Gene replacement of [alpha]-globin with [beta]-globin restores hemoglobin balance in [beta]-thalassemia-derived hematopoietic stem and progenitor cells

[beta]-Thalassemia pathology is due not only to loss of [beta]-globin (HBB), but also to erythrotoxic accumulation and aggregation of the [beta]-globin-binding partner, [alpha]-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:[alpha]-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing [beta]-thalassemia.