Induction of therapeutic levels of HbF in genome-edited primary beta(0)39-thalassaemia haematopoietic stem and progenitor cells

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of beta-thalassaemia. The homozygous mutation c.-196 C>T in the A gamma-globin (HBG1) promoter, which causes Sardinian delta beta(0)-thalassaemia, is able to completely rescue the beta-major thalassaemia phenotype caused by the beta(0)39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from beta(0)-thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous beta(0)39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of beta-thalassaemia irrespective of the beta-globin gene (HBB) mutations.