Dual α-globin and truncated erythropoietin receptor knock-in restores hemoglobin production in α-thalassemia major-derived hematopoietic stem and progenitor 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 -globin genes, and disease severity is directly related to the number of allelic copies compromised. The most severe form, -thalassemia major (TM), results from loss of all four copies of -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 -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 -thalassemia. To address this, we designed a novel Cas9/AAV6-mediated genome editing strategy that integrates a functional -globin gene into the {beta}-globin locus in TM patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the -globin integration cassette dramatically increased erythropoietic output from edited HSPCs and led to the most robust production of -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 -thalassemia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/555926v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1b63dacorg.highwire.dtl.DTLVardef@18b25a1org.highwire.dtl.DTLVardef@53835corg.highwire.dtl.DTLVardef@d52bf5_HPS_FORMAT_FIGEXP M_FIG C_FIG