Dual alpha-globin and truncated EPO receptor knockin restores hemoglobin production in alpha-thalassemia-derived red blood cells
Alpha-thalassemia is an autosomal recessive disease with increasing worldwide prevalence. The molecular basis is due to mutation or deletion of one or more duplicated alpha-globin genes, and disease severity is directly related to the number of allelic copies compromised. The most severe form, alpha-thalassemia major (alphaTM), results from loss of all four copies of alpha-globin and has historically resulted in fatality in utero. However, in utero transfusions now enable survival to birth. Postnatally, patients face challenges similar to beta-thalassemia, including severe anemia and erythrotoxicity due to imbalance of beta-globin and alpha-globin chains. While curative, hematopoietic stem cell transplantation (HSCT) is limited by donor availability and potential transplant-related complications. Despite progress in genome editing treatments for beta-thalassemia, there is no analogous curative option for patients suffering from alpha-thalassemia. To address this, we designed a novel Cas9/AAV6-mediated genome editing strategy that integrates a functional alpha-globin gene into the beta-globin locus in alphaTM patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the alpha-globin integration cassette dramatically increased erythropoietic output from edited HSPCs and led to the most robust production of alpha-globin, and consequently normal hemoglobin. By directing edited HSPCs toward increased production of clinically relevant RBCs instead of other divergent cell types, this approach has the potential to mitigate the limitations of traditional HSCT for the hemoglobinopathies, including low genome editing and low engraftment rates. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for alpha-thalassemia.