Correcting a pathogenic mouse mitochondrial DNA mutation by base editing

Primary mitochondrial disorders are most often caused by deleterious mutations in the mtDNA. Here, we harnessed a mitochondrial base editor, DdCBE, to introduce a compensatory edit (m.5081G￫A) in a mouse model that carries the pathological m.5024C￫T mutation in the mitochondrial tRNAAla gene. For this, the DdCBE gene construct was packaged in recombinant AAV9 and systemically injected into mice. We found that total mt-tRNAAla levels, which are drastically reduced by the mutation, are restored by the m.5081G￫A edit in a dose-dependent manner. However, an excessive expression of DdCBE also induces extensive mtDNA off-target editing, counteracting this positive outcome. To address this, we optimized the dosage to maximize the amount of compensatory edit generated with minimal off-target editing. These results show that mitochondrial base editors are promising candidates for gene therapy for mitochondrial disorders, but their expression need to be carefully controlled. tRNA-Seq datasets were generated from mouse embryonic fibriblasts (MEF) and mouse tissues investigate the impact of correcting a pathogenic mutation in the mitochondrial tRNA-Ala gene by base editing.