Recent clinical trials of severe combined immunodeficiency (SCID) gene therapy have highlighted both the therapeutic potential and the risks of retroviral vector (RV)-mediated gene transfer into hematopoietic stem cell (HSC). Objective of this project is to improve the safety and efficiency of transduction, and advance the prospective application of HSC-based gene therapy to the hemoglobinopathies. Although closely related to RV, advanced generation lentiviral vectors (LV) may have safety features and risk factors not present in RV. We will comparatively assess RV and LV integration, and explore strategies to improve its safety. In cell culture studies, we will assess integration site selection and transcriptional perturbation at the integration for both vector types. By transducing and transplanting HSC obtained from leukemia-prone Cdkn2alpha deficient mice, we will determine the oncogenic potential of each vector in vivo. Using these assays as readouts, we will then explore the possibility of generating safer integrating vectors, either by incorporation of chromatin insulator elements or by engineering lentiviral vectors for specific integration at defined chromatin sites. Concerning the efficiency of HSC transduction, a major factor in the successful reconstitution of the hematopoietic system by gene-corrected cells in the SCID trials was the selective growth advantage conferred in vivo to transduced cells, that enabled amplification of even a small input of transduced HSC. To broaden significantly the scope of HSC-based gene therapy, however, transduction of the majority of HSC in a transplant would be required. In addition, to apply such strategy to the therapy of hemoglobinopathies, high-level, erythroid lineage-specific gene expression must be reached. LV may provide the means to achieve these ambitious goals. We will identify critical factors affecting LV gene transfer into human HSC from different sources, i.e.. cord blood, bone marrow and mobilized peripheral blood. We will investigate whether the more efficient transduction obtained with cytokines is offset by decreased engraftment or long-term repopulation capacity of the transduced cells using the NOD/SCID, SCID/hu, and RAG2-/-gammac-/- double mutant mouse xenotransplant models. Testing different protocols and envelopes, we will aim at limiting the average vector integration level per cell while reaching a high transduction frequency. All these studies will identify the safest and most efficient vector and transduction protocol to evaluate beta globin gene transfer. In such optimized conditions, we will test different types of expression cassettes for efficient and lineage-specific beta globin expression, and test the therapeutic potential of LV-corrected cells in a preclinical lethal model of the disease, corresponding to human severe beta/0-thalassemia.
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