Gene therapy for mitochondrial disorders.

In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno-associated virus-based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator-like effector nucleases, zinc-finger nucleases, and meganucleases (mitoARCUS). rAAV-based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR-Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR-free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design.

Severe neonatal onset neuroregression with paroxysmal dystonia and apnoea: Expanding the phenotypic and genotypic spectrum of CARS2-related mitochondrial disease.

Disorders of mitochondrial function are a collectively common group of genetic diseases in which deficits in core mitochondrial translation machinery, including aminoacyl tRNA synthetases, are key players. Biallelic variants in the CARS2 gene (NM_024537.4), which encodes the mitochondrial aminoacyl-tRNA synthetase for cysteine (CARS2, mt-aaRScys; MIM*612800), result in childhood onset epileptic encephalopathy and complex movement disorder with combined oxidative phosphorylation deficiency (MIM#616672). Prior to this report, eight unique pathogenic variants in the CARS2 gene had been reported in seven individuals. Here, we describe a male who presented in the third week of life with apnoea. He rapidly deteriorated with paroxysmal dystonic crises and apnoea resulting in death at 16 weeks. He had no evidence of seizure activity or multisystem disease and had normal brain imaging. Skeletal muscle biopsy revealed a combined disorder of oxidative phosphorylation. Whole-exome sequencing identified biallelic variants in the CARS2 gene: one novel (c.1478T>C, p.Phe493Ser), and one previously reported (c.655G>A, p.Ala219Thr; rs727505361). Northern blot analysis of RNA isolated from the patient's fibroblasts confirmed a clear defect in aminoacylation of the mitochondrial tRNA for cysteine (mt-tRNACys). To our knowledge, this is the earliest reported case of CARS2 deficiency with severe, early onset dystonia and apnoea, without epilepsy.

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing.

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

The human mitochondrial genome contains a second light strand promoter.

The human mitochondrial genome must be replicated and expressed in a timely manner to maintain energy metabolism and supply cells with adequate levels of adenosine triphosphate. Central to this process is the idea that replication primers and gene products both arise via transcription from a single light strand promoter (LSP) such that primer formation can influence gene expression, with no consensus as to how this is regulated. Here, we report the discovery of a second light strand promoter (LSP2) in humans, with features characteristic of a bona fide mitochondrial promoter. We propose that the position of LSP2 on the mitochondrial genome allows replication and gene expression to be orchestrated from two distinct sites, which expands our long-held understanding of mitochondrial gene expression in humans.

In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue.

Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.

DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.

YbeY is required for ribosome small subunit assembly and tRNA processing in human mitochondria.

Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes.

Elongational stalling activates mitoribosome-associated quality control.

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo-electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.

A homozygous MRPL24 mutation causes a complex movement disorder and affects the mitoribosome assembly.

Mitochondrial ribosomal protein large 24 (MRPL24) is 1 of the 82 protein components of mitochondrial ribosomes, playing an essential role in the mitochondrial translation process. We report here on a baby girl with cerebellar atrophy, choreoathetosis of limbs and face, intellectual disability and a combined defect of complexes I and IV in muscle biopsy, caused by a homozygous missense mutation identified in MRPL24. The variant predicts a Leu91Pro substitution at an evolutionarily conserved site. Using human mutant cells and the zebrafish model, we demonstrated the pathological role of the identified variant. In fact, in fibroblasts we observed a significant reduction of MRPL24 protein and of mitochondrial respiratory chain complex I and IV subunits, as well a markedly reduced synthesis of the mtDNA-encoded peptides. In zebrafish we demonstrated that the orthologue gene is expressed in metabolically active tissues, and that gene knockdown induced locomotion impairment, structural defects and low ATP production. The motor phenotype was complemented by human WT but not mutant cRNA. Moreover, sucrose density gradient fractionation showed perturbed assembly of large subunit mitoribosomal proteins, suggesting that the mutation leads to a conformational change in MRPL24, which is expected to cause an aberrant interaction of the protein with other components of the 39S mitoribosomal subunit.

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila.

Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research.

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing.

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism.

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.

Enhanced Manipulation of Human Mitochondrial DNA Heteroplasmy In Vitro Using Tunable mtZFN Technology.

As a platform capable of mtDNA heteroplasmy manipulation, mitochondrially targeted zinc-finger nuclease (mtZFN) technology holds significant potential for the future of mitochondrial genome engineering, in both laboratory and clinic. Recent work highlights the importance of finely controlled mtZFN levels in mitochondria, permitting far greater mtDNA heteroplasmy modification efficiencies than observed in early applications. An initial approach, differential fluorescence-activated cell sorting (dFACS), allowing selection of transfected cells expressing various levels of mtZFN, demonstrated improved heteroplasmy modification. A further, key optimization has been the use of an engineered hammerhead ribozyme as a means for dynamic regulation of mtZFN expression, which has allowed the development of a unique isogenic cellular model of mitochondrial dysfunction arising from mutations in mtDNA, known as mTUNE. Protocols detailing these transformative optimizations are described in this chapter.

Mitochondrial transcription and translation: overview.

Mitochondria are the major source of ATP in the cell. Five multi-subunit complexes in the inner membrane of the organelle are involved in the oxidative phosphorylation required for ATP production. Thirteen subunits of these complexes are encoded by the mitochondrial genome often referred to as mtDNA. For this reason, the expression of mtDNA is vital for the assembly and functioning of the oxidative phosphorylation complexes. Defects of the mechanisms regulating mtDNA gene expression have been associated with deficiencies in assembly of these complexes, resulting in mitochondrial diseases. Recently, numerous factors involved in these processes have been identified and characterized leading to a deeper understanding of the mechanisms that underlie mitochondrial diseases.

NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction.

The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.

Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized.

In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial.

Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome.

Defects in nuclear-encoded proteins of the mitochondrial translation machinery cause early-onset and tissue-specific deficiency of one or more OXPHOS complexes. Here, we report a 7-year-old Italian boy with childhood-onset rapidly progressive encephalomyopathy and stroke-like episodes. Multiple OXPHOS defects and decreased mtDNA copy number (40%) were detected in muscle homogenate. Clinical features combined with low level of plasma citrulline were highly suggestive of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was excluded. Targeted exome sequencing of genes encoding the mitochondrial proteome identified a damaging mutation, c.567 G > A, affecting a highly conserved amino acid residue (p.Gly189Arg) of the MRM2 protein. MRM2 has never before been linked to a human disease and encodes an enzyme responsible for 2'-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA. We generated a knockout yeast model for the orthologous gene that showed a defect in respiration and the reduction of the 2'-O-methyl modification at the equivalent position (U2791) in the yeast mitochondrial 21S rRNA. Complementation with the mrm2 allele carrying the equivalent yeast mutation failed to rescue the respiratory phenotype, which was instead completely rescued by expressing the wild-type allele. Our findings establish that defective MRM2 causes a MELAS-like phenotype, and suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation.

Macropinocytic entry of isolated mitochondria in epidermal growth factor-activated human osteosarcoma cells.

Mammalian mitochondria can be transferred between cells both in culture and in vivo. There is evidence that isolated mitochondria enter cells by endocytosis, but the mechanism has not been fully characterised. We investigated the entry mechanism of isolated mitochondria into human osteosarcoma (HOS) cells. Initially we confirmed that respiratory-competent cells can be produced following incubation of HOS cells lacking mitochondrial DNA (mtDNA) with functional exogenous mitochondria and selection in a restrictive medium. Treatment of HOS cells with inhibitors of different endocytic pathways suggest that uptake of EGFP-labelled mitochondria occurs via an actin-dependent endocytic pathway which is consistent with macropinocytosis. We later utilised time-lapse microscopy to show that internalised mitochondria were found in large, motile cellular vesicles. Finally, we used confocal imaging to show that EGFP-labelled mitochondria colocalise with a macropinocytic cargo molecule during internalisation, HOS cells produce membrane ruffles interacting with external mitochondria during uptake and EGFP-labelled mitochondria are found within early macropinosomes inside cells. In conclusion our results are consistent with isolated mitochondria being internalised by macropinocytosis in HOS cells.