作者
Gregory A. Newby,Jonathan Yen,Kaitly J. Woodard,Thiyagaraj Mayuranathan,Cícera R. Lazzarotto,Yichao Li,Heather Sheppard-Tillman,Shaina N. Porter,Yu Yao,Kalin Mayberry,Kelcee A. Everette,Yoonjeong Jang,Christopher J. Podracky,Elizabeth Thaman,Christophe Lechauve,Akshay Sharma,Jordana M. Henderson,Michelle F. Richter,Kevin T. Zhao,Shannon M. Miller,Tina Wang,Luke W. Koblan,Anton P. McCaffrey,John F. Tisdale,Theodosia A. Kalfa,Shondra M. Pruett-Miller,Shengdar Q. Tsai,Mitchell J. Weiss,David R. Liu
摘要
Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar β-globin (HBBG), a non-pathogenic variant4,5. 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 HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 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 HBBS-to-HBBG 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 HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.