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

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB(1). We used a custom adenine base editor (ABE8e-NRCH)(2,3) to convert the SCD allele (HBB(S)) to Makassar β-globin (HBB(G)), a non-pathogenic variant(4,5). 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% HBB(S)-to-HBB(G) conversion. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, HBB(G) frequency was 68% and bone marrow reticulocytes demonstrated a 5-fold decrease in hypoxia-induced sickling, indicating durable editing. To assess the physiological effects of HBB(S) base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse(6), followed by transplantation into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-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 ≥20% HBB(S)-to-HBB(G) 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 HBB(S), generates benign HBB(G), and minimizes undesired consequences of double-strand DNA breaks