Correction of B-thalassemia by CRISPR/Cas9 editing of the a-globin locus in human hematopoietic stem cells. Homo sapiens

B-thalassemias (B-thal) are a group of blood disorders caused by mutations in the B-globin gene (HBB) cluster. B-globin associates with a-globin to form adult hemoglobin (HbA, a2B2), the main oxygen-carrier in erythrocytes. When B-globin chains are absent or limiting, free a-globins precipitate and damage cell membranes causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of B-thal correlates with the number of inherited a-globin genes (HBA1 and HBA2), with a-globin gene deletions having a beneficial effect for patients.Here, we describe a novel strategy to treat B-thal based on genome editing of the a-globin locus in human hematopoietic stem/progenitor cells (HSPCs).Using CRISPR/Cas9, we combined two therapeutic approaches: i) a-globin downregulation, by deleting the HBA2 gene to recreate an a-thalassemia trait and ii) B-globin expression, by targeted integration of a B-globin transgene downstream the HBA2 promoter.First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of B-thal (HUDEP-2 B0). Then, we edited healthy donor hematopoietic stem/progenitor cells (HSPCs) and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice.To assess the clinical potential of this approach, we next edited B-thal HSPCs and achieved correction of a/B globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing more precise editing with no InDels.Overall, we described an innovative CRISPR/Cas9 approach to improve a/B globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for B-thal.