Abstract
Gene therapy might fall short in achieving a complete reversion of the β-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 β-globin, impairing the therapeutic potential of the treatment. Our aim was to evaluate whether, in absence of complete reversion of the β-globin phenotype upon gene transfer, it is possible to use fetal hemoglobin induction to eliminate the residual α-globin aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines and erythroid precursor cells from β039-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 β-globin gene, showing that γ-globin mRNA expression and HbF production can be induced in the presence of high levels of β-globin gene expression and HbA accumulation. We then treated erythroid progenitor cells from β-thalassemic patients with T9W, which expresses the human β-globin gene and mithramycin separately or in combination. When transduction with our lentiviral vector is insufficient to completely eliminate the unpaired α-globin chains, combination of β-globin gene transfer therapy together with fetal hemoglobin induction might be very efficacious to remove the excess of α-globin proteins in thalassemic erythroid progenitor cells.
Highlights
In β-thalassemias, mutations of the β-globin gene or its regulatory regions cause absence (β0 phenotype) or reduced synthesis (β+ phenotype) of β-globin chains [1,2,3,4], and this impairment leads to an excess of the complementary αglobin chains [1]
We used K562 cellular clones described in Guerrini et al [43] and Lampronti et al [44] and transduced the cells with the recombinant pCCL.Promβ.HcRed1.Promγ.EGFP vector, carrying a green fluorescent protein (EGFP) and a red fluorescent protein (RFP) genes under the control of γ-globin and β-globin promoters, respectively [43, 44]
Using this experimental cellular system, the increase of green signal is consistent with a γ-globin promoter-driven transcriptional activity, while the increase of the far red (RFP) signal is associated with β-globin promoter activity [43]
Summary
In β-thalassemias, mutations of the β-globin gene or its regulatory regions cause absence (β0 phenotype) or reduced synthesis (β+ phenotype) of β-globin chains [1,2,3,4], and this impairment leads to an excess of the complementary αglobin chains [1]. Retroviral- or lentiviral-mediated insertion of single and multiple copies of the β-globin gene in ErPCs has been reported in many studies to demonstrate the feasibility of the gene therapy approach for the cure of β-thalassemia [11,12,13,14,15,16,17] This approach, while straightforward in its principle, exhibits several critical issues, the major being the control of transgene expression, which needs to be: (a) erythroid-specific, (b) differentiation- and stage-restricted, (c) elevated, (d) position-independent, (f) sustained over time and (g) independent from the patient's genotype [18,19,20,21]. Clinical trials based on gene therapy on β-thalassemic patients have been initiated, this therapeutic intervention was used on a restricted number of patients
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