Correcting a pathogenic mitochondrial DNA mutation by base editing in mice

Primary mitochondrial disorders are most often caused by deleterious mutations in the mitochondrial DNA (mtDNA). Here, we used a mitochondrial DddA-derived cytosine base editor (DdCBE) to introduce a compensatory edit in a mouse model that carries the pathological mutation in the mitochondrial transfer RNA (tRNA) alanine (mt-tRNA^Ala) gene. Because the original m.5024C→T mutation (G→A in the mt-tRNA^Ala) destabilizes the mt-tRNA^Ala aminoacyl stem, we designed a compensatory m.5081G→A edit (C→T in the mt-tRNA^Ala) that could restore the secondary structure of the tRNA^Ala aminoacyl stem. For this, the DdCBE gene construct was initially tested in an m.5024C→T mutant cell line. The reduced mt-tRNA^Ala amounts in these cells were increased after editing up to 78% of the mtDNA. Then, DdCBE was packaged in recombinant adeno-associated virus 9 (AAV9) and intravenously administered by retro-orbital injections into mice. Expression of the transduced DdCBE was observed in the heart and skeletal muscle. Total mt-tRNA^Ala amounts were restored in heart and muscle by the m.5081G→A edit in a dose-dependent manner. Lactate amounts, which were increased in the heart, were also decreased in treated mice. However, the highest dose tested of AAV9-DdCBE also induced severe adverse effects in vivo because of the extensive mtDNA off-target editing that it generated. These results show that although DdCBE is a promising gene therapy tool for mitochondrial disorders, the doses of the therapeutic constructs must be carefully monitored to avoid deleterious off-target editing.