A combined approach for β-thalassemia based on gene therapy-mediated adult hemoglobin (HbA) production and fetal hemoglobin (HbF) induction

Gene therapy might fall short in achieving a complete reversion of the beta-thalassemic phenotype due to current limitations in vector design and myeloablative regimen. Following gene transfer, all or a large proportion of erythroid cells might express suboptimal levels of beta-globin, impairing the therapeutic potential of the treatment. Our aim was to evaluate whether, in absence of complete reversion of the beta-globin phenotype upon gene transfer, it is possible to use fetal hemoglobin induction to eliminate the residual alpha-globin aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines and erythroid precursor cells from beta(0)39-thalassemia patients were employed. Gene therapy was performed with the lentiviral vector T9W. Induction of fetal hemoglobin was obtained using mithramycin. Levels of mRNA and hemoglobins were determined by qRT-PCR and HPLC. First, we analyzed the effect of mithramycin on K562 transgenic cell lines harboring different copies of a lentiviral vector carrying the human beta-globin gene, showing that gamma-globin mRNA expression and HbF production can be induced in the presence of high levels of beta-globin gene expression and HbA accumulation. We then treated erythroid progenitor cells from beta-thalassemic patients with T9W, which expresses the human beta-globin gene and mithramycin separately or in combination. When transduction with our lentiviral vector is insufficient to completely eliminate the unpaired alpha-globin chains, combination of beta-globin gene transfer therapy together with fetal hemoglobin induction might be very efficacious to remove the excess of alpha-globin proteins in thalassemic erythroid progenitor cells.