MitoTALENs: A general approach to reduce mutant mtDNA loads and restore oxidative phosphorylation function in mitochondrial diseases

We have designed mitochondrially targeted Transcription Activator-Like Effector Nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA^(Lys) gene mutation associated with Myoclonic Epilepsy with Ragged-Red Fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh Syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analysed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials

Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin

Mitochondrial DNA Base Editing: Good Editing Things Still Come in Small Packages

The collaborative work of two HHMI groups, one at the University of Washington and the other at the Broad Institute of MIT and Harvard, led to the development of a novel molecular tool to edit single bases in the mtDNA (Mok et al., 2020). The collaborative work of two HHMI groups, one at the University of Washington and the other at the Broad Institute of MIT and Harvard, led to the development of a novel molecular tool to edit single bases in the mtDNA (Mok et al., 2020)

Cybrid technology

The study of the mitochondrial DNA (mtDNA) has been hampered by the lack of methods to genetically manipulate the mitochondrial genome in living animal cells. This limitation has been partially alleviated by the ability to transfer mitochondria (and their mtDNAs) from one cell into another, as long as they are from the same species. This is done by isolating mtDNA-containing cytoplasts and fusing these to cells lacking mtDNA. This transmitochondrial cytoplasmic hybrid (cybrid) technology has helped the field understand the mechanism of several pathogenic mutations. In this chapter, we describe procedures to obtain transmitochondrial cybrids

Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks

To investigate mtDNA recombination induced by multiple double strand breaks (DSBs) we used a mitochondria-targeted form of the Sca I restriction endonuclease to introduce DSBs in heteroplasmic mice and cells in which we were able to utilize haplotype differences to trace the origin of recombined molecules. Sca I cleaves multiple sites in each haplotype of the heteroplasmic mice (five in NZB and three in BALB mtDNA) and prolonged expression causes severe mtDNA depletion. After a short pulse of restriction enzyme expression followed by a long period of recovery, mitochondrial genomes with large deletions were detected by PCR. Curiously, we found that some Sca I sites were more commonly involved in recombined molecules than others. In intra-molecular recombination events, deletion breakpoints were close to or upstream of Sca I cleavage sites, confirming the recombinogenic character of DSBs in mtDNA. A region adjacent to the D-loop was preferentially involved in recombination of all molecules. Sequencing through NZB and BALB haplotype markers in recombined molecules enabled us to show that in addition to intra-molecular mtDNA recombination, rare inter-molecular mtDNA recombination events can also occur. This study underscores the role of DSBs in the generation of mtDNA rearrangements and supports the existence of recombination hotspots

Decreased mitochondrial tRNALys steady-state levels and aminoacylation are associated with the pathogenic G8313A mitochondrial DNA mutation.

Mutations in human mitochondrial tRNA genes cause a number of multisystemic disorders. A G-to-A transition at position 8313 (G8313A) transition in the mitochondrial tRNALys gene has been associated with a childhood syndrome characterized by gastrointestinal-system involvement and encephaloneuropathy. We have used transmitochondrial cybrid clones harbouring patient-derived mitochondrial DNA with the G8313A mutation for the study of the molecular pathogenesis. Our results showed that mutant mitochondrial cybrids respired poorly, and had severely defective mitochondrial protein synthesis and respiratory-chain-enzyme activity. Mutant cybrids also showed a marked decrease in tRNALys steady-state levels and aminoacylation, suggesting that these molecular abnormalities may underlie the pathogenesis of the mitochondrial G8313A mutation

Targeted Mitochondrial Genome Elimination

Mitochondrial diseases are a heterogeneous group of pathologies, presenting mitochondrial oxidative phosphorylation (OXPHOS) deficiency and subsequent deterioration of affected tissues, usually the ones with high energy demand. Nuclear and mitochondrial DNA (mtDNA) mutations have been associated with mitochondrial disorders, and only palliative therapies are available, with no approach available to correct the underlining genetic problem. Our and other groups, taking advantage of mtDNA heteroplasmy, were able to develop gene therapy strategies to target the mtDNA, inducing the increase of the wild-type haplotype. The gene editing tools used by our group and others such as specific endonucleases targeted to the mitochondria, and more recently mitoTALENs and mitochondrial Zinc-Finger Nucleases were shown to effectively shift mtDNA heteroplasmy. This change towards an increase in wild-type mtDNA molecules occurred when the nuclease was specifically designed to cleave DNA regions harboring point mutations or the common deletion. In this chapter we will focus on strategies to target specific mtDNA haplotypes to shift heteroplasmy in vitro and in vivo. A brief overview on heteroplasmic animal models and viral delivery will be introduced, as these will be critical for future translation of this approach into human gene therapy

Manipulating mitochondrial genomes in the clinic: playing by different rules

Recently, several publications have surfaced describing methods to manipulate mitochondrial genomes in tissues and embryos. With them, a somewhat sensationalistic uproar about the generation of children with ‘three parents’ has dominated the discussion in the lay media. It is important that society understands the singularities of mitochondrial genetics to judge these procedures in a rational light, so that this ongoing discussion does not preclude the helping of patients and families harboring mutated mitochondrial genomes

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double strand breaks (DSB) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In the present work we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results demonstrate the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders