Introducing a G-Makassar variant in HSCs by in vivo base editing treats sickle cell disease in mice. Homo sapiens

Precise repair of the pathogenic mutation in hematopoietic stem cells (HSCs) represents an ideal cure for patients with sickle cell disease (SCD). Here, we demonstrated correction of the SCD phenotype by converting the sickle mutation codon (GTG) into a benign G-Makassar variant (GCG) using in vivo base editing in HSCs. We demonstrated successful production of helper-dependent adenoviral vectors expressing an all-in-one base editor mapping to the sickle mutation site. In CD34+ cells from SCD patients, transduction with the base editing vector in vitro resulted in 35% GTG > GCG conversion and phenotypic improvements of derived red blood cells. In an ex vivo setting using lineage minus cells from a SCD mouse model, we achieved an average of 88% editing at the target site after ex vivo transduction with ensuing transplantation. Importantly, in vivo HSC base editing followed by selection generated 24.5% Makassar variant in long-term repopulating HSCs of SCD mice. The treated animals demonstrated correction of disease hallmarks without showing noticeable side effects. Off-target analyses at top-scored genomic sites revealed no off-target editing. Our in vivo approach requires one non-integrating vector, only intravenous/subcutaneous injections, and minimal in vivo selection. This technically simple approach has the potential for scalable applications in resource-limiting regions where SCD is prevalent.