Objective: Recent improvements in human beta-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 beta-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 beta-globin (beta87(+)) 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 beta87(+)-globin expression assessed.
Results: Analysis of the proportion of infected cells that became GFP(+) showed that this virus transduced approximately 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. beta87(+)-globin transcripts were readily detected in erythroblasts generated from primitive transduced CB cells and SCD progenitors. Evidence of beta87(+)-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 beta-globin protein expression in the erythroid progeny of transplantable human precursors.