Expression of an anti-sickling β-globin in human erythroblasts derived from retrovirally transduced primitive normal and sickle cell disease hematopoietic cells

Objective Recent improvements in human β-globin vector design have fueled interest in gene therapy approaches to the treatment of human thalassemia and sickle cell disease (SCD). The present study was undertaken to determine whether human β-globin mRNA and protein could be obtained in the erythroid progeny of more primitive human target cells transduced with a retrovirus containing murine stem cell virus long terminal repeats, a phosphoglycerate kinase promoter driving the expression of a green fluorescence protein (GFP) cDNA, and an anti-sickling β-globin (β87⁺) gene under the control of an HS2, HS3, HS4 enhancer cassette. Materials and Methods A two-step pseudotyping strategy was devised to obtain useful preparations of this virus. Primitive cells present in normal human cord blood (CB) and adult SCD patients' blood samples were infected and the level of gene transfer (% GFP⁺ cells) and erythroid-specific β87⁺-globin expression assessed. Results Analysis of the proportion of infected cells that became GFP⁺ showed that this virus transduced ∼50% of initial CD34⁺ CB and SCD cells and up to 23% of cells able to regenerate both lymphoid and myeloid cells in sublethally irradiated primary and secondary NOD/SCID mice. β87⁺-globin transcripts were readily detected in erythroblasts generated from primitive transduced CB cells and SCD progenitors. Evidence of β87⁺-derived protein in transduced CB cell-derived erythroblasts also was obtained. Conclusion These findings demonstrate that retroviral vector-based gene transfer approaches can be used to achieve human β-globin protein expression in the erythroid progeny of transplantable human precursors.