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 ) into Makassar β-globin ( HBB G ), a non-pathogenic variant 4 , 5 . Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB S to HBB G . Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBB G was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene 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 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBB S -to- HBB G editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBB S , generates benign HBB G , and minimizes the undesired consequences of double-strand DNA breaks.