ABSTRACT
Mutations within mtDNA frequently give rise to severe encephalopathies. Given that a majority of these mtDNA defects exist in a heteroplasmic state, we harnessed the precision of mitochondrial-targeted TALEN (mitoTALEN) to selectively eliminate mutant mtDNA within the CNS of a murine model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T). This targeted approach was accomplished by the use of AAV-PHP.eB and a neuron-specific synapsin promoter for effective neuronal delivery and expression of mitoTALEN. We found that most CNS regions were effectively transduced and showed a significant reduction in mutant mtDNA. This reduction was accompanied by an increase in mitochondrial tRNA alanine levels, which are drastically reduced by the m.5024C>T mutation. These results showed that mitochondrial-targeted gene editing can be effective in reducing CNS-mutant mtDNA in vivo, paving the way for clinical trials in patients with mitochondrial encephalopathies.
ABSTRACT
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.
Subject(s)
DNA, Mitochondrial , MELAS Syndrome , Humans , Animals , Mice , DNA, Mitochondrial/genetics , MELAS Syndrome/genetics , MELAS Syndrome/metabolism , Mitochondria/genetics , Mitochondria/metabolism , MutationABSTRACT
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.
Subject(s)
DNA, Mitochondrial , Heteroplasmy , Humans , Animals , Mice , DNA, Mitochondrial/genetics , DNA Copy Number Variations , Mitochondria/genetics , Polymerase Chain ReactionABSTRACT
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.
Subject(s)
DNA Restriction Enzymes/metabolism , DNA, Mitochondrial/metabolism , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Mitochondrial Diseases/therapy , Animals , DNA Restriction Enzymes/genetics , DNA, Mitochondrial/genetics , Dependovirus/genetics , Disease Models, Animal , Fibroblasts , Gene Editing/methods , Genetic Vectors/genetics , HeLa Cells , Humans , Mice , Mice, Transgenic , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Point Mutation , Primary Cell Culture , RNA, Transfer, Ala/geneticsABSTRACT
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).
Subject(s)
Cytidine Deaminase , DNA, Mitochondrial , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Mitochondria/geneticsABSTRACT
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.
Subject(s)
Cytological Techniques/methods , Cytoplasm/metabolism , Hybrid Cells/metabolism , Animals , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Humans , Mice , Mitochondria/metabolismABSTRACT
Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.
Subject(s)
DNA, Mitochondrial/genetics , Genes, Mitochondrial , Heteroplasmy/genetics , Animals , COS Cells , Chlorocebus aethiops , Female , HeLa Cells , Humans , Mice , Mutation/genetics , Plasmids/genetics , Transcription Activator-Like Effector Nucleases , Zinc Finger Nucleases/metabolismABSTRACT
Myopathies are common manifestations of mitochondrial diseases. To investigate whether gene replacement can be used as an effective strategy to treat or cure mitochondrial myopathies, we have generated a complex I conditional knockout mouse model lacking NDUFS3 subunit in skeletal muscle. NDUFS3 protein levels were undetectable in muscle of 15-day-old smKO mice, and myopathy symptoms could be detected by 2 months of age, worsening over time. rAAV9-Ndufs3 delivered systemically into 15- to 18-day-old mice effectively restored NDUFS3 levels in skeletal muscle, precluding the development of the myopathy. To test the ability of rAAV9-mediated gene replacement to revert muscle function after disease onset, we also treated post-symptomatic, 2-month-old mice. The injected mice showed a remarkable improvement of the mitochondrial myopathy and biochemical parameters, which remained for the duration of the study. Our results showed that muscle pathology could be reversed after restoring complex I, which was absent for more than 2 months. These findings have far-reaching implications for the ability of muscle to tolerate a mitochondrial defect and for the treatment of mitochondrial myopathies.
Subject(s)
Electron Transport Complex I/genetics , Genetic Therapy , Mitochondrial Myopathies , Animals , Electron Transport Complex I/deficiency , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria , Mitochondrial Myopathies/genetics , Mitochondrial Myopathies/metabolism , Muscle, Skeletal/metabolism , NADH Dehydrogenase/geneticsABSTRACT
In the version of this article originally published, there was an error in Fig. 1a. The m.5024C>T mutation, shown as a green T, was displaced by one base. The error has been corrected in the print, HTML and PDF versions of this article.
ABSTRACT
Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system1. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules1. Using a mouse model with a heteroplasmic mtDNA mutation2, we tested whether mitochondrial-targeted TALENs (mitoTALENs)3,4 could reduce the mutant mtDNA load in muscle and heart. AAV9-mitoTALEN was administered via intramuscular, intravenous, and intraperitoneal injections. Muscle and heart were efficiently transduced and showed a robust reduction in mutant mtDNA, which was stable over time. The molecular defect, namely a decrease in transfer RNAAla levels, was restored by the treatment. These results showed that mitoTALENs, when expressed in affected tissues, could revert disease-related phenotypes in mice.
Subject(s)
Heart/physiopathology , Mitochondrial Diseases/genetics , Muscle, Skeletal/physiopathology , Transcription Activator-Like Effector Nucleases/genetics , Animals , DNA, Mitochondrial/genetics , Disease Models, Animal , Humans , Mice , Mitochondria, Heart/genetics , Mitochondria, Heart/pathology , Mitochondria, Muscle/genetics , Mitochondria, Muscle/pathology , Mitochondrial Diseases/physiopathology , Mitochondrial Diseases/therapy , Oxidative Phosphorylation , Point Mutation/genetics , Transcription Activator-Like Effector Nucleases/therapeutic useABSTRACT
Pathogenic mitochondrial DNA (mtDNA) mutations often co-exist with wild-type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70-90%). Previously, our laboratory showed that mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY-YIG homing nuclease from T4 phage (I-TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev-TALEs) could specifically bind and cleave mtDNA of patient-derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged-red fibers (MERRF). We tested two mitoTev-TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev-TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev-TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues.
Subject(s)
DNA, Mitochondrial/metabolism , Endonucleases/metabolism , Molecular Targeted Therapy/methods , Recombinant Proteins/metabolism , Transcription Activator-Like Effector Nucleases/metabolism , Viral Proteins/metabolism , Cells, Cultured , DNA Repair , Endonucleases/genetics , Humans , Hydrolysis , MERRF Syndrome/drug therapy , Protein Binding , Recombinant Proteins/genetics , Transcription Activator-Like Effector Nucleases/genetics , Viral Proteins/geneticsABSTRACT
Double-strand breaks in the mitochondrial DNA (mtDNA) result in the formation of linear fragments that are rapidly degraded. However, the identity of the nuclease(s) performing this function is not known. We found that the exonuclease function of the mtDNA polymerase gamma (POLG) is required for this rapid degradation of mtDNA fragments. POLG is recruited to linearized DNA fragments in an origin of replication-independent manner. Moreover, in the absence of POLG exonuclease activity, the prolonged existence of mtDNA linear fragments leads to increased levels of mtDNA deletions, which have been previously identified in the mutator mouse, patients with POLG mutations and normal aging.
Subject(s)
DNA Polymerase gamma/metabolism , DNA, Mitochondrial/metabolism , Mitochondria/metabolism , Sequence Deletion , Animals , Base Sequence/genetics , Cells, Cultured , DNA Breaks, Double-Stranded , DNA, Mitochondrial/genetics , Fibroblasts , Mice , Mice, Transgenic , Mutation , Primary Cell CultureABSTRACT
We observed that the transient induction of mtDNA double strand breaks (DSBs) in cultured cells led to activation of cell cycle arrest proteins (p21/p53 pathway) and decreased cell growth, mediated through reactive oxygen species (ROS). To investigate this process in vivo we developed a mouse model where we could transiently induce mtDNA DSBs ubiquitously. This transient mtDNA damage in mice caused an accelerated aging phenotype, preferentially affecting proliferating tissues. One of the earliest phenotypes was accelerated thymus shrinkage by apoptosis and differentiation into adipose tissue, mimicking age-related thymic involution. This phenotype was accompanied by increased ROS and activation of cell cycle arrest proteins. Treatment with antioxidants improved the phenotype but the knocking out of p21 or p53 did not. Our results demonstrate that transient mtDNA DSBs can accelerate aging of certain tissues by increasing ROS. Surprisingly, this mtDNA DSB-associated senescence phenotype does not require p21/p53, even if this pathway is activated in the process.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA, Mitochondrial/metabolism , Tumor Suppressor Protein p53/metabolism , Acetylcysteine/pharmacology , Aging , Animals , Apoptosis , Cell Cycle Checkpoints/drug effects , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Breaks, Double-Stranded/drug effects , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mifepristone/toxicity , Phenotype , Reactive Oxygen Species/metabolism , Thymocytes/cytology , Thymocytes/drug effects , Thymocytes/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
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 analyzed 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 will improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials.
Subject(s)
Genetic Vectors , Mutation , Oxidative Phosphorylation , Animals , Cell Line , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Deoxyribonucleases/metabolism , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Gene Dosage , Gene Expression , Gene Order , Genetic Therapy , Genetic Vectors/genetics , Humans , Hydrolysis , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/therapy , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Point Mutation , Protein Transport , Transcription Factors/metabolismABSTRACT
Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.
Subject(s)
Gene Targeting , Mitochondrial Diseases/genetics , Animals , Cell Fusion , DNA, Mitochondrial , Embryo, Mammalian/metabolism , Endonucleases/metabolism , Female , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NZB , Mitochondrial Diseases/prevention & control , Mutation , Oocytes/metabolismABSTRACT
For more than a decade, mitochondria-targeted nucleases have been used to promote double-strand breaks in the mitochondrial genome. This was done in mitochondrial DNA (mtDNA) homoplasmic systems, where all mtDNA molecules can be affected, to create models of mitochondrial deficiencies. Alternatively, they were also used in a heteroplasmic model, where only a subset of the mtDNA molecules were substrates for cleavage. The latter approach showed that mitochondrial-targeted nucleases can reduce mtDNA haplotype loads in affected tissues, with clear implications for the treatment of patients with mitochondrial diseases. In the last few years, designer nucleases, such as ZFN and TALEN, have been adapted to cleave mtDNA, greatly expanding the potential therapeutic use. This chapter describes the techniques and approaches used to test these designer enzymes.
Subject(s)
DNA, Mitochondrial/metabolism , Endonucleases/metabolism , Mitochondria/genetics , Molecular Biology/methods , Recombinant Proteins/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , DNA, Mitochondrial/analysis , Disease Models, Animal , Endonucleases/genetics , Humans , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Molecular Sequence Data , Mutation , Oxidative Phosphorylation , Recombinant Proteins/genetics , Zinc FingersABSTRACT
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.
Subject(s)
DNA, Mitochondrial/genetics , Evolution, Molecular , Genome, Mitochondrial/genetics , Mitochondria/genetics , Animals , Humans , Patient RightsABSTRACT
Mitochondrial diseases are commonly caused by mutated mitochondrial DNA (mtDNA), which in most cases coexists with wild-type mtDNA, resulting in mtDNA heteroplasmy. We have engineered transcription activator-like effector nucleases (TALENs) to localize to mitochondria and cleave different classes of pathogenic mtDNA mutations. Mitochondria-targeted TALEN (mitoTALEN) expression led to permanent reductions in deletion or point-mutant mtDNA in patient-derived cells, raising the possibility that these mitochondrial nucleases can be therapeutic for some mitochondrial diseases.
Subject(s)
DNA, Mitochondrial/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Mitochondrial Diseases/genetics , Osteosarcoma/genetics , Amino Acid Sequence , Cell Line, Tumor , Deoxyribonucleases, Type II Site-Specific/pharmacology , Genome, Mitochondrial , Humans , Mitochondria/genetics , Mitochondrial Diseases/drug therapy , Molecular Sequence Data , Mutation , Osteosarcoma/drug therapyABSTRACT
Mitochondrial diseases are very heterogeneous and can affect different tissues and organs. Moreover, they can be caused by genetic defects in either nuclear or mitochondrial DNA as well as by environmental factors. All of these factors have made the development of therapies difficult. In this review article, we will discuss emerging approaches to the therapy of mitochondrial disorders, some of which are targeted to specific conditions whereas others may be applicable to a more diverse group of patients.
Subject(s)
Developmental Disabilities/therapy , Mitochondrial Diseases/therapy , Adult , DNA Mutational Analysis , DNA, Mitochondrial/genetics , Developmental Disabilities/genetics , Genetic Carrier Screening , Genetic Therapy/methods , Humans , Mitochondrial Diseases/genetics , Oxidative PhosphorylationABSTRACT
To investigate mtDNA recombination induced by multiple double strand breaks (DSBs) we used a mitochondria-targeted form of the ScaI 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. ScaI 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 ScaI sites were more commonly involved in recombined molecules than others. In intra-molecular recombination events, deletion breakpoints were close to or upstream of ScaI 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.