Base editing of hematopoietic stem cells rescues sickle cell disease in mice

Sickle cell disease (SCD) is caused by a mutation in the beta-globin gene HBB. We used a custom adenine base editor (ABE8e-NRCH) to convert the SCD allele (HBBS) to Makassar beta-globin (HBBG), a non-pathogenic variant. Ex vivo delivery of mRNA encoding base editor with a targeting guide RNA into hematopoietic stem and progenitor cells (HSPCs) from SCD patients resulted in 80% HBBS-to-HBBG conversion. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, HBBG frequency was 68 percent and bone marrow reticulocytes demonstrated a 5-fold decrease in hypoxia-induced sickling, indicating durable editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse, followed by transplantation into irradiated mice. After sixteen weeks, Makassar beta-globin represented 79 percent of beta-globin protein in blood and hypoxia-induced sickling was reduced 3-fold. Mice receiving base-edited HSPCs showed rescue of hematologic parameters to near-normal levels and reduced splenic pathology compared to unedited controls. Secondary transplantation of edited bone marrow confirmed durable editing of long-term hematopoietic stem cells and revealed that greater than 20 percent HBBS-to-HBBG editing is sufficient for phenotypic rescue. Base editing of human HSPCs avoided p53 activation and larger deletions observed following Cas9 nuclease treatment. These findings suggest a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes undesired consequences of double-strand DNA breaks.