vectors displayed a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation, with respect to untransduced cells. Transduction of CD34+ cells isolated from the bone marrow of a WAS patient was also performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. Efficacy and safety of gene therapy in a murine model of WAS were also investigated. WAS−/− bone marrow-derived HSC untransduced or transduced with a human WAS promoter/cDNA encoding lentiviral vector at low or high multiplicity of infection was injected into sub-lethally irradiated WAS−/− recipient mice. Mice were sacrificed 12 months after gene therapy. WASP expression was detected in bone marrow CD45+ cells and splenic myeloid cells, and in significantly higher percentages in splenic B, NK, and T cells. Gene therapy completely restored T cell function and B cell and platelet counts were increased in peripheral blood of mice treated with high MOI. Safety of gene therapy was also demonstrated with long-term survival of treated mice and no increase in tumor incidence. In conclusion, our data demonstrate that the WAS promoter/ cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in cells from WAS patients and can safely correct the immunodeficiency in the preclinical model. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients. doi:10.1016/j.bcmd.2007.10.072

62 Evidence for efficacy and safety of lentiviral mediated gene transfer in Wiskott–Aldrich syndrome Maria Grazia Roncarolo1,2, Marita Bosticardo1, Francesco Marangoni1,2, Samantha Scaramuzza1, Sara Trifari1, Anne Galy3, Loïc Dupré4, Luigi Naldini1,2, Alessandro Aiuti1, Anna Villa1,5 1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 2 Università Vita-Salute San Raffaele, Milan, Italy 3 Généthon, Evry, France 4 INSERM U563, Toulouse, France 5 ITB-CNR, Milan, Italy Wiskott–Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of the autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal

63 Random and targeted integration of adeno-associated virus vectors David W. Russell Division of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA Unlike retroviral vectors, AAV vectors do not contain an integrase or nuclease, so they rely entirely on host cell factors for transduction, leading to integration at sites of chromosomal DNA damage such as double strand breaks. Although less efficient than retroviral integration, a significant number of AAV vector integrations still occur after in vivo administration. In collaboration with Mark Sands, we have found that AAV vector integration at a specific locus on chromosome 12 in murine hepatocytes is associated with hepatocellular carcinoma. This is the first example of genotoxicity due to AAV and raises important safety concerns for this vector. One potential approach for avoiding insertional mutagenesis is to target specific chromosomal locations. AAV vectors are able to efficiently modify chromosomal target sites by