Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice

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.