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

Prime editing can efficiently correct the sickle-cell allele to produce wild-type haemoglobin in patient haematopoietic stem cells that engraft efficiently in mice, yielding erythrocytes resistant to hypoxia-induced sickling. Sickle-cell disease (SCD) is caused by an A center dot T-to-T center dot A transversion mutation in the beta-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBBS) to wild type (HBBA) 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 HBBA 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 HBBA, exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBBA-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 HBBS to HBBA, does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.

Automated summary

Prime editing efficiently corrects the sickle-cell allele in patient haematopoietic stem cells, resulting in erythrocytes resistant to hypoxia-induced sickling. This correction was achieved at frequencies of 15%-41% in patients with sickle-cell disease. After transplantation into mice, the prime-edited cells maintained normal gene expression and displayed similar differentiation and maturation as unedited stem cells from healthy donors.