70. Long Term Correction of beta-Thalassemia by Transplantation of Transduced Hematopoietic Stem Cells

Abstract

Gene therapy for beta-thalassemia requires stable transfer of the beta-globin gene into hematopoietic stem cells (HSCs) and high and regulated Hb expression in the erythroblastic progeny. We developed a novel high-titer LV to express therapeutic level of Hb in one of the most severe murine model of beta-thalassemia. We cloned the human beta globin gene under the control of a minimal promoter and 2 elements of the LCR (HS2 and HS3), in the context of a SIN-18 LV. We evaluated the efficiency of this LV to correct the thalassemic phenotype in the th3/+ mouse mutant, that exhibit chronic anemia, anomalies in red cell size and shape, splenomegaly, extramedullary hematopoiesis (EMH) and iron accumulation in liver and spleen. The beta globin transgene expression was analyzed in the differentiated progeny of transduced HSCs, after transplantation into lethally irradiated thalassemic mice. Transplantation of LV- transduced HSCs lead to a high proportion (79%|[ndash]|100%) of donor RBCs expressing human beta globin, which remained stable up to 9 months after transplantation. HPLC analysis allowed the detection of the human beta globin chain, accounting for up to 43% of endogenous murine beta globin chains. The hematocrit, red blood cell count and hemoglobin level were markedly improved (HCT 44.90|[plusmn]|6.10; RBC 9.15|[plusmn]|1.22, HGB 12.07|[plusmn]|1.75) in comparison to the hematological parameters in control mice transplanted mock- transduced th3/+ cells (HCT 29.33|[plusmn]|2.08; RBC 5.79|[plusmn]|0.45, HGB 8.03|[plusmn]|1.12). The vector derived beta-globin synthesis was able to strongly reduced abnormalities in RBC morphology in mice transplanted with LV-transduced cells. FACS analysis of the erythroid TER119+ bone marrow subpopulations showed the restoration of normal erythroblastic maturation in the gene therapy- treated mice. Finally, the histopathological analysis demonstrated the correction of splenomegaly, EMH and drastic reduction of iron deposition in the spleen and liver. Quantitative PCR analysis revealed that the average proviral copy number ranged from 0.3 to 2.2, showing a direct correlation with the degree of phenotype correction. Analysis of single erythroid CFU-S, derived from secondary recipients, confirmed that the level of vector-derived human beta-globin was copy number dependent. Moreover, Southern Blot analysis of DNA extracted from transduced bone marrow samples indicated that the engraftment of a relatively small number of transduced HSCs was sufficient to fully correct the thalassemic phenotype in the th3/+ model. Finally, we demonstrated that LV-derived human beta globin expression was persistent and rescued the thalassemic phenotype in secondary transplanted th3/+ mice. Experiments are ongoing to test the therapeutic potential of the described LV to correct thalassemia major.