Lentiviral vector-mediated human ^Aγ-globin expression in erythrocytes derived from transplanted, β-thalassemic repopulating cells transduced with the d432-β-^Aγ lentiviral vectors.

(A) FACS analysis for human ^Aγ-globin expression (HbF-PE fluorescence) in β-thalassemic red cells 15 weeks following transplantation with γ-globin vector-transduced β-thalassemic BM cells. At top is the histogram from the β-thalassemic/γ-globin transgenic control animal. Below are histograms of γ-globin expression from representative mice (mouse numbers indicated) that received transplants of genetically modified cells (solid lines). The percentage of red cells with unequivocal γ-globin expression is shown at the right of each histogram along with the average PBL vector copy (VC) number. The dotted line indicates the staining profile of red cells from a mouse that received transplants of mock-transduced β-thalassemic BM cells. (B) Cellulose acetate Hb electrophoresis gels were used to separate the different Hb species of red cell lysates as indicated by the arrow to the right. This β-thalassemic mouse strain has the “diffuse” Hb pattern characterized by an uppermost mα₂mβ$2minor$ species and a faster migrating mα₂β$2maj$ species. Chimeric mα₂hγ₂ molecules migrate faster than the endogenous murine Hb species as demonstrated in the β-thalassemic/γ-globin transgenic mouse lane (Tg). No endogenous murine “single” Hb molecules, which migrate between the “diffuse” and chimeric species, were observed, indicating full donor engraftment. M indicates mouse that received transplants of mock-transduced β-thalassemic BM cells; numbered lanes represent samples from representative mice that received transplants of γ-globin vector-transduced β-thalassemic BM cells. % F indicates the quantity of the chimeric mα₂hγ₂species, estimated by densitometry, as a proportion of all Hb species. % F cells indicates the proportion of red cells staining for human γ-globin by FACS analysis. Vector copy number is the average copy number in PBLs as estimated by DNA PCR. For reference, the uppermost mα₂mβ$2minor$ band makes up approximately 20% of total mouse Hb.