Key Points: CRISPR/Cas9-mediated disruption of the β-globin locus architecture reactivates fetal γ-globin expression in adult erythroblasts. Fetal γ-globin reactivation and sickle β-globin downregulation leads to the amelioration of the SCD cell phenotype.

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) β chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.

Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the −200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy – even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies.

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.

Microparticles (MPs) are submicron extracellular vesicles exposing phosphatidylserine (PS) detected in large quantities in the circulation of sickle cell anemia (SS) patients. Several groups studied the biological effects of MPs generated ex vivo. Here, we analyzed for the first time the impact of circulating MPs of 60 sickle cell disease (SCD) patients on endothelial cells (ECs). (1) SCD MPs were collected cross-sectionally, and compared to MPs isolated from healthy individuals (AA); whereas other plasma MPs were purified in a longitudinal manner, from SS patients (2) before and 2 years after hydroxyurea (HU) treatment onset, or (3) during a vaso-occlusive crisis and at steady state. SS MPs increased EC ICAM-1 mRNA and protein levels, as well as neutrophil adhesion, compared to AA MPs. We showed that ICAM-1 overexpression was mainly caused by MPs derived from erythrocytes, rather than from platelets, and that it was abolished by MPs PS capping using annexin V. MPs from SS patients treated with HU were less potent to induce a pro-inflammatory phenotype in ECs, compared to MPs collected before therapy. In contrast, MPs released during crisis increased ICAM-1 and neutrophil adhesion levels, in a PS-dependent manner, compared to MPs collected at steady state. Furthermore, neutrophil adhesion was abolished by a blocking anti-ICAM-1 antibody. Our study provides evidence that MPs play a key role in SCD pathophysiology by triggering a pro-inflammatory phenotype of endothelial cells. We also uncover a new mode of action for HU and identify potential therapeutics, namely annexin V and anti-ICAM-1 antibodies.