Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo

Abstract

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

Mitochondrial Retinopathies

“…Idebenone overcomes mitochondrial complex I respiratory chain deficiency in patients with LHON by transferring electrons directly to mitochondrial complex III (by-passing complex I), thereby restoring cellular energy (ATP) production and re-activating inactive-but-viable RGCs, which ultimately prevents further vision loss and promotes vision recovery [ 170 ]. The approval of idebenone in the treatment of LHON was based on the overall data from a randomized clinical trial [ 171 ], retrospective assessment of off-label treated patients [ 172 ] and real-world data obtained from an expanded-access program [ 173 ]. Taken together, these studies provide convincing evidence that oral idebenone 900 mg/day for 24 weeks has persistent beneficial effects in preventing further vision impairment and promoting vision recovery in about 50% of patients with LHON relative to the natural course of the disease.…”

Recent Advances in Modeling Mitochondrial Cardiomyopathy Using Human Induced Pluripotent Stem Cells

“…This disadvantage motivated to develop alternative protein-only genome editing tools fused to a mitochondrial targeting signal, which enable to selectively modify mtDNA. Although these mitochondrial-specific transcription activator-like effector nucleases (mitoTALEN), zinc finger nucleases (mtZFN) and meganucleases (mitoARCUS) have not yet been tested in the human cardiac setting, these systems demonstrated certain success in selective degradation of mutant mtDNA by nuclease-mediated cleavage of mtDNA in both patients’ iPSCs ( Yahata et al, 2017 ; Yang et al, 2018 ) as well as in vivo ( Gammage et al, 2018b ; Zekonyte et al, 2021 ), thereby resulting in heteroplasmic shifts by decreasing the pathological heteroplasmy levels. Importantly, classical DNA repair mechanisms are limited in mitochondria ( Fu et al, 2020 ), which promotes major effects in edited cells, such as: 1) mitochondrial repair pathways of introduced double stand breaks in the mtDNA typically result in deletions that can manifest in a pathological phenotype, 2) nuclease-induced mtDNA linearization is rapidly recognized leading to rapid degradation of mtDNA and depletion of mitochondria ( Peeva et al, 2018 ), and 3) ineffective mtDNA repair may trigger a toxic accumulation of mitochondrial content in host cells, therefore, might enhance intrinsic immune surveillance mechanisms ( Tigano et al, 2021 ).…”

Interrogating Mitochondrial Biology and Disease Using CRISPR/Cas9 Gene Editing

“…More recently, Zekonyte and colleagues had shown effective application of the I-CreI meganuclease-based ARCUS gene editing platform, successfully shifting mtDNA heteroplasmy in a wide range of tissues to nearly wild type levels, except those in the central nervous system due to limitations of adeno-associated virus (AAV9) delivery. The mitoARCUS system is advantageous due to its smaller size and homodimeric nature, which ease vector packaging, as well as due to its high specificity with no nuclear off-target edits observed in tissues harvested from mice model tested compared to mitoZFNs and mitoTALENs [36].…”

“…As mitochondrial disease encompasses a heterogenous group of disorders, it can affect various cell or tissue types, which hampers the practical application of genome editing tools in the treatment of human mitochondrial diseases. Nonetheless, several nucleasemediated genome editing approaches, i.e., mitoZFNs, mitoTALENs, and mitoARCUS, have demonstrated robust pre-clinical evidence of cell-or tissue-specific gene targeting to correct pathogenic mtDNA variants in vivo using mouse models [34,36,72]. These genome editing tools show robust pre-clinical evidence for mtDNA heteroplasmy shifting both in vitro and in vivo, and together with the newly emerging mitochondrial base editors, are revealing promising avenues for translation into mitochondrial medicine.…”