To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated selfinactivating (SIN) lentiviral vector, termed GGHI, carrying the A c-globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of A c-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with b-thalassemia. Our results show that GGHI increased the production of c-globin by 32.9% as measured by high-performance liquid chromatography ( p = 0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of c-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.
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