Functional correction and genome integrity with duplex base editing of ß-thalassemic hematopoietic stem cells
ß-thalassemia is a prevalent monogenic disorder. Editing genetic modifiers such as the BCL11A erythroid enhancer and HBG promoters increases fetal hemoglobin (HbF) expression and represents a promising therapeutic strategy. Double-strand-break (DSB)–independent base editors (BEs) may offer safety advantages over CRISPR/Cas approaches, but comprehensive assessment of on- and off-target effects remains essential. Using primary patient-derived CD34? cells from three donors, we evaluated simplex and duplex BE targeting the BCL11A erythroid enhancer and the BCL11A binding site in the HBG promoter. Molecular and functional outcomes were assessed at the DNA, RNA, protein, and morphological levels and directly compared to DSB-based editing. CAST-seq was used to detect recombination events. Duplex BE achieved maximal HBG induction, robust ?-globin and HbF expression, and improved functional correction compared with simplex editing, while showing low levels of genomic alterations. RNA-seq analyses revealed comparable apoptotic and immune response signatures across editing strategies. Duplex BE targeting of BCL11A and HBG enables efficient functional correction while preserving genome integrity, supporting its therapeutic potential for ß-thalassemia.