Long-term efficacy and safety of hematopoietic stem cell gene therapy mediated by lentiviral vectors in the murine model of Wiskott-Aldrich syndrome

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haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA−CD62L−, CD28±CD27+, IL-2± IFN-γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA−CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generate central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHagspecific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect. doi:10.1016/j.bcmd.2007.10.021

12 Cancer stem cells: New therapeutic targets? Dominique Bonnet Cancer Research UK, London Research Institute, London, UK Acute myeloid leukemia (AML) is a clonal disorder defined by the accumulation of abnormally differentiated myeloid blasts. Because leukemic blasts have very limited proliferative capacity, it is believed that leukemic clone is maintain by a rare

population of leukemic stem cells (LSC) that have extensive proliferation and self-renewal capacities. Elucidating the nature of the target cell that undergoes leukemic transformation and characterizing the LSC is essential for both the understanding of the leukemogenic process and for the design of effective therapies. The development of an in vivo model that replicates many aspects of human AML had provide a mean to identify leukemic stem cells (termed the SCID-Leukemia Initiating Cells, SL-IC). SL-IC is defined by the ability of that cell to initiate AML in NOD/SCID mice. This in vivo assay provides the foundation of an assay to define the biological and molecular properties of such leukemic stem cells (LSC). Despite the clear importance of the LSC in the genesis and perpetuation of leukemic disease, little is currently known about the biological and molecular properties that make LSCs distinct from normal hematopoietic stem cells. The presentation will summarize the work done using the xenograft system to characterise the nature of the leukemic clone and will specifically highlights the advances made in phenotypically, molecularly and functionally defining LSC. It will also discuss why leukemic stem cells represent new therapeutic targets and what means we might be able to use to targets them. doi:10.1016/j.bcmd.2007.10.022

13 Long-term efficacy and safety of hematopoietic stem cell gene therapy mediated by lentiviral vectors in the murine model of Wiskott-Aldrich syndrome Marita Bosticardo1,∗, Francesco Marangoni1,2,∗, Michela Locci2, Elena Draghici2, Samantha Scaramuzza2, Cristina Panaroni2, Anne Galy3, Luigi Naldini1,2, Alessandro Aiuti2, Loic Dupré2,4, Maria Grazia Roncarolo1,2, Anna 1 Vita-Salute San Raffaele University, Milan, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 3 Généthon UMR CNRS 8115, Evry, France 4 INSERM U563, Toulouse, France 5 Istituto di Tecnologie Biomediche, Segrate, Milan, Italy *Contributed equally to this work. Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by thrombocytopenia, eczema and increased risk of autoimmunity and malignancies. HLAmatched bone marrow transplantation is curative; however, hematopoietic stem cell (HSC) gene therapy could represent an alternative treatment for patients lacking a suitable donor. The aim of the present study was to investigate the long-term efficacy and safety of gene therapy in a large cohort of WAS−/− mice. WAS−/− bone marrow-derived HSC untransduced or transduced with a human WAS promoter/cDNA encoding lentiviral vector at low or high multiplicity of infection (MOI 10–20 or 200) were injected into sub-lethally irradiated WAS−/−

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

recipient mice (untransduced n = 24; low MOI n = 48; high MOI n = 20). Mice were sacrificed 12 months after gene therapy. Full donor engraftment was detected in all groups. WASP expression was detected in 15–30% of bone marrow CD45+ cells and splenic myeloid cells, and in significantly higher percentages (up to 70%) of splenic B, NK, and T cells, thus indicating a selective advantage for lymphoid cells. Gene therapy completely restored T cell proliferation, IL-2 production, secretion of Th1 and Th2 cytokines, and B cell migration to CXCL13. Finally, B cell and platelet counts were increased in peripheral blood of mice treated with high MOI. Safety of gene therapy was also demonstrated. Long-term survival of mice treated with low or high MOI was comparable to that of control mice. Tumors arose in the same percentage (20%) in gene therapy and control groups, and they were all of host origin. In conclusion, we demonstrated efficacy and safety in a mouse model of WAS gene therapy, up to 12 months after treatment. Experiments aimed at correcting platelet defects and in vivo immune responses by WAS gene therapy are ongoing. Results from these studies will contribute to the design of a clinical trial for Wiskott-Aldrich syndrome. doi:10.1016/j.bcmd.2007.10.023

14 Initial evaluation of oncoretroviral vectors carrying HIV-1 inhibitor gene into rhesus CD34+ cells and/or CD4+ T cells: An in vivo model for the gene therapy of AIDS Stephen E. Braun1, Fay Eng Wong1, Michelle Connole1, Ran Taube2, Akikazu Murakami2, Julianna Lisziewicz3, Wayne A. Marasco2, R. Paul Johnson1 1 Division of Immunology, NEPRC, Harvard Medical School, Southborough, MA, USA 2 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA 3 Research Institute for Genetic and Human Therapy, Washington, DC, USA Despite the initial success of highly active antiretroviral therapy in inhibiting viral replication in HIV-infected individuals, there is a compelling need for the development of complementary therapies. The protection of susceptible cells from viral replication may be achieved with inhibitor genes by transduction and adoptive transfer of CD4+ T cells or by transduction of hematopoietic stem cells and differentiation into CD4+ T cells. Studies in nonhuman primates offer the opportunity to experimentally address basic questions regarding stem cell gene therapy for AIDS in an in vivo disease model. For the adoptive T cell studies, we optimized transduction of primary rhesus CD4+ T cells with 3 days of anti-CD3/antiCD28 stimulation and one exposure to 2 MOIs of viral particles on Retronectin; marking was routinely between 60% to 80% in vitro. After leukapheresis and isolation of 135 and 116 × 106 CD4+ T cell from two animals, we stimulated and transduced

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the CD4+ T cells with the therapeutic and the control vectors, and then expanded and reinfused them on Day 10 or 9 without further conditioning. Gene marking peaked in PBMC day at 0.6% and 6.8% after 1 day and was detected in the LN at 0.2% and 2.8% on day 7. In the first animal, gene marking was only detected in one lymph node at 0.007% and in the spleen at 0.034% after 26 weeks. For the autologous stem cell transplantation studies, we optimized transduction of rhesus CD34+ bone marrow cells with GaLV- and amphotropicpseudotyped vectors and induced CD4+ T cell differentiation during thymic stroma culture in vitro. With 2 days of prestimulation (Tpo, Flt3L, SCF) and exposure to viral particles preloaded on Retronectin (3 days); transduction of bulk CD34+ cells ranged from 1.8 to 8.2 copies per cell, CFU ranged from 60% to 90% positive, and CD4+ T cells ranged from 0.4 to 0.9 copies per cell. After priming two animals with G-CSF and SCF, BM cells were aspirated or mPB cells were collected by leukapheresis. The rhesus CD34+ cells were isolated, transduced with the therapeutic and the control vectors, and reinfused 1 day after 400 rads TBI. Gene marking in granulocytes peaked at 8.8% and 14% after 2 weeks and leveled out at 0.14% and 0.01% after 6 weeks. Gene marking in PBMC reached 3.5% and 6.5% after 2 weeks and leveled out at 0.61% and ∼ 0.04% at 10 weeks. Marking in sorted CD4+ and CD8+ T cell populations ranged from 0.25% and 0.46%, with 90% of the marking in naive cells versus memory cells. In conclusion, these studies indicate that the in vitro gene transfer frequencies are high, but that stable long-term in vivo engraftment of transduced cells may require additional conditioning regimens. These studies in the rhesus macaque model will have important implications in advancing stem cell gene therapy for AIDS. doi:10.1016/j.bcmd.2007.10.024

15 UCOE (ubiquitous chromatin opening element) mediates copy dependent expression of gp91phox in lentiviral vectors Christian Brendel1, Stefan Stein1, Michael Antoniou2, Manuel Grez1 1 Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, 60596 Frankfurt, Germany 2 Nuclear Biology Group, Department of Medical and Molecular Genetics, Kings College London School of Medicine, Guy’s Campus, London, SE1 9RT, UK Chronic Granulomatous Disease (X-CGD) is a rare inherited immunodeficiency caused by mutations in any of the subunits of the phagocytic NADPH oxidase. Due to the lack of NAPDH oxidase activity, neutrophils of CGD patients are unable to generate superoxide which is required for the killing of intracellular pathogens. As a consequence, CGD patients suffer from severe and recurrent life-threatening infections. One strategy for the treatment of patients lacking a matched donor for bone marrow transplantation is a gene replacement