Evaluation of the effect of ALS20αI transduction on α-globin–KO cells. (A) Chromatograms from cation-exchange HPLC of hemolysates from nontransduced and ALS20αI–transduced HUDEP-αKO cells (clone 9). In nontransduced HUDEP-αKO cells, only a single peak corresponding to HbH (β₄) is visible, because of the lack of all α-globin chains. After transduction, HbA (α₂β₂) and HbF (α₂γ₂) peaks become visible, whereas HbH decreases. (B) Correlation between VCN and percent HbA + HbF + HbA₂ tetramers in HUDEP-αKO clones and α-globin-KO CD34⁺–derived erythroid cells. (C) Correlation between VCN and percent α-globin chains (top); correlation between VCN and percent single β-globin-like chains (β + γ + δ; bottom) detected by reversed-phase HPLC in hemolysates from terminally differentiated primary α-KO CD34⁺ cells or immortalized HUDEP-αKO erythroblast untreated or treated with ALS20αI. Gray bars represent values in healthy human erythroblasts. The values normalize at VCN of ∼4 for both α-globin–KO CD34⁺ and HUDEP-αKO cells, indicating that 1 copy of ALS20αI can generate as many α-globin chains as 1 endogenous α-globin gene.