Recapitulating HPFH by CRISPR-Cas9 editing of γ-globin regulators to reactivate γ-globin expression

Hereditary persistence of fetal hemoglobin (HPFH) represents a clinically proven therapeutic model for β-hemoglobinopathies, where in persistent γ-globin (HBG1/HBG2) production compensates for impaired β-globin synthesis. To capitalize on this naturally occurring mechanism, we engineered a precise Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) strategy targeting HBG regulatory elements to therapeutically reactivate fetal hemoglobin. Through optimized Cas9: sgRNA ribonucleoprotein (RNP) delivery, we implemented a dual-editing approach: (1) B-cell lymphoma/leukemia 11A (BCL11A) binding site disruption to alleviate transcriptional repression and (2) HPFH-associated deletion recapitulation. This strategy achieved robust fetal hemoglobin (HbF) reactivation in multiple experimental systems, including K562 erythroleukemia cells and primary hematopoietic stem/progenitor cell (HSPC)-derived erythroid cultures. The successful translation of this approach could provide a transformative treatment paradigm for β-thalassemia and sickle cell disease patients.