Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice
Kelcee A. Everette, Gregory A. Newby, Rachel M. Levine, Kalin Mayberry, Yoonjeong Jang, Thiyagaraj Mayuranathan, Nikitha Nimmagadda, Erin Dempsey, Yichao Li, Senthil Velan Bhoopalan, Xiong Liu, Jessie R. Davis, Andrew T. Nelson, Peter J. Chen, Alexander A. Sousa, Yong Cheng, John F. Tisdale, Mitchell J. Weiss, Jonathan Yen, David R. Liu
Abstract
Abstract Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the β -globin gene ( HBB ). Here we show that prime editing can correct the SCD allele ( HBB S ) to wild type ( HBB A ) at frequencies of 15%–41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBB A levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBB A , exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBB A -derived adult haemoglobin at 28%–43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBB S to HBB A , does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.