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

ABSTRACT: ?-thalassemias (?-thal) are a group of blood disorders caused by mutations in the ?-globin gene (HBB) cluster. ?-globin associates with ?-globin to form adult hemoglobin (HbA, ?2?2), the main oxygen-carrier in erythrocytes. When ?-globin chains are absent or limiting, free ?-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of ?-thal correlates with the number of inherited ?-globin genes (HBA1 and HBA2), with ?-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat ?-thal based on genome editing of the ?-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) ?-globin downregulation, by deleting the HBA2 gene to recreate an ?-thalassemia trait, and (2) ?-globin expression, by targeted integration of a ?-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of ?-thalassemia (human erythroid progenitor cell [HUDEP-2] ?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 ?-thal HSPCs and achieved correction of ?/? 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 ?/? globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for ?-thal.

SUBMITTER: Pavani G 

PROVIDER: S-EPMC7948300 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature