Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.

Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.

Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR.

Mitochondrial DNA (mtDNA) is present in multiple copies and phenotypic consequences of mtDNA mutations depend on the mutant load surpassing a specific threshold. Additionally, changes in mtDNA copy number can impact mitochondrial ATP production, resulting in disease. Therefore, the precise determination of mtDNA heteroplasmy and copy number is crucial to the study of mitochondrial diseases. However, current methods can be imprecise, and quantifying small changes in either heteroplasmy or copy number is challenging. We developed a new approach to measure mtDNA heteroplasmy using a single digital PCR (dPCR) probe. This method is based on the observation that fluorescent-labeled probes in dPCR exhibit different intensities depending on the presence of a single nucleotide change in the sequence bound by the probe. This finding allowed us to precisely and simultaneously determine mtDNA copy number and heteroplasmy levels using duplex dPCR. We tested this approach in two different models (human and mouse), which proved faster and more internally controlled when compared to other published methods routinely used in the mitochondrial genetics field. We believe this approach could be broadly applicable to the detection and quantification of other mixed genetic variations.