Efficient reversion of the IVS1-110 (G>A) mutation in BT HSPCs corrects ineffective erythropoiesis. (A-C) Frequency of CD36⁺ (A), CD71⁺ (B), and GPA⁺ (C) cells at days 13, 16, and 20 of erythroid differentiation, as measured by flow cytometry analysis. Control BT or HD cells were either electroporated with TE buffer (TE), or only with SpRY-ABE8e mRNA (BE). Data are expressed as mean ± SEM (3 patients with BT and 3 HDs). ∗P ≤ .05; ∗∗P ≤ .01 (paired t test; BT-BE vs BT-cor). (D) Mean Fluorescence Intensity (MFI) in ROS-containing cells (DCFDA⁺) for control and edited (cor) BT samples. For the control, we pooled data obtained in RBCs derived from BT HSPCs electroporated with TE or with SpRY-ABE8e mRNA only (3 patients with BT). ∗P ≤ .05 (paired t test). (E) Flow cytometry histograms showing the frequency of apoptotic cells (annexin V⁺ cells) in the 7AAD⁻ cell population in unstained (Uns), BT and HD samples at day 13 of erythroid differentiation (3 patients with BT and 3 HDs). (F) Frequency of enucleated cells at days 13, 16, and 20 of erythroid differentiation, as measured by flow cytometry analysis of cells stained with the DRAQ5 nuclear dye (3 patients with BT and 3 HDs). Data for HD samples are expressed as mean ± SEM. (G) Cell size of enucleated cells at days 13, 16, and 20 of erythroid differentiation, as measured by flow cytometry analysis of the median FSC-A intensity (3 patients with BT and 3 HDs). Data for HD samples are expressed as mean ± SEM. ∗P ≤ .05; ∗∗P ≤ .01 (paired t test; BT-BE vs BT-cor). (H) Single RBC parameters (perimeter, surface, optical volume, dry mass, and surface density) evaluated by quantitative phase image microscopy in RBCs obtained from BT HSPCs (cor). As controls, we used RBCs derived from BT or HD HSPCs electroporated only with SpRY-ABE8e mRNA (BE). Data are expressed as mean ± SEM (3 patients with BT and 2 HDs). ∗∗∗∗P ≤ .0001 (Ordinary one-way ANOVA). cor, corrected; ctr, control; D, day.