Correcting a pathogenic mitochondrial DNA mutation by base editing in mice
Jose Domingo Barrera-Paez, Sandra R. Bacman, Till Balla, Derek J. Van Booven, Durga Prasad Gannamedi, James B. Stewart, Beverly Mok, David R. Liu, David B. Lombard, Anthony J. Griswold, Danny D. Nedialkova, Carlos T. Moraes
Abstract
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.