Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells

beta-thalassemias (beta-thal) are a group of blood disorders caused by mutations in the beta-globin gene (HBB) cluster. beta-globin associates with alpha-globin to form adult hemoglobin (HbA, alpha(2)beta(2)), the main oxygen-carrier in erythrocytes. When beta-globin chains are absent or limiting, free alpha-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of beta-thal correlates with the number of inherited alpha-globin genes (HBA1 and HBA2), with alpha-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat beta-thal based on genome editing of the alpha-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) alpha-globin downregulation, by deleting the HBA2 gene to recreate an alpha-thalassemia trait, and (2) beta-globin expression, by targeted integration of a beta-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of beta-thalassemia (human erythroid progenitor cell [HUDEP-2] beta(0)). Then, we edited healthy donor 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 beta-thal HSPCs and achieved correction of alpha/beta globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve alpha/beta globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for beta-thal.