Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

The promising ability to genetically modify hematopoietic stem and progenitor cells (HSPCs) by precise gene editing remains challenging due to their sensitivity and poor permissiveness. This represents the first evidence of implementing a gene editing strategy in a murinesafe harborlocus that phenotypically corrects primary cells derived from a mouse model of Fanconi anemia (FA).By co-delivering TALENs and a donor therapeuticFANCAcassette template to theMbs85locus (ortholog of the hAAVS1 safe harborlocus), we achieved efficient gene targeting (23%) in FA mouse embryonic fibroblasts (MEFs). This resulted in the phenotypic correction of these cells, as revealed by the improvement of their hypersensitivity to mitomycinC. Moreover, robust evidence of targeted integration was observed in murine WT and FA-A hematopoietic progenitor cells (HPC) reaching mean targeted integration values of 20.98% and 16.33% respectively, with phenotypic correction of FA HPCs. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy in a murinesafe harborlocus, such as theMbs85gene, of MEFs and murine HPC from a FA mouse model.