Base editing mediated correction of severe ß0 thalassemia mutations

ß-thalassemia is a highly prevalent monogenic recessive disease caused by mutations affecting the synthesis of the adult hemoglobin ß-chains. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE), a new CRISPR/Cas9 derived genome editing tool, allows the effective introduction of point mutations at precise locations within the genome without generating double strand breaks. The two ß0 mutations CD39 (CAG>TAG) and IVS2-1 (G>A) are among the most common and severe ß-thalassemia mutations in the Mediterranean area and Middle East. We exploited the capacity of BE to correct these mutations in HSPCs from ß-thalassemia patients. We demonstrated that red blood cells in vitro derived from edited HSPCs exhibited high ß-globin levels and that the delayed erythroid differentiation typically observed in ß-thalassemic cell cultures was corrected by our treatment. Finally, xenotransplantation experiments showed base editing in HSCs and correction of the ß-thalassemic phenotype in vivo. Overall, our study provides in vitro and in vivo proof of efficacy of a BE approach to treat patients with prevalent and severe ß-thalassemia mutations. Overall design: Human beta-thalassemia HSPCs carrying the CD39 mutation were transfected using an adenine base editor to correct the CD39 mutation. As control, each of the 3 donors was mock-transfected with TE buffer. Bulk RNAseq was performed at 48h to evaluate the impact of the editing procedure at the transcriptomic level.