Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells

{beta}-thalassemia pathology is not only due to loss of {beta}-globin (HBB), but also erythrotoxic accumulation and aggregation of the {beta}-globin binding partner, -globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in {beta}-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize {beta}-globin:-globin mRNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in mice, establishing proof-of-concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing {beta}-thalassemia.