ABE-mediated correction of the IVS1-110 (G>A) mutation in BT HSPCs restores normal Hb production in their erythroid progeny. (A) The frequency of corrected alleles (BE) and indels as assessed by Sanger sequencing in BTHSPCs. Data are expressed as mean ± SEM (3 donors). The frequency of corrected alleles in the cells obtained from the compound heterozygous patient (BT2) was corrected considering only the alleles harboring the IVS1-110 (G>A) mutation. The dotted line represents the maximum background noise of indels calculated by TIDE. (B) RT-qPCR using primers detecting exclusively correctly spliced β-globin mRNAs in erythroid cells derived from BT HSPCs (cor). β-globin expression was normalized to α-globin. Data are expressed as mean ± SEM. The dotted lines indicate the maximum and minimum values observed in HD cells. (C) RT-PCR using primers amplifying a region spanning the HBB exon 1-exon 2 junctions. cDNA was obtained from erythroid cells derived from BT HSPCs (cor). (D) Expression of β-, ^Gγ-, ^Aγ- and δ- globin chains measured by RP-HPLC in BT and HD RBCs. β-like-globin expression was normalized to α-globin. The α-/non–α-globin ratio is reported on top of the graph. RBCs were obtained from BT HSPCs (cor). Data are expressed as mean ± SEM. (E) Analysis of HbA, HbF, and HbA₂ by CE-HPLC in BT and HD RBCs. We calculated the percentage of each Hb type over the total Hb tetramers. RBCs were obtained from corrected BT HSPCs (cor). (B-C) As controls, we used erythroid cells derived from BT or HD HSPCs electroporated only with SpRY-ABE8e mRNA (BE) (3 patients with BT and 2 HDs). (D-E) As controls, we pooled data obtained in RBCs derived from BT or HD HSPCs electroporated with TE or with SpRY-ABE8e mRNA only (3 patients with BT and 2 HDs). Data are expressed as mean ± SEM. cor, corrected; ctr, control.