Understanding the molecular basis and pathogenesis of hereditary optic neuropathies: towards improved diagnosis and management

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques 1,2 . Since previously described cytidine deaminases operate on single-stranded nucleic acids 3 , their use in base editing requires the unwinding of double-stranded DNA (dsDNA), for example, by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria 4 . Here we describe an interbacterial toxin, which we named DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

Mitochondria form a highly interconnected tubular network throughout the cell via a dynamic process, with mitochondrial segments fusing and breaking apart continuously. Strong evidence has emerged to implicate disturbed mitochondrial fusion and fission as central pathological components underpinning a number of childhood and adult-onset neurodegenerative disorders. Several proteins that regulate the morphology of the mitochondrial network have been identified, the most widely studied of which are optic atrophy 1 and mitofusin 2. Pathogenic mutations that disrupt these two pro-fusion proteins cause autosomal dominant optic atrophy and axonal Charcot-Marie-Tooth disease type 2A, respectively. These disorders predominantly affect specialized neurons that require precise shuttling of mitochondria over long axonal distances. Considerable insight has also been gained by carefully dissecting the deleterious consequences of imbalances in mitochondrial fusion and fission on respiratory chain function, mitochondrial quality control (mitophagy), and programmed cell death. Interestingly, these cellular processes are also implicated in more-common complex neurodegenerative disorders, such as Alzheimer disease and Parkinson disease, indicating a common pathological thread and a close relationship with mitochondrial structure, function and localization. Understanding how these fundamental processes become disrupted will prove crucial to the development of therapies for the growing number of neurodegenerative disorders linked to disturbed mitochondrial dynamics.

Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.