Targeted integration of a β^AS3transgene in the α-globin locus corrects thalassemic phenotype in HUDEP-2 β⁰cells. (A) AAV6 donors used for KI experiments. Both vectors contain a promoterless β^AS3 transgene, followed by a phosphoglycerate kinase (PGK) promoter with a GFP reporter and simian virus pA. This cassette is flanked by 250-bp homology arms (homology) to gRNA genomic target. ITR, inverted terminal repeats. Top: β^AS3 cDNA followed by the woodchuck posttranscriptionally regulatory element (WPRE) and SV40 pA (β^AS3 cDNA); bottom: β^AS3 transgene that includes endogenous introns, 3′UTR and pA (β^AS3 full). A 1-kb scale bar is indicated at top. (B) Schematic representation of HUDEP-2 β⁰ targeting experiments. (C) KI efficiency of β^AS3 cDNA (blue) or β^AS3 full (red) in HUDEP-2 β⁰ cells measured by on-target ddPCR before and after sorting. (D) β^AS3 transcript upregulation in targeted HUDEP-2 β⁰ upon erythroid differentiation (qPCR, n = 2, mean ± SD). (E-F) HPLC analysis of globin monomers in differentiated HUDEP-2 β⁰. Representative chromatograms (E) and relative quantification (F) of β-like subunits are shown; (mean ± SD; n = 3). (G-H) HPLC analysis of globin tetramers in differentiated HUDEP-2 β⁰. Representative chromatograms (G) and HbA (α₂β₂) quantification (H) are shown (mean ± SD, n = 3; **P < .01; ANOVA, Tukey test). ns, not significant.