CRISPR editing of HPFH3 genotype induces γ-globin expression and reverses sickle cell disease and β-thalassemia phenotypes.

backgroundHereditary persistence of Fetal Hemoglobin (HPFH) is a benign condition known to mitigate symptoms in individuals with co-inherited β-hemoglobinopathies, such as β-thalassemia (BT) and sickle cell disease (SCD), through the reactivation of fetal hemoglobin (HbF). HPFH typically arises from deletions of varying sizes affecting the β-globin gene cluster or point mutations in the promoters of the γ-globin genes. While the therapeutic benefits of point mutations have been extensively studied, the potential of deletional forms of HPFH remains underexplored in preclinical settings.

methodIn this study, we generated benign deletional HPFH3 genotype in SCD and BT patient-derived HSPCs using CRISPR/Cas9 and showed that therapeutically relevant levels of HbF reactivation result in the alleviation of the pathological phenotypes.

resultsIn edited cells derived from SCD patients, we observed reduced sickling and oxidative stress, while in edited from BT cells, restoration of the α-globin/β-globin ratio improved erythroid lineage maturation and reduced ROS levels. Importantly, HPFH3-edited HSPCs retained their genome integrity and showed no detrimental effect on their regeneration or differentiation into erythroid, myeloid, T, and B cell lineages in immunodeficient NBSGW mice post-xenotransplantation. Additionally, we showed a reduced interaction between the LCR and HBB, suggesting that the HPFH3 deletion specifically promoted LCR interactions with HBG1/2, likely due to the absence of the HBB locus.

conclusionsCollectively, our preclinical findings suggest that the generation of the HPFH3 genotype has the potential to significantly enhance HbF levels, offering a promising universal therapeutic strategy for treating both SCD and β-thalassemia.