Increased HbF and reduced HbS synthesis after treatment with pCL-ZF-Ldb1. (A) Lentiviral construct carrying ZF-Ldb1 and the GFP genes expressed under the ankyrin promoter (Pr) via an internal ribosomal entry site (IRES). (B) Percentage of HbF, HbS, and HbA₂ measured by high-performance liquid chromatography (HPLC) in representative erythroid cells untreated (top) or treated with ZF-Ldb1 (bottom). The transduced cells have, on average, 0.7 copies of viral molecules integrated per genome. (C) Changes in percentage of cells expressing HbF (left panels) and GFP (right panels), measured by flow cytometry. (D) Overall percentages of HbF, HbS, and HbA₂ changes in SCD samples treated with pCL-ZF-Ldb1 (n = 10, 5 samples were duplicated; average VCN = 1.2). (E) Hb (F + S + A₂) content in erythroid cells with or without pCL-ZF-Ldb1. (F) Ratio of β- or γ-globin chains over α-globin chains. The quantity of the globin chains (in μg) is measured via liquid chromatography under denaturing conditions (**P < .05). (G) Ratio of γ/β-like chains measured in 250 000 erythroid sickle cells untreated or treated with pCL-ZF-Ldb1 (n = 5, VCN = 1.6; ***P < .005 and **P < .05). FSC-H, forward scatter height; LTR, long terminal repeat; pA, polyadenylation signal; pCL, vector backbone; VCN, vector copy number; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.