A cure roadmap for MT-ATP6 Leigh Syndrome
This is the second cure roadmap developed for the United Mitochondrial Disease Foundation in partnership with a Leigh Syndrome pioneer family. We've already commenced a yeast drug repurposing project.
In collaboration with
Prepared for Michael and Amy Hall and the United Mitochondrial Disease Foundation
MT-ATP6 Leigh Syndrome: Cure Roadmap
Vision
Towards a future where people with MT-ATP6 Leigh Syndrome (atpLS) will lead healthy, interactive, and fulfilled lives. This multi-year, multi-modality roadmap describes the scientific and commercial landscape for finding a treatment for atpLS patients. Based on the current knowledge of the pathogenesis of this disorder and the urgency of finding a new treatment, we recommend moving forward with a drug repurposing approach. In parallel, the document further discusses other exploratory therapeutic modalities. We explore the clinical development landscape and the current status of endpoint development and biobanking. Finally, we encourage the establishment of a drug repurposing consortium that will bring together academic and industrial researchers and clinicians to facilitate transfer of knowledge, patients’ information, and biological samples to accelerate the process of drug discovery and improve the lives of atpLS patients.
In 4-6 months, a drug repurposing screen consisting of 1,500-2,000 compounds could be completed in a yeast patient avatar, providing an initial dataset of rescue mechanisms and bioactive compounds to consider for treatment.
In 6-12 months, validation of drugs that had positive effect in the repurposing screen can be tested in model organisms (C. elegans and/or Drosophila).
In approximately 12 months, a drug repurposing screen could be tested on neuronal populations derived from Nina Hall’s samples. The screen may also test a few compounds that according to our expert interviews and literature overview show promise for treatment of atpLS. Alternatively, a repurposing screen or validation experiments could also be performed in iPSC-derived cardiomyocytes.
In 12-24 months, we seek to generate preclinical data packages based on our drug repurposing results that will form the basis for tackling fundraising and partnership opportunities with biotech and pharmaceutical companies.
Authors:
Justin Donnely (PhD candidate, Stanford University, Cure Guide, Perlara, PBC)
Shiri Zakin, PhD (Cure Guide, Perlara, PBC)
Ethan O. Perlstein, PhD. CEO of Perlara, PBC & Maggie’s Pearl, LLC
Reviewers:
Philip Yeske, PhD (Science and Alliance Officer, UMDF)
Alessandro Prigione, PhD (Heinrich Heine University)
Albert Quintana, PhD (Universitat Autònoma de Barcelona)
Greg Enns, MD (Stanford University)
Tamas Kozicz, MD, PhD (Mayo Clinic)
Executive Summary
The goal of the Leigh syndrome Cure Roadmap is to accelerate identification of a treatment for Nina Hall, whose family is a partner in the development of this Roadmap. We also hope and expect that developments that arise from this work will produce new treatment options for other Leigh syndrome patients, and perhaps for other mitochondrial diseases more generally.
Our key recommendations for the next 12-24 months are:
Obtain an existing yeast model of Nina’s MT-ATP6 pathogenic variant from Drs. Kucharczyk and di Rago and perform a drug repurposing screen using a galactose challenge approach.
Initiate a drug repurposing screen on Nina’s iPSC-derived neural cells in collaboration with Dr. Prigione and/or iPSC-derived cardiomyocytes with Dr. Kozicz.
Collaborate with Dr. Prigione and Dr. Minzuck to test base editors to correct Nina’s pathogenic variant (m.9176 T>C).
Collaborate with Dr. Samantha Lewis to develop a C. elegans model of ATP6 that can be used to validate hits from the drug repurposing screen.
Connect with key mitochondrial biotech companies to support the development of technologies that would treat Leigh syndrome (and potentially other mitochondrial disorders) by streamlining access to patient samples and cultivating an engaged, trial-ready patient community.
Perlara can provide scientific consulting, project management, and data analysis services to ensure efficient coordination and execution of research efforts across those different programs.
Introduction
Disease Overview: Deficiencies in the Mitochondrial Electron Transport Chain
While most genetic information in a mammalian cell is stored in nuclear DNA (nDNA), mitochondria are unique among our organelles in that they also carry some of their own genetic information. Included in these 37 genes are the codes for several critical proteins in the mitochondrial electron transport chain (ETC), which transfers electrons derived from glucose to molecular oxygen (O2) via a series of steps. At each step, energy released by these chemical reactions is harnessed to drive protons out of the mitochondrial matrix, creating an electrochemical gradient. This gradient, in turn, is used to produce adenosine triphosphate (ATP) via the activity of ATP synthase (also known as Complex V).
Deficiencies in the ETC often cause Leigh syndrome (LS), a rare clinically-defined syndrome with multiple etiologies. The most commonly mutated of the mitochondrially encoded proteins causing LS is MT-ATP6, a subunit of the F0 domain of ATP synthase [1,2]. Historically, the research community has assumed that the major pathological mechanism of MT-ATP6 pathogenic variants, as well as other ETC deficiencies, was the bioenergetic deficit caused by malfunctioning mitochondria [3] Interestingly, while pathogenic mutations in MT-ATP6 disrupt the activity of the mitochondrial ATP synthase and reduce the enzyme’s rate of ATP synthesis [3,4], they do not significantly impact the cell’s overall ATP synthesis rate [5]. This finding, along with subtle differences in the phenotypes of different ETC deficiencies, and the significant symptomological divergence of non-ETC mitochondrial insufficiencies [6] have led scientists to question whether a bioenergetic defect is, in fact, the phenotypic driver of mitochondrial diseases [5,7,8].
The molecular effects of ATP synthase deficiency caused by pathogenic mutations in MT-ATP6 trigger a series of cellular changes. Cells compensate for reduced mitochondrial ATP output via up-regulation of other pathways, such as glycolysis [5,6]. A reduction in proton flow through ATP synthase results in a buildup of protons in the mitochondrial inter-membrane space and their depletion in the matrix - as a result, Complex V-deficient mitochondria exhibit elevated membrane potential [3,9] which can also secondarily inhibit the activity of upstream ETC complexes [10]. Altered membrane potential also impacts other cell systems such as calcium homeostasis, a key signaling ion that, among other roles, is critical for neuronal function [11] and involved in the regulation of cell death [12]. Inhibition of the mitochondrial calcium uniporter can also protect against ischemia-reperfusion injury [13]. It has been shown that deficiencies in the oxidative, proton-pumping complexes of the ETC lead to impaired calcium buffering, while deficiencies in ATP synthase lead to a reduction in calcium release [14] - each of these disruptions can result in pathology. Aside from calcium dysregulation, failure to convert this electrochemical energy to ATP can result in hyperthermia [10]. Additionally, the systems that pump protons out of the matrix, mitochondrial Complexes I, III, and IV, become “backed-up” by the elevated electrochemical gradient potential, leading to a decreased NAD+/NADH ratio, which can impact mitochondrial metabolism as well as cellular resistance to oxidative stress [15,16]. These significant alterations in mitochondrial homeostasis, when compared to the relatively mild impact of MT-ATP6 disruption on ATP level, together indicate that the phenotype of MT-ATP6 deficient Leigh Syndrome (atpLS) is driven more by the compensatory mechanisms triggered by ATP deficiency rather than the bioenergetic deficit itself.
Disease Overview: Clinical Features
Unlike the nuclear genome, which is usually present in just one or two copies in healthy adult somatic cells and whose replication is strictly regulated as part of the cell cycle, the average mitochondrial DNA (mtDNA) copy number per cell in an adult human is ~1000 and can vary widely. Moreover, all human mitochondrial genomes, including healthy ones, exhibit some degree of heterogeneity in the sequences of different mtDNA copies - even within a single cell [17]. This is referred to as heteroplasmy, and the fraction of mtDNA copies containing a specific mutation is referred to as the degree of heteroplasmy or percent heteroplasmy. If all mtDNA copies are identical, or all contain the same mutation, this state is referred to as homoplasmy. The degree of heteroplasmy can vary widely between patients in a patient population, though in atpLS it tends to be relatively consistent throughout the body [18]. Heteroplasmy of the pathogenic mutation is believed to be related to disease severity. In atpLS for example, patients tend to exhibit their mutation with a mean of 95% heteroplasmy - interestingly, unaffected carriers of these mutations average 73% heteroplasmy, indicating that only a small quantity of functional MT-ATP6 protein in the mitochondrial network is necessary to eliminate disease phenotypes [9,18]. In fact, only 5 out of 70 patients diagnosed with atpLS exhibited heteroplasmy levels below 90% [18]. However, environmental factors often play an outsized role in the pathogenesis of atpLS. Physiological stresses, such as infection, often precipitate metabolic crises that cause the basal ganglia lesions commonly observed [1,6,9].
Like other mitochondrial diseases, atpLS presents with highly variable severity in different patients. The age range of disease onset is vast - some patients experience disease onset as late as 75 years of age - but more than half of patients experience disease onset before the age of one [18]. Age of onset is believed to be a prognostic indicator of disease, with earlier-onset patients experiencing more severe disease than later-onset patients [19]. The most common symptoms (in decreasing order of frequency) include: ataxia, cognitive dysfunction, neuropathy, seizures, and retinopathy[18]. Cardiomyopathy is also commonly observed [20]. The most common causal pathogenic variant of atpLS is m.8993 T>G. However, significant clinical variability exists within a given genotype, and the causal factors relating to differential presentation have not been fully elucidated [18].
While all cells in an atpLS patient experience the effects of ATP synthase deficiency, different cell types and organs have different susceptibility to this disruption [7,10]. Most patients with atpLS exhibit irreparable lesions in the brainstem and basal ganglia, and these are the most common cause of death in patients who exhibit shortened lifespan [6,18]. Brainstem lesions are typically associated with respiratory depression and failure, while lesions in the basal ganglia - particularly in the globus pallidi and putamen - cause movement disorders, which can be either hyperkinetic or hypokinetic in nature [15]. Dystonia and chorea are more frequently reported in children, while Parkinsonism arises more frequently in adults [21]. The nervous system is believed to be most heavily affected because of neurons’ high energy demand and particular reliance on oxidative phosphorylation (OXPHOS) to meet their energy requirements [1], but it is not known why the brain stem is more severely impacted than the cortex [10]. Interestingly, in Complex I-deficient models of LS, it was indicated that Complex I deficiency in glutamatergic neurons in the basal ganglia was responsible for movement and breathing impairments, while loss of this complex in GABAergic neurons resulted in epilepsy [22]. Moreover, it has been observed that damage in these respective cell types is also caused by different mechanisms: in glutamatergic neurons, oxidative stress in the form of reactive oxygen species (ROS) is responsible for neuronal damage, and this damage can be rescued by antioxidant treatment; in GABAergic neurons, damage is inflammatory in nature [7].
Nina Hall, whose family is a partner of Perlara in the development of this document, is a patient living with atpLS. Nina was born premature and required NICU care. From birth she exhibited lack of responsiveness and engagement and was slightly delayed with gross motor development. Her first signs of regression occurred at around 11 months, and a hospitalization around this time marked the beginning of her diagnostic journey. Her abnormal EEG readings led to an incorrect initial diagnosis of myasthenia gravis, an autoimmune disease. She experienced a series of significant respiratory illnesses, including RSV at age 3. It is likely that these infections played a role in her severe basal ganglia lesions. In 2013, her parents sought a second clinical opinion, which led to an MRI and a correct diagnosis of LS. Subsequent biopsies established her ETC deficiency and genotype, the m.9176 T>C pathogenic variant [23], which is the third most common causative pathogenic variant of atpLS [6].
Today, at 11 years old, Nina exhibits severe dystonia and chorea. She has not independently walked or stood due to the limitations on her strength and coordination. Oral motor skills are a significant challenge - she has difficulty swallowing, verbalizing, and breathing. Her difficulties with eating have resulted in challenges maintaining a healthy body weight. She must be fed through a G tube and has difficulty passing bowel movements without the aid of a laxative or suppository. She is easily fatigued and has visual issues including ptosis and an inability to focus her vision. According to her family, Nina’s issues with mobility and coordination as well as management of feeding and hygiene have the most significant impact on her quality of life, as she requires direct assistance from others to accomplish these everyday tasks. These difficulties are compounded by her difficulties communicating, although her parents note that the physical act of verbalizing poses a greater challenge than her conceptual understanding of what she wants to communicate [23]. These understandings from the perspective of the patient family offer key insight into what effects a treatment must accomplish in order to be considered a success.
Before she was two years old, Nina was enrolled in a clinical trial of the experimental drug EPI-743, a quinone medication that works by targeting ferroptosis, which improves redox balance and increases cellular resistance to oxidative stress [15]. While her clinicians believe the medicine has helped her, the open-label nature of this trial makes it difficult to say for certain. Given her early diagnostic age, she would be statistically expected to be doing worse than she is today - but this is far from conclusive causal evidence [6,15]. Overall, the EPI-743 trial failed to meet its clinical endpoints [24], but many patients have continued treatment on the basis of compassionate use [15]. Its sponsor, PTC Therapeutics, is currently using EPI-743 in a clinical trial to treat mitochondrial epilepsy, with other trials planned [15].
Disease Overview: Impaired Stress Tolerance Leads to Metabolic Crisis
The most common progression of atpLS occurs rapidly due to the onset of a metabolic crisis triggered by environmental stress, often in the form of an infection, which leads to catabolic illness [1,6]. Patients rapidly deteriorate, and then plateau, often for long periods. The symptoms that result from the patient’s metabolic crisis are due to irreparable neurological injury, usually to the basal ganglia. While it may be a long time before further brain injury occurs again, many patients die of complications from these lesions such as central apnea. Damage to posture and coordination, as well as muscular atrophy, can also lead to long-term consequences[6].
Research efforts to investigate the mechanistic basis of these crisis events are ongoing. It is known that increased oxidative stress precedes neural injury. This increase in neuronal ROS levels leads to activation of transcription factors JNK and SREBP, which in turn elevate lipid synthesis and lipid droplet formation in neurons. Along with elevated ROS, this increased lipogenesis leads to increased lipid peroxidation, which accelerates cell damage [25] . Many LS patients also exhibit evidence of lipid droplet accumulation in their muscles [26]. In Complex I-deficient models, antioxidant treatment and over-expression of lipases, which clear lipid droplets, delayed onset of neurodegeneration [25]. Additionally, brain lesions in this model are infiltrated with macrophages, indicating a central role for inflammation in this process [8]. Unfortunately, to our knowledge, similar experiments have not been performed in a Complex V-deficient background.
While the various ETC deficiencies manifest in a broadly similar manner, the subtle differences highlight key points that must be considered in therapeutic development. For example, atpLS patients uniquely exhibit elevated C5-hydroxy acylcarnitine, along with the reduced citrulline levels typically observed in mitochondrial disease [6]. When interpreted with data showing that ATP levels are only minorly reduced in atpLS patients [5,10], this indicates that these patients compensate for the reduction in mitochondrial ATP generation in part by up-regulating β-oxidation and amino acid catabolism. This indicates that the bioenergetic defect may not be the main pathological mechanism at play in atpLS. The “triggering” pattern observed in atpLS patients also points to two crucial and distinct roles that therapeutics must play. Firstly, therapeutics are needed that can prevent irreversible brain injury during metabolic crises, and secondly, interventions that alleviate the effects of brain lesions after they occur are also required [6].
Mounting evidence indicates that impaired ATP production is not necessarily the primary driver of pathology in mitochondrial disease, and atpLS is no exception. While the details of how molecular changes like altered mitochondrial inner membrane potential or NADH/NAD+ ratio manifest as the symptoms observed in atpLS are not completely clear yet, it is readily apparent that effective drug-development strategies will need to move beyond this outdated paradigm of simple bioenergetic deficit. The selection of effective models that capture these changes in cellular homeostasis for research and drug screening will be a key step in identifying and testing these new treatments.
Disease Models: Cellular Models
A key step in the cure development process is the establishment of disease models that can be used to investigate mechanistic hypotheses or test new potential therapies. Evidence of safety and efficacy in a trustworthy disease model is a key prerequisite to clinical investigation of any new therapeutic. The first of these models are cellular models, which can be cultured in a dish, making the process of generating and handling them relatively simple. Often, these models are patient-derived, rather than generated in a lab, so they already carry the patient’s pathologic mutation as well as an identical genetic background. Cellular disease models are key for performing large-scale screens for new therapeutics as well as answering important questions about their activity and mechanism of action, but selection of an appropriate model is key. Even with an identical genome, different cell types vary widely in how effectively they model disease.
The most common cellular models in patient-centric drug discovery are based on patient-derived fibroblasts, commonly obtained via a skin biopsy. These models have the advantage of being relatively easy to obtain as well as proliferative, enabling a single source to be renewed for an extended period of time. Such models are widely used in mitochondrial disease, including to model atpLS [1]. However, in most cases, results obtained using these models have failed to translate into patients [10]. Because they are proliferative and produce large quantities of protein, fibroblasts do not rely heavily on mitochondria for energy production, instead favoring more carbon-intensive pathways such as glycolysis. In fact, cultured fibroblasts have relatively few mitochondria compared to most other human cells - as a result, cellular models that rely more heavily on mitochondrial function for their overall health are more likely to effectively model disease [9,10].
In order to expand access to new kinds of human cellular models, in the early part of this century scientists developed methods to convert patient-derived cells, including fibroblasts, into induced pluripotent stem cells (iPSCs). Scientists have already demonstrated that MT-ATP6 pathogenic variants can be maintained at homoplasmic levels in iPSCs [27]. Furthermore, iPSCs have the advantage of being able to be converted into almost any other type of human cell - this works by changing the genes the cell is expressing at a given time. Although the iPSCs themselves are actively dividing and predominantly glycolytic, they can be differentiated into cells that rely much more heavily on mitochondrial function, such as neurons or cardiomyocytes [9,10,28]. In atpLS pathogenesis and fatality, iPSC-derived cellular models of the brain are likely best to investigate mechanisms of and potential therapeutics for the human condition. iPSC-derived neurons are readily obtained within 5-15 days from progenitors and can be easily produced at quantities sufficient for drug screening [9]. The Prigione lab at the Heinrich Heine University (HHU) in Düsseldorf, Germany and the Kozicz lab at Mayo Clinic in Minnesota both have experience running drug screens in these cells.
Not all brain pathologies arise from neurons, however. There are many examples of neuronal death caused by the activity of other cell types in the brain, collectively known as glia. Microglia (brain-resident macrophages), for example, play a central role in neuroinflammation, which can lead to the kind of neural damage observed in atpLS [8,29]. For this reason, it may also be useful to utilize iPSC-derived models that encompass multiple brain cell types. These models are also known as brain organoids. Although these models are challenging to work with and cannot be produced at scales necessary for drug screening, they provide unprecedented insight into how the interactions between different cell types in the brain give rise to atpLS pathology [9]. For example, iPSC-derived brain organoids carrying mutations associated with LS showed defects in neuronal morphogenesis and corticogenesis that were not observed in simpler models [11,30]. For this reason, such models will likely also play a key role in mechanistic validation of hits that arise from screening in simpler model systems.
Disease Models: Whole-Organism Models
Whole-organism models of mitochondrial diseases provide a powerful tool for investigating the molecular basis of these pathologies and for developing potential treatments. By studying the in vivo effects of genetic and environmental perturbations on the mitochondrial network, these models can identify the pathways and processes that are dysregulated in disease and reveal potential therapeutic strategies. A key challenge in the production of mitochondrial disease models is the relative difficulty of creating stable genetic alterations in mtDNA compared to nDNA. Nevertheless, many in vivo models for mitochondrial diseases have been developed by the research community and characterized through basic research.
Saccharomyces cerevisiae: The yeast Saccharomyces cerevisiae is an excellent single cell model to study the biology of complex eukaryotic organisms. Despite their simplicity, yeast cells are similar to those in higher eukaryotes, and many cellular activities are conserved from yeast to humans including mitochondria. One of the major challenges of studying the pathogenesis of a certain mitochondrial mutation is that it may affect a fraction of the mitochondrial genomes, and not all of them. However, in S. cerevisiae it is possible to create a homoplasmic population [31], facilitating clearer understanding of disease pathogenesis. In the presence of fermentable sugars, yeasts have the ability to survive even in the absence of OXPHOS by relying on glycolysis. Inhibition of the glycolytic pathway (using non-fermentable sugars as carbon source) allows for the identification of mechanisms that restore OXPHOS. Prof. Roza Kucharczyk of The Polish Academy of Sciences has developed several different models of yeast MT-ATP6 pathogenic variants, including m.9176 T>C. When grown on non-fermentable carbon source, yeasts harboring the T9176C mutation are more sensitive to oligomycin [32]. This phenotype can be used to screen for compounds that restore the ability to grow on non-fermentable carbon source in the presence of oligomycin.
Caenorhabditis elegans: The nematode C. elegans is a powerful multicellular model organism for studying human mitochondrial diseases, as well as an ideal system for drug screening. This is due to its small size, short life-cycle and fully sequenced genome that is amenable to genetic manipulation. C. elegans has several types of differentiated tissues such as muscle, dermal, nervous and intestinal tissue. It has also been shown that defects in their OXPHOS systems lead to phenotypes such as neuromuscular deficits, developmental delay, altered anesthetic sensitivity, and increased lactate levels, making them a valuable tool in the understanding of human mitochondrial diseases. Mitochondrial dysfunction in C. elegans follows a threshold-dependent pattern similar to that observed in humans, where a certain level of dysfunction must be exceeded in order for the pathological phenotype to appear. High levels of mitochondrial dysfunction lead to reduced fitness and shortened lifespan [33]. While still uncharacterized, a C. elegans model of atpLS exists in Professor Sam Lewis’s lab [34]. It is also possible to construct a C. elegans model of MT-ATP6 9176 T>C for high-throughput screening of a multitude of compounds to determine their efficacy in restoring mitochondrial function. Such a model could also be utilized to validate drug repurposing results obtained from patient-derived cells.
Drosophila melanogaster: In recent years, the fruit fly Drosophila melanogaster has emerged as a powerful tool to study mitochondrial diseases. Nuclear and mitochondrial genes are highly conserved between humans and Drosophila, and Drosophila mtDNA contains the same complement of genes as that of vertebrates. In addition, Drosophila have a complex brain system, developed skeletal muscles, and a cardiac muscle. In contrast to mammalian mtDNA manipulation, which poses a significant challenge, the tools to allow for modification of Drosophila mtDNA have been developed. These factors make fruit flies an attractive model for studying human mitochondrial diseases. Celotto et al have shown that Drosophila carrying an MT-ATP6 pathogenic variant recapitulated key phenotypic features of the human mutations, such as shortened lifespan, locomotor impairment and myodegeneration [35].
Mus musculus: Due to their high genetic homology with humans, the use of mouse models is essential for investigating the pathological mechanisms of mitochondrial disorders, as well as for identifying potential therapeutic strategies. Several mouse models of LS have been developed. Mouse models of LS caused by nuclear gene mutations in Complexes I, II, and IV, has enabled researchers to investigate the progression of the disease, elucidate the pathogenic mechanisms, and assess potential therapeutic interventions [22,36–39]. The most prevalent of these models is the Complex I-deficient Ndufs4 knockout mouse, which replicates numerous characteristics of human LS. Hyperoxia is quite toxic to these animals, for example, which may indicate a reduced tolerance to oxidative stress [40]. However, it should be noted that because this mouse line is based on knockout of a nuclear gene encoding a subunit of Complex I, it may not be perfectly representative of atpLS [39]. Recently, Yuan et al. reported that a mouse with an MT-ATP6 pathogenic variant m.8993 T>G developed several of the hallmarks of Leigh syndrome: multiple premature death, paralysis, vision loss, seizures and cardiomegaly [41].
Therapeutic Readiness
There are several therapeutic modalities that could potentially be applied to intervene in the currently-understood mechanisms of atpLS to benefit patients. There is significant variation in development strategy, precedence, timeline, and likelihood of success for these different modalities. In this section, we outline these modalities and situate them in the context of an overall treatment strategy for Nina as well as other patients suffering from atpLS. The first of these is drug screening, which Perlara is currently implementing in collaboration with academic partners (see Biotechnology Readiness). The second involves rational development of existing molecules that intervene in pathological processes of known relevance to atpLS. The third modality, mitochondrial transplantation, is in clinical development by a biotechnology company and could potentially be accessed by compassionate use. Finally, we explore two forms of gene therapy that are currently under development - while their development timelines are longer, they are useful starting points to understand how the atpLS treatment arsenal will expand in the next decade. The end of each subsection highlights key challenges for each approach and, when possible, potential short-term opportunities to gain greater insight into their feasibility.
Modalities: Drug Repurposing Screen
Drug repurposing of FDA approved drugs offers a fast, efficient, and economical way to discover new treatments for untreated diseases. One of the major challenges to finding treatments to mitochondrial disease in general, and LS specifically, is the fact that pathogenesis is not fully understood. This is further complicated by the fact that different cell types are affected by various mechanisms [7,22]. We therefore recommend an unbiased drug repurposing screen for atpLS disease-suppressing compounds - such a screen could provide a rich dataset of potential new therapies as well as identify important disease mechanisms. In addition, during our informational interviews, several drugs have been proposed as potential treatments, such as tetracycline, rapamycin and hypoxia-inducing pharmacological agents (see Modalities: Repurposing/Developing Drugs Targeting Known Pathological Mechanisms). These could be added to the unbiased screen to study their therapeutic potential. When designing a drug repurposing screen, there are important questions that require consideration. These include which model to utilize, how to measure rescue (readouts), and how to validate the hits that are generated.
Selecting an appropriate model system for LS is essential when searching for compounds with therapeutic potential. The use of somatic cell reprogramming to produce patient-derived iPSCs and their subsequent differentiation into specific lineages has proven to be a valuable technique to model mitochondrial disease. Work done in the last few years have shown that neural progenitor cells (NPCs) derived from human iPSCs maintain the same mtDNA profile as their parental iPSCs [11,14,42,43].
We recommend conducting a drug repurposing screen on cells derived from Nina's biopsy samples in order to maximize the probability of discovering a successful treatment for her. In previous work, we’ve engaged Dr. Tamas Kozicz at the Mayo clinic to run a drug repurposing screen for a patient with MELAS. Dr. Kozicz uses human iPSC-derived neurons as disease models for studying mitochondrial dysfunction and its effect on neural structure and function [10]. Another possible partner for such a study is Prof. Alessandro Prigione at HHU. Dr. Prigione uses patient-derived iPSCs to identify compounds to treat LS, and his screens are based on an assay that measures mitochondrial membrane potential and neuronal branching [9,44,45]. Alessandro recommends using iPSC-derived neural progenitors or neurons for the initial drug screening phase, followed by hit validations using various types of iPSC-derived neurons - including dopaminergic neurons, which represent a cell population that is destroyed in the basal ganglia of LS patients - and brain organoids [9,11].
Another critical consideration when designing a drug screening campaign is finding the most relevant readouts - how would we know that a certain drug has a beneficial effect? The pathogenesis of atpLS is multifaceted, with energy deprivation, ROS, neuroinflammation, and perturbations in calcium homeostasis all contributing to the progression of the disease. One approach to examine mitochondrial energy production is to measure ATP levels or the ATP/ADP ratio. In order to use this readout, we would first need to determine whether Nina’s basal ATP production was impacted by her pathogenic variant. If this is the case, we may be able to screen on this basis.
When glucose is not a limiting factor, cells with deficiencies in the oxidative phosphorylation (OXPHOS) system may still stay viable by relying on glycolysis for ATP production. However, if the cells are shifted to a galactose-containing growth medium, they depend on OXPHOS for energy production. This approach is known as a galactose challenge assay [46]. Cells with a defect in their ETC have a growth defect in galactose media, and compounds that can rescue these phenotypes can be identified by screening. Lorenz et al. showed that NPCs from atpLS patients carrying m.9185 T>C pathogenic mutation have an increased membrane potential and impaired mitochondrial calcium homeostasis. It is therefore possible to screen for compounds that would ameliorate the increased membrane potential [9,11].
Finally, it has been demonstrated that neuroinflammation is a major contributor to the neurodegeneration observed in LS and depletion of leukocytes has been shown to rescue some of the major phenotypic manifestations of LS in Ndufs4 knockout mouse [29,47]. A possible explanation for the neuroinflammation is that the mitochondria are intracellular organelles with bacterial origins: when damaged, leaking of the mitochondrial content could be a damage signal that activates innate immune response [8]. However, the exact mechanistic details of this process remain unclear. There are two possible in-vitro systems that can be used in the search for drug that protects neurons from inflammation induced damage:
Direct protection of neurons from inflammation-induced damage: macrophage culture treated with lipopolysaccharide (LPS), a compound known to activate macrophages, is used to produce cell media with inflammatory mediators [48,49]. The inflammatory media is collected and is added to the neuronal culture during the drug screen to simulate the inflammatory conditions found in the brains of atpLS patients. We would look for compounds that can decrease or prevent neuronal damage. A possible readout for this assay would be assessment of live/dead cells. Since the exact cause of neuronal death following neuroinflammation is currently unclear, another option will be to measure biomarkers associated with programmed cell death (apoptosis), such as caspase activation and DNA fragmentation.
Prevention of leukocyte activation: this screen searches for compounds that prevent the secretion of inflammatory mediators when the culture is treated with LPS. The readout in this case is more straightforward: reduction of proinflammatory cytokines such as IL-1, IL-6 and TNFɑ. These data can be determined using a variety of methods, such as enzyme-linked immunosorbent assay (ELISA).
Once drug candidates are identified, they can be validated in an orthogonal system such as yeast or C. elegans. These model systems will also provide opportunities to better understand how they could exhibit a disease-suppressive effect in humans.
Modalities: Repurposing/Developing Drugs Targeting Known Pathological Mechanisms
AMPK activators: 5’ adenosine monophosphate-activated protein kinase (AMPK) agonists including metformin, resveratrol, and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) have been explored for the treatment of mitochondrial disease with mixed results. AMPK is an enzyme that detects states of cellular energetic stress by sensing the level of adenosine monophosphate (AMP), and it responds by activating downstream factors that increase the deployment of alternative energy sources such as fatty acids and upregulate mitochondrial biogenesis. In principle, drugs that increase the activity of AMPK could activate compensatory pathways that reduce cellular energetic stress in mitochondrial disease. However, metformin, resveratrol, and AICAR themselves are not suitable for atpLS. Metformin and resveratrol activate AMPK by inducing the upstream energetic stress that precipitates high AMP, which AMPK responds to, while AICAR is an endogenous metabolite with poor pharmacological properties [15,50].
Because of these limitations, researchers have searched for molecules that allosterically activate AMPK in an AMP- and substrate-independent manner. One compound that has been identified in such a search, PT1, rescued viability in patient-derived fibroblasts with SURF1 (Complex IV)-deficiency and polymerase γ (POLG)-deficiency, suggesting that it may be generally mitoprotective. Experiments demonstrated an increase in ATP level and a reduction in ROS in response to treatment with PT1. Further, in a drug-induced mouse model of mitochondrial disease, PT1 protected against retinal degeneration. A company has been formed to develop this class of molecules further [15,50].
mTOR inhibitors: PI3K-Akt-mechanistic (or mammalian) target of rapamycin (mTOR) is a highly conserved protein-kinase involved in a variety of biological activities including cellular proliferation, survival, metabolism, autophagy, and immunity. In a series of studies done in an Ndufs4 KO (Complex I) mouse model, inhibition of mTOR using rapamycin attenuates neurodegeneration and increases life-span. The authors showed that rapamycin rescued the metabolic imbalance as shown by an increase in amino acids and their metabolites, nucleotide catabolism, and free fatty acids. Moreover, glycolysis, which is increased to adapt to mitochondrial dysfunction [51,52], was reduced. Importantly, this beneficial effect was independent of mitochondrial function[53].
Later studies aimed at elucidating the rescue mechanism of mTOR inhibitors in the Ndufs4 KO model showed that targeting an upstream protein in the mTOR signaling pathway mimicked the beneficial effect of rapamycin. Interestingly, this effect was mediated by leukocyte depletion, indicating an effect on inflammation. They further demonstrated that specific depletion of macrophages (including microglia) using pexidartinib, an inhibitor of colony-stimulating factor 1 receptor (CSF1R), resulted in increased survival, reduced disease progression and neurologic sign of disease, and prevention of CNS lesion formation. Other disease manifestations such as hyperlactemia, seizures, hypoglycemia, and anesthetic responses were also rescued. While Pexidartinib is an hepatotoxic compound, a drug screen could include non-toxic inhibitors of macrophage proliferation or activation [29].
A different mechanism of mTOR inhibition was proposed by Zheng et al. In their study, in an iPSC-derived neuron model harboring the MT-ATP6 m.8993 T>G pathogenic variant, treatment with rapamycin resulted in increased cellular ATP. The authors concluded that inhibition of protein synthesis, an ATP consuming process, is responsible for the increase in ATP [54]. It should be noted that the reduction in ATP synthesis by MT-ATP6 in the m.8993 T>G mutant is more severe than in m.9176 T>C, and this could affect treatment efficacy [55].
Initial clinical data shows that the response to rapamycin (or its analogues) is mixed. A patient with Ndufs4-deficient LS responded very well to the treatment, with a reversal of brain lesions and striking recovery of gross motor function which persisted through ~ 20 months of therapy. Another patient with MELAS (m.3243 A>G) did not respond to treatment [56]. Administration of rapamycin to kidney transplant recipients suffering from MELAS (m.3243 A>G) to induce immunosuppression post-transplant was associated with clinical improvement without affecting leukocyte heteroplasmy [57].
These studies and clinical data point to rapamycin as a potential treatment for LS, but more clinical data are necessary to determine whether the treatment could benefit atpLS patients. Disease severity as well as the specific mutation may affect the response to mTOR inhibitors as well as the mechanism mediating any beneficial effect.
Hypoxia: A series of publications by Vamsi Mootha’s group show that in a mouse model of Leigh syndrome (Ndufs4 deficiency), continued breathing of 11% oxygen (hypoxia) prevents neurodegeneration and leads to a dramatic extension in lifespan. This can be attributed to two mechanisms: decreased ROS production and activation of hypoxia induced signaling pathways. Hypoxia also decreases the organism's reliance on mitochondrial OXPHOS. While hypoxia on its own is not a treatment, understanding what happens on the molecular level when oxygen is limited suggests exploring pharmacological agents that mimic hypoxia [40,58,59].
Tetracyclines (doxycycline): Tetracyclines are a class of broad-spectrum antibiotics used to treat various bacterial infections. Tetracyclines interact with the bacterial ribosome and prevent protein synthesis [60]. Perry et al performed a drug repurposing screen using MELAS (m.3243A>G) cybrid cells that showed that tetracyclines promoted cell survival and proliferation. The beneficial effect also extended to other mitochondrial disease models (such as complex III and complex I deficiencies). Doxycycline slows down mitochondrial protein expression machinery leading to normalization of redox balance, reduction of pro-inflammatory gene expression, and increased cell survival. The authors further show that in a mouse model of Leigh syndrome (Ndufs4 KO), doxycycline decreases the expression of inflammatory genes and increases the expression of anti-inflammatory ones. Similarly, to Rapamycin, Doxycycline also suppress the innate immune response which is associated with the pathogenesis of Leigh syndrome[61].
Modalities: Mitochondrial Transplantation Therapy
Another exciting therapeutic approach involves mitochondrial transplantation. The principle behind this approach is that, rather than degrading damaged mtDNA, promoting clearance of abnormal mitochondria, or compensating for impaired mitochondrial function, new healthy mitochondria can simply be added to the system. This approach is being pioneered by Minovia Therapeutics, which is a biotechnology company developing a strategy to produce healthy mitochondrial populations and administer them to patients. In a preliminary exchange, Minovia expressed openness to treating patients under compassionate use (see Biotechnology Readiness). However, a key limitation of this approach is that it has not yet been demonstrated for use in brain mitochondrial disease - it is currently limited to use in peripheral mitochondrial disease, such as Pearson syndrome [15].
In Minovia’s approach, mitochondria isolated from maternal peripheral blood mononuclear cells (PBMCs) or healthy donor term placenta are added to patient-derived human CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from the patients (either from the bone marrow aspiration, umbilical cord blood, or mobilized peripheral blood). The mitochondria are taken up by the patients HSPCs (a process called mitochondrial augmentation therapy or MAT). The HSPCs enriched with healthy mitochondria are then transplanted back into the patient. Pre-clinical results from a POLG mutant mouse model with a higher mtDNA mutation rate indicate transfer of donor mitochondria into recipient host hematopoietic cells. Donor mitochondria were also present in myeloid cells, as well as B cells in the peripheral blood [62]. A clinical trial evaluating the safety and therapeutic effects of transplantation of Minovia mitochondria in pediatric patients with Pearson syndrome was recently completed, however the results have not been published yet [63].
Another clinical trial aimed evaluating the safety of infusion of MNV-BM-PLC (autologous CD34+ cells enriched with placenta-derived allogeneic mitochondria) in patients with primary mitochondrial diseases associated with mtDNA mutation or deletion is expected to start in the near future [64]. Additionally, it should be noted that while Minovia is the most well-developed company in this space, others, such as Mitrix Biosciences, are also developing mitochondrial transplantation approaches, offering further evidence in favor of the feasibility of and interest in this approach.
Modalities: Gene Replacement Therapy
Gene therapy focuses on the genetic manipulation of cells to elicit a therapeutic effect or to treat diseases by repairing or replacing damaged genetic material. In recent years, the FDA has approved several gene therapy products such as Hemgenix (for treatment of Hemophilia B caused by a deficiency in factor IX), Zolgensma (for the treatment of spinal muscular atrophy, caused by defective or missing SMN1 gene), Zynteglo (for the treatment of β-Thalassemia caused by a deficiency or complete absence of the β-globin gene). Allotopic expression of mitochondrial genes is a form of gene replacement therapy in which a functional mitochondrial gene is expressed as a nuclear gene. The resulting polypeptide is then transported back into the mitochondria. Pioneering work by Manfredi at al. showed that allotopic expression of MT-ATP6 in cytoplasmic hybrids (cybrids) increased ATP production and improved cell growth rate in restrictive media (galactose challenge).
While this method shows great promise for treatment of mitochondrial disease in general, the authors also noted that the mitochondrial incorporation of the translated MT-ATP6 was relatively low, probably due to the hydrophobic nature of the protein preventing efficient translocation through the cytoplasm [65,66]. Later reports used MT-ATP6 mRNA with a mitochondrial targeting signal (MTS) and the 3’ UPR of MT-ATP6 to efficiently target the mRNA to the mitochondrial membrane, and its uptake by the mitochondria lead to synthesis of the protein in skin fibroblasts harboring the neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) m.8993 T>G ATP6 pathogenic variant. Fibroblasts allotopically expressing MT-ATP6 were able to survive for 21 days in a galactose challenge [67]. However, caution should be exercised when attempting to allotopically express a mitochondrial gene, as too much expression can lead to aggregation and cellular toxicity [65,68]. Further, while these ex vivo results are promising, further work will be required before this strategy will be applicable to brain mitochondrial diseases such as atpLS.
This form of therapy is also tested for treatment of Leber's hereditary optic neuropathy (LHON), a rare, maternally inherited mitochondrial genetic disease caused by a dysfunction of complex I, leading to blindness. In this case, an adeno-associated virus (AAV), which is a nonpathogenic virus, is used to introduce a normal copy of the gene. The results of the initial phase I/II studies showed that the treatment was well-tolerated and bilateral visual improvement following unilateral intravitreal treatment was observed. This is probably due to transfer of the viral vector DNA from the injected eye to the untreated eye [69,70]. A phase III clinical trial for LHON is ongoing [71].
Modalities: Base Editors
In most mtDNA disorders, a pathological phenotype will manifest only when a certain threshold (usually 60-90%) of heteroplasmy is exceeded. Therefore, a reasonable therapeutic aim would be to shift the heteroplasmy level to below that threshold. There are several systems for sequence-specific editing of genomic DNA. The most widely used method is CRISPR-Cas9, which allows for changes to be made to an organism's genetic code. It can be used to add, delete, or modify DNA. Cas9 is an enzyme that acts like a pair of molecular scissors and can be directed to a specific location in the genome by a guide RNA (gRNA) molecule [72,73]. The gRNA molecule is a short piece of RNA that binds to a specific sequence of DNA and directs the Cas9 enzyme to that location. Once Cas9 binds to the DNA, it makes a double-stranded break in the DNA, allowing scientists to insert, delete, or modify the genetic material. While a very powerful tool for research and therapeutics, sequence-specific editing of mtDNA using CRISPR-Cas9 system is not possible as the gRNA cannot be imported into the mitochondria. Modification of the mtDNA relies on the use of RNA-free programmable nucleases, such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFN). Similar to CRISPR-Cas9, both methods are used to target the mutated sequence specifically and induce double strand break (DSB).
There are, however, differences in the downstream effects of DSBs in nuclear and mitochondrial DNA. In nDNA, DSBs result in mutation (insertions or deletions) by the cell’s own repair system. In mitochondria, on the other hand, DSBs result in a linearized DNA molecule which is rapidly degraded [74], leaving only the uncut (normal) mtDNA. This can shift the heteroplasmy to below the pathogenicity threshold [75]. Unfortunately, TALENs and ZFNs are unable to introduce specific mtDNA changes and they cannot be used to target near-homoplasmic mtDNA mutations (as in atpLS), since this would lead to the destruction of all the mtDNA.
Recently, new tools for precise genome editing in the mitochondria have been developed to overcome the limitations of the previous methods. These are referred to as base editors. There are two types of DNA base editors: cytosine base editors (CBEs) which catalyze the conversion of a C-G base pair to a T•-A base pair, and adenine base editors (ABEs) which facilitate the transformation of an A-T base pair to a G-C base pair. Together, CBEs and ABEs are capable of mediating all four transition mutations (C to T, A to G, T to C, and G to A) [76–78]. Base editors are composed of a split interbacterial toxin, DddAtox, a mitochondrial targeted TALE motif to confer sequence specificity, and a uracil glycosylase inhibitor. In the case of CBEs, binding of two monomers of DddAtox to their target sequence leads to deamination of cytosine (C) into uracil, which is later converted to thymidine (T). Similarly, when two monomers of ABE bind to their target sequences, adenine (A) is deamidated into inosine which is replaced [74,76,77] with guanine (G) during replication [77,79]. Although still an exploratory therapeutic modality, Nina's MT-ATP6 T to C conversion is a prime candidate for CBE technology, as the tools to create this genetic change are among the most robust [6]. CONFIDENTIAL: Nina’s fibroblasts will be sent to Prof. Alessandro Prigione’s lab to study the feasibility of CBE to correct her mutation.
One of the major safety concerns of base editors (and gene therapy in general) is the possibility of off-target effect, which could lead to genome-wide modifications. These could arise from spontaneous assembly of DddA monomers or nonspecific binding of the TALE protein to DNA. Several mechanisms have been proposed and are being tested to reduce off-target activity in base editors. These include enzyme design, improved delivery methods for the base editor, and improved selection of editing sites [80,81], and continued refinement of these methods should be supported.
Clinical Research and Development Readiness
The development of new therapeutics, particularly in rare disease, requires hand-in-glove coordination between clinicians and researchers – even at the earliest stage. Having reviewed the scientific landscape, laboratory model systems, and promising technologies that define the space of possible therapeutic candidates, here we explore the readiness of the clinical healthcare system to support development of these candidates as well as their evaluation in human patients – the final, costliest, and riskiest stage of the drug development process.
Clinical Research: Patient Identification and Community Engagement
Engagement of a patient community in the drug development process is crucial. Enrollment of a sufficient number of patients in clinical trials for FDA approval is a massive bottleneck in the drug development pipeline. This bottleneck is a major contributor to the reality that the clinical development phase is often the most time-consuming in the entire drug development pipeline. As a result, early efforts to diagnose, identify, and register patients can pay significant dividends, both financially and for the health of the patient community.
LS is a clinically heterogeneous disease, with significant variation between patients with respect to age of onset, age of death, and symptomatology. Diagnosis of LS is made when a neurodegeneration is present and accompanied by (1) brainstem and/or basal ganglia dysfunction (evident by an MRI or CT scan), (2) intellectual and motor developmental delay or regression, and (3) abnormal metabolism characterized by a severe defect in oxidative phosphorylation (OXPHOS) or pyruvate dehydrogenase complex activity, a molecular diagnosis in a gene related to mitochondrial energy generation, or elevated lactate in the serum or CSF. Where patients do not fulfill these stringent criteria, a diagnosis of Leigh-like syndrome can be considered, particularly in patients with atypical neuroradiology or normal lactate levels [2,82]. While the general diagnostic guidelines are clear, variability in disease presentation involving the age of onset as well as symptoms that are shared by many other mitochondrial and some non-mitochondrial diseases leads to a long diagnostic odyssey for many patients. Nina, for example, was initially misdiagnosed with myasthenia gravis, an autoimmune disease [23]. Increased awareness of mitochondrial DNA tests on the part of doctors and access to them on the part of payers will certainly help. Less invasive diagnostic strategies or biomarkers to suggest whether a biopsy to look for mtDNA lesions is necessary could also facilitate more accurate diagnoses (see Biomarkers).
Despite these challenges, the North American Mitochondrial Disease Consortium (NAMDC), an organization funded by the National Institutes of Health (NIH), has established a registry of patients. Additionally, the United Mitochondrial Disease Foundation (UMDF), a privately funded non-profit and a partner in the development of this Roadmap, engages patients by helping connect them to clinical, scientific, and community resources. Since many factors influence the specific number of patients necessary to power a given clinical trial (see Clinical Development), these ongoing efforts to identify patients and keep them informed of and invested in the advancement of therapeutic candidates through the drug development pipeline must be continued. Strong patient engagement will be a key advantage with the potential to cut years from these candidates’ clinical development timelines.
Clinical Research: Natural History
A detailed understanding of the natural history of a disease is fundamentally important to a successful drug development campaign, particularly in the case of a rare disease. There are two major reasons for this: first, the natural progression of a disease offers clues as to how a drug that corrects it might work; second, a robust natural history study has significant regulatory implications. As part of a modernization of its handling of the regulation of new medicines for rare diseases, FDA now accepts natural history as a control group on a case-by-case basis. Much as a strong patient network can accelerate a clinical development timeline by facilitating enrollment, a natural history control group accelerates timelines by reducing the total number of patients required to produce a signal of efficacy that FDA requires in order for a drug to be marketed. This also has the advantage of reducing trial costs and addresses ethical concerns associated with withholding a potentially effective drug from a patient, often a child, with severe disease in order to recapitulate a natural history that we already understand (see Regulatory Landscape and Strategy).
A longitudinal natural history study of LS patients has been published recently. The authors used the Newcastle Paediatric Mitochondrial Disease Scale (NPMDS) score to quantify LS progression. The NPMDS is a tool that is used to assess the severity of LS in children. The NPMDS score is based on their current function, the specific systems involved, clinical assessment, and quality of life. The key findings of this study indicate that the onset of disease before the age of 6 months is associated with an increased likelihood of developing severe disease burden. Additionally, the presence of pathogenic variants in SURF1, bilateral caudate involvement, and generalized atrophy were found to be associated with faster disease progression [2].
There are, however, a few limitations to this study. Disease progression was only evaluated at two timepoints. In addition, the study had a higher survival rate compared to other studies. This may be attributed to the fact that the study design focused on children who have likely already survived acute crises in their early years. Many times, LS patients do not have a second crisis [6]. That would imply that the study population was biased toward the variants with slower progression. This study highlights the importance of conducting large-scale international research involving a greater number of patients, including those with less common genetic profiles. With such a study, scientists could evaluate a variety of outcome measures that will enable better design of future clinical trials, particularly when it is not possible to have a control group. In addition, modern laboratory techniques such as single-cell genetic analysis, proteomics, and metabolomics can be useful for identifying surrogate endpoints and regulatory strategies. Finally, it warrants the identification of molecular biomarkers indicative of disease progression. All of these will support and complement the use of the NPMDS score (see Biomarkers).
Clinical Research: Biomarkers and Candidate Surrogate Endpoints
To date, there are no FDA approved biomarkers for use as primary surrogate endpoints in LS (or other mitochondrial diseases). This is a significant bottleneck to clinical readiness for an atpLS therapeutic. FDA will require significant evidence that any surrogate biomarker serving as a primary endpoint in a clinical trial is highly representative of the desired clinical outcome. As such, research toward the identification and validation of potential surrogate biomarker candidates and their specific relevance to atpLS disease progression should be prioritized and supported.
For a therapeutic intervention to be effective in preventing further deterioration once the initial crisis has appeared, it is crucial that clinicians are able to diagnose the diseases as early as possible. In the case of atpLS, children develop normally for the first 1-2 years of life, with a small fraction of them presenting some non-specific symptoms such as motor or developmental delays. Symptoms usually manifest following an acute infection [6]. In Nina’s case, she had an RSV infection prior initiation of the symptoms [23]. The lack of early signs that could aid in her atpLS diagnosis highlights the need to establish biomarkers that would accelerate diagnosis and hopefully prevent further deterioration.
Existing research into biomarkers for mitochondrial disease yields some interesting starting points. Serum lactate levels have been proposed as a biomarker for mitochondrial function, as lactate is produced via anaerobic ATP production [83]. However, data offer mixed support for the applicability of lactate as a biomarker. For LS in general, increased lactate in the CSF has been observed to be correlated with a more severe disease course [84]; however, another study disputes this finding [2]. Additionally, not all patients with atpLS have high lactate levels [84]. Furthermore, increased lactate can also be caused by other medical issues, including cardiac dysfunction, sepsis, liver disease and thiamine (B1) deficiency [2,85,86]. Blood lactate levels can also continue to change in blood after it is drawn, introducing technical variability. Other potentially useful metabolic biomarkers include blood gasses, pyruvate, glucose, electrolytes, GSH/GSSG ratio, and amino acids, which should be monitored as part of the routine course of LS treatment [1,15]. Glutaric acidemia can result in similar brain injury as seen in atpLS, and also deserves investigation [6]. Although none of these biomarkers themselves are strong candidates for surrogate biomarkers, they point to mitochondrial function as an important phenotype associated with mitochondrial disease severity and progression.
Peretz et al reported on 6 cases that were found via newborn screening to have low citrulline (a non-essential, non-proteinogenic amino acid) and elevated C5-hydroxy acylcarnitine (C5-OH, a carrier for the transport activated long chain fatty acids into mitochondria) levels. These individuals were later diagnosed with atpLS. The authors suggest that this combination is specific for atpLS over other forms of LS [6,87]. Biomarkers are also being explored to report on cells’ redox activity and health[15]. The usefulness of these biomarkers in combination should be further assessed as potential prognostic and diagnostic tools for LS.
Two protein-based biomarkers have been identified to be upregulated in skeletal muscle tissue in patients exhibiting mitochondrial insufficiency. These are the growth factors FGF-21 and GDF-15 [1]. As protein biomarkers, these have the advantage of being detectable via ELISA (rather than mass spectrometry, which is far more expensive and requires specialized operators). They are also less likely to break down after being sampled [83]. Considerable evidence has accumulated to support the predictive value of GDF-15, in particular, for the severity of a different rare mitochondrial disease, TK2-deficient myopathy [88], GDF-15 is currently being evaluated in a clinical trial for this disease [89]. In a recent study evaluating GDF-15 and FGF-21 as biomarkers for primary mitochondrial disorders (PMDs) in children, the authors found that GDF15 and FGF21 are both helpful, non-invasive predictors for children with PMDs regardless of phenotype and genotype. GDF15 was superior to previous biomarkers and FGF21 in screening for the diagnosis of PMDs; FGF-21 was found to be useful as a biomarker for monitoring disease severity [90]. These biomarkers are excellent candidates for surrogate endpoints, even though they are not specific for atpLS.
Another potential molecular biomarker that has been considered is the heteroplasmy rate. In the case of mitochondrially-inherited LS, there appears to be a significant correlation between the heteroplasmy level and clinical severity. White et al. showed that in the case of MT-ATP6 m.8993 T>G, mutant loads of approximately 70% to 90% lead to the development of neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), while mutant loads of 90% or greater result in the more severe atpLS phenotype [26]. Different heteroplasmy levels resulting from mitochondrially inherited ATP6 mutations vs mutations in the nuclear gene SURF1 may potentially explain why SURF1 LS patients have a faster and more linear illness progression [6]. However, much as with metabolites, variability in the measurement of heteroplasmy rate - between patients as well as between individual biopsies of the same patient [89]- complicates its use as a surrogate biomarker. While monitoring heteroplasmy rate may be useful with some therapeutic approaches, such as base-editing or mitochondrial transplantation, it is worth mentioning that some therapeutic strategies may have a positive effect on disease progression without correcting the mitochondrial defect or the heteroplasmy. For such strategies, such as protecting from ROS or neuroinflammation, different biomarkers need to be used to assess the effectiveness of the treatment [8].
Clinical Research: Biobanking
Reliable patient samples are fundamental components to multiple ongoing efforts necessary to develop a LS therapeutic (see Models, Natural History). The core of patient sample collection, cataloging, and distribution to researchers is the biobank or biorepository. Fortunately, NAMDC has also begun the process of building a centralized mitochondrial diseases biobank. They currently have samples, primarily blood samples, from approximately 1800 US patients. Since Nina’s initial diagnosis of atpLS, several biopsies have been utilized to establish cell lines, including a fibroblast line that is housed at NAMDC and available to researchers [23]. Some smaller additional biobanks exist, but NAMDC is likely to constitute the primary resource for drug developers working in this space.
In service of the production of useful cellular models for research and screening, the samples most likely to be useful to biobank include blood and skin biopsies. Overall, in order to maximize the representativeness of a patient-derived model for a given individual’s disease, cellular models should be generated from tissues most highly affected by the disease whenever possible [90,91]. In the case of atpLS, this would primarily involve brain cells: neurons and glia.
Clinical Development: Regulatory Landscape and Strategy
The FDA plays a key role in the healthcare system by serving as gatekeeper to the pharmaceutical marketplace - for access, any new therapeutic must demonstrate both safety and efficacy in human clinical data. FDA is also tasked with ethically evaluating and approving the clinical trials necessary to generate these safety and efficacy data based on the risks and potential benefits to the patients involved in the study. As such, a well-developed regulatory strategy is an indispensable component to a successful drug development campaign.
Traditionally, FDA has operated with a degree of aloofness toward drug developers in service of its role as a neutral gatekeeper. However, driven in particular by the need for innovative regulatory approaches to ensure drug approvals for patients suffering from rare diseases, FDA can now be engaged at a much earlier stage in the process and in many cases can operate as a partner and resource. Additionally, there are a series of regulatory designations that FDA now offers to facilitate the development of drugs in therapeutic areas that are traditionally underserved by the biopharmaceutical industry. Fortunately, therapeutic candidates for atpLS are likely to be eligible for multiple such designations - their specific details, as well as their implications for the overall development process, are explored here.
The Orphan Drug Act (ODA), passed by Congress in 1983, grants FDA the authority to provide specific incentives for the development of drugs for diseases affecting fewer than 200,000 patients per year in the United States. atpLS falls into this category [84]. The most significant outcome of ODA designation is a seven-year period of market exclusivity which is awarded to the entity that submits the drug for FDA approval. As market exclusivity, traditionally secured by patents, is the key incentive driving private investment in the high upfront development cost of drugs, the introduction of the ODA designation has radically shifted the calculus around whether a drug development program has the potential to be profitable, particularly if the underlying asset is difficult to protect through patents (as is often the case with repurposed drugs). Because of this, the certainty of an ODA designation from FDA will be a major advantage in securing private, for-profit funding to finance clinical development of any new therapeutic candidates identified through a repurposing screen.
Any new therapeutic candidate for atpLS will also be eligible for Fast Track designation, as there are no current atpLS therapeutics available - this is known as an unmet need. Fast track designees have the advantage of more frequent meetings and communication with FDA, which increases the probability of success of a clinical trial. A similar designation known as Priority Review can accelerate the timeline on which FDA reviews the final application for approval - any new atpLS therapeutic with significant efficacy has a strong likelihood of receiving this designation. Again, because they shorten the clinical development timeline, these designations can both reduce development costs and help patients benefit from new treatments faster.
Two other FDA designations exist that warrant attention at this stage: Accelerated Approval and the Breakthrough Designation. Accelerated approval allows for a drug to be approved earlier on the basis of a surrogate endpoint - essentially, this is a biomarker that is not itself a measure of clinical benefit but is thought to be a strong predictor of it. Clinical endpoints often require long observation times and have more variability due to their more subjective nature than biomarkers measured in a laboratory. As such, the ability to conditionally approve a drug based on a surrogate endpoint can also significantly reduce the clinical development timeline. Evaluation on the basis of clinical endpoints then continues while the drug is conditionally approved. If these surrogate endpoint data suggest that a drug is in fact achieving a substantial benefit in a serious condition, that drug will be eligible for Breakthrough designation, which offers all Fast Track features as well as intensive guidance from senior FDA managers - drugs that receive this designation receive the full support of FDA to ensure that a drug’s safety and efficacy is evaluated quickly, efficiently, and reliably. In order to maximize the probability of accessing these two valuable FDA designations, the establishment of a validated surrogate endpoint will be critical (see Biomarkers).
Clinical Development: Clinical Endpoints
In nearly all cases, the primary relevant outcome to FDA (and, generally, to patients) is whether the drug produces some kind of clinical benefit that is meaningful. Even if surrogate endpoints are used to approve a drug, ongoing investigation to demonstrate such clinical benefit will continue, and if there is no evidence of clinical benefit the drug can be removed from the marketplace. Furthermore, nearly all clinical trials in mitochondrial disorders have failed to meet clinical endpoints, even if they had an impact on surrogate endpoints and the patient or their family self-reported improvements in quality of life [10]. As such, even though defining strong surrogate endpoints is key at this stage, it is important to consider clinical endpoints as well.
Clinical endpoints are generally more challenging, expensive, and time-consuming to measure than biomarker endpoints. Clinical endpoints are more subjective, measured over longer periods of time, and capture more inherent biological variability than biomarker endpoints do. Thus, reaching a clinical endpoint is a high bar that most therapeutic candidates do not achieve - but to offer a patient anything less than this high standard would be unacceptable.
Several clinical endpoints have been defined for mitochondrial diseases in general and their use is precedented in clinical trials for LS. The primary clinical endpoint for most mitochondrial diseases is a change in the NPMDS score. Depending on the drug’s activity, other clinical endpoints may include myopathy, dystonia, ataxia, retarded motor development, or reduced activities of daily living [92]. However, it will be critical to extend the set of options available for clinical endpoints by working in close collaboration with a clinician who has experience with atpLS patients and clinical development of drugs in mitochondrial disease. It would also be prudent to develop more patient-reported outcome measures, so that clinical trial data are more reflective of the quality of life improvements that a drug can bring to patients and their caregivers [10].
Biotechnology Ecosystem Readiness
Having explored the scientific landscape and considerations for clinical development of a therapeutic candidate, we turn our attention in this final section of the Roadmap to the biotechnology ecosystem, encompassing academia, industrial biotechnology, and public and private funding sources. The successful development of new therapies nearly always requires engagement of multiple players in this ecosystem. In this section, we explore potential partners who can facilitate development of the various modalities previously discussed (see Therapeutic Readiness) as well as patient access to those therapies. Perlara offers scientific consulting, project management, and data analysis services to ensure efficient coordination and execution of research efforts through these partnerships.
In the course of preparing this Roadmap, we’ve identified potential academic partners with knowledge and expertise that will enable accelerating the discovery of potential treatments and cures. Alessandro Prigione at the HHU in Düsseldorf could develop drug screening assays for mitochondrial disease in Nina’s iPSC-derived neuronal cells.
Simon Johnson at the University of Northumbria at Newcastle has expressed interest in developing drug screening assays for compounds that will modulate the neuroinflammation involved in the pathogenesis of LS[8]. Additionally, Samantha Lewis of UC Berkeley has developed C. elegans models of MT-ATP6 deficiency. Her lab could also help develop screening assays methodologies for validating screening hits in an animal model [34].
A potential industrial partner is Minovia Therapeutics, which is developing mitochondrial transplantation therapy. They are currently the most advanced of these possible partners (see Modalities). Preliminary data from their compassionate-use mitochondrial augmentation treatment in six children with Pearson syndrome or Kearns-Sayre syndrome (a pair of mitochondrial DNA deletion diseases) showed some promising results, including decreased heteroplasmy in the peripheral blood, increased ATP production, and improvement in most patients' quality of life [93]. While this research was conducted on patients with a single large DNA deletion syndrome (see MDDD Cure Roadmap), it provides the groundwork for clinical trials of mitochondrial augmentation therapy for the treatment of patients with additional primary mtDNA abnormalities, such as Leigh syndrome, and offers an excellent complement or alternative to a drug-repurposing approach[3,94] .
References
1. Schubert, M. B., & Vilarinho, L. (2020). Molecular basis of Leigh syndrome: A current look. In Orphanet Journal of Rare Diseases (Vol. 15, Issue 1). BioMed Central. https://doi.org/10.1186/s13023-020-1297-9
2. Lim, A. Z., Ng, Y. S., Blain, A., Jiminez-Moreno, C., Alston, C. L., Nesbitt, V., Simmons, L., Santra, S., Wassmer, E., Blakely, E. L., Turnbull, D. M., Taylor, R. W., Gorman, G. S., & McFarland, R. (2022). Natural History of Leigh Syndrome: A Study of Disease Burden and Progression. Annals of Neurology, 91(1), 117–130. https://doi.org/10.1002/ana.26260
3. Carrozzo, R., Tessa, A., Vázquez-Memije, M. E., Piemonte, F., Patrono, C., Malandrini, A., Dionisi-Vici, C., Vilarinho, L., Villanova, M., Schägger, H., Federico, A., Bertini, E., & Santorelli, F. M. (2001). The T9176G mtDNA mutation severely affects ATP production and results in Leigh syndrome. Neurology, 56(5), 687–690. https://doi.org/10.1212/wnl.56.5.687
4. Kucharczyk, R. (n.d.). Personal Communication. Dec 2022.
5. Bakare, A. B., Lesnefsky, E. J., & Iyer, S. (2021). Leigh Syndrome: A Tale of Two Genomes. Frontiers in Physiology, 12, 693734. https://doi.org/10.3389/fphys.2021.693734
6. Larson, A. (n.d.). Expert Interview. 23 Nov 2022.
7. Quintana, A. (n.d.). Expert Interview. 11 Nov 2022.
8. Simon Johnson. (n.d.). Expert Interview. 23 Nov 2022.
9. Prigione, A. (n.d.). Expert Interview. 1 Dec 2022.
10. Kozicz, T. (n.d.). Expert Interview. 29 Nov 2022.
11. Lorenz, C., Lesimple, P., Bukowiecki, R., Zink, A., Inak, G., Mlody, B., Singh, M., Semtner, M., Mah, N., Auré, K., Leong, M., Zabiegalov, O., Lyras, E. M., Pfiffer, V., Fauler, B., Eichhorst, J., Wiesner, B., Huebner, N., Priller, J., … Prigione, A. (2017). Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders. Cell Stem Cell, 20(5), 659-674.e9. https://doi.org/10.1016/j.stem.2016.12.013
12. Giorgi, C., Baldassari, F., Bononi, A., Bonora, M., de Marchi, E., Marchi, S., Missiroli, S., Patergnani, S., Rimessi, A., Suski, J. M., Wieckowski, M. R., & Pinton, P. (2012). Mitochondrial Ca(2+) and apoptosis. Cell Calcium, 52(1), 36–43. https://doi.org/10.1016/j.ceca.2012.02.008
13. Rasmussen, T. P., Wu, Y., Joiner, M. A., Koval, O. M., Wilson, N. R., Luczak, E. D., Wang, Q., Chen, B., Gao, Z., Zhu, Z., Wagner, B. A., Soto, J., McCormick, M. L., Kutschke, W., Weiss, R. M., Yu, L., Boudreau, R. L., Abel, E. D., Zhan, F., … Anderson, M. E. (2015). Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart. Proceedings of the National Academy of Sciences of the United States of America, 112(29), 9129–9134. https://doi.org/10.1073/pnas.1504705112
14. Galera-Monge, T., Zurita-Díaz, F., Canals, I., Hansen, M. G., Rufián-Vázquez, L., Ehinger, J. K., Elmér, E., Martin, M. A., Garesse, R., Ahlenius, H., & Gallardo, M. E. (2020). Mitochondrial Dysfunction and Calcium Dysregulation in Leigh Syndrome Induced Pluripotent Stem Cell Derived Neurons. International Journal of Molecular Sciences, 21(9). https://doi.org/10.3390/ijms21093191
15. Enns, G. (n.d.). Expert Interview. 14 Nov 2022.
16. Stein, L. R., & Imai, S. (2012). The dynamic regulation of NAD metabolism in mitochondria. Trends in Endocrinology and Metabolism: TEM, 23(9), 420–428. https://doi.org/10.1016/j.tem.2012.06.005
17. Jeedigunta, S. P., Minenkova, A. v, Palozzi, J. M., & Hurd, T. R. (2021). Avoiding Extinction: Recent Advances in Understanding Mechanisms of Mitochondrial DNA Purifying Selection in the Germline. Annual Review of Genomics and Human Genetics, 22, 55–80. https://doi.org/10.1146/annurev-genom-121420-081805
18. Stendel, C., Neuhofer, C., Floride, E., Yuqing, S., Ganetzky, R. D., Park, J., Freisinger, P., Kornblum, C., Kleinle, S., Schöls, L., Distelmaier, F., Stettner, G. M., Büchner, B., Falk, M. J., Mayr, J. A., Synofzik, M., Abicht, A., Haack, T. B., Prokisch, H., … ATP6 Study Group. (2020). Delineating MT-ATP6-associated disease: From isolated neuropathy to early onset neurodegeneration. Neurology. Genetics, 6(1), e393. https://doi.org/10.1212/NXG.0000000000000393
19. Hong, C.-M., Na, J.-H., Park, S., & Lee, Y.-M. (2020). Clinical Characteristics of Early-Onset and Late-Onset Leigh Syndrome. Frontiers in Neurology, 11. https://doi.org/10.3389/fneur.2020.00267
20. Ganetzky, R. D., Stendel, C., McCormick, E. M., Zolkipli-Cunningham, Z., Goldstein, A. C., Klopstock, T., & Falk, M. J. (2019). MT-ATP6 mitochondrial disease variants: Phenotypic and biochemical features analysis in 218 published cases and cohort of 14 new cases. Human Mutation, 40(5), 499–515. https://doi.org/10.1002/humu.23723
21. Musumeci, O., Oteri, R., & Toscano, A. (2020). Spectrum of movement disorders in mitochondrial diseases. Journal of Translational Genetics and Genomics. https://doi.org/10.20517/jtgg.2020.22
22. Bolea, I., Gella, A., Sanz, E., Prada-Dacasa, P., Menardy, F., Bard, A. M., Machuca-Márquez, P., Eraso-Pichot, A., Mòdol-Caballero, G., Navarro, X., Kalume, F., & Quintana, A. (2019). Defined neuronal populations drive fatal phenotype in a mouse model of Leigh syndrome. ELife, 8. https://doi.org/10.7554/eLife.47163
23. Hall, A., & Hall, M. (n.d.). Family Interview. 26 Oct 2022.
24. PTC Therapeutics. (n.d.). Long-Term Safety and Efficacy Evaluation of EPI-743 in Children With Leigh Syndrome. ClinicalTrials.gov Identifier: NCT02352896. Last updated: November 18, 2022. Retrieved January 1, 2023, from https://clinicaltrials.gov/ct2/show/NCT02352896
25. Liu, L., Zhang, K., Sandoval, H., Yamamoto, S., Jaiswal, M., Sanz, E., Li, Z., Hui, J., Graham, B. H., Quintana, A., & Bellen, H. J. (2015). Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration. Cell, 160(1–2), 177–190. https://doi.org/10.1016/j.cell.2014.12.019
26. Thorburn, D. R., Rahman, J., & Rahman, S. (2003). Mitochondrial DNA-Associated Leigh Syndrome and NARP.In: GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993. 2003 Oct 30 [updated 2017 Sep 28].
27. Lorenz, C., Zink, A., Henke, M.-T., Staege, S., Mlody, B., Bünning, M., Wanker, E., Diecke, S., Schuelke, M., & Prigione, A. (2022). Generation of four iPSC lines from four patients with Leigh syndrome carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6. Stem Cell Research, 61, 102742. https://doi.org/10.1016/j.scr.2022.102742
28. Inak, G., Rybak-Wolf, A., Lisowski, P., Pentimalli, T. M., Jüttner, R., Glažar, P., Uppal, K., Bottani, E., Brunetti, D., Secker, C., Zink, A., Meierhofer, D., Henke, M. T., Dey, M., Ciptasari, U., Mlody, B., Hahn, T., Berruezo-Llacuna, M., Karaiskos, N., … Prigione, A. (2021). Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-22117-z
29. Stokes, J. C., Bornstein, R. L., James, K., Park, K. Y., Spencer, K. A., Vo, K., Snell, J. C., Johnson, B. M., Morgan, P. G., Sedensky, M. M., Baertsch, N. A., & Johnson, S. C. (2022). Leukocytes mediate disease pathogenesis in the Ndufs4(KO) mouse model of Leigh syndrome. JCI Insight, 7(5). https://doi.org/10.1172/jci.insight.156522
30. Romero-Morales, A. I., Robertson, G. L., Rastogi, A., Rasmussen, M. L., Temuri, H., McElroy, G. S., Chakrabarty, R. P., Hsu, L., Almonacid, P. M., Millis, B. A., Chandel, N. S., Cartailler, J.-P., & Gama, V. (2022). Human iPSC-derived cerebral organoids model features of Leigh syndrome and reveal abnormal corticogenesis. Development, 149(20). https://doi.org/10.1242/dev.199914
31. Okamoto, K., Perlman, P. S., & Butow, R. A. (1998). The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud. The Journal of Cell Biology, 142(3), 613–623. https://doi.org/10.1083/jcb.142.3.613
32. Kucharczyk, R., Ezkurdia, N., Couplan, E., Procaccio, V., Ackerman, S. H., Blondel, M., & di Rago, J. P. (2010). Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase. Biochimica et Biophysica Acta - Bioenergetics, 1797(6–7), 1105–1112. https://doi.org/10.1016/j.bbabio.2009.12.022
33. Ventura, N., & Rea, S. L. (2007). Caenorhabditis elegans mitochondrial mutants as an investigative tool to study human neurodegenerative diseases associated with mitochondrial dysfunction. Biotechnology Journal, 2(5), 584–595. https://doi.org/10.1002/biot.200600248
34. Lewis, S. (n.d.). Personal Communication. Nov 2022.
35. Celotto, A. M., Frank, A. C., McGrath, S. W., Fergestad, T., van Voorhies, W. A., Buttle, K. F., Mannella, C. A., & Palladino, M. J. (2006). Mitochondrial encephalomyopathy in Drosophila. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 26(3), 810–820. https://doi.org/10.1523/JNEUROSCI.4162-05.2006
36. Reynaud-Dulaurier, R., Benegiamo, G., Marrocco, E., Al-Tannir, R., Surace, E. M., Auwerx, J., & Decressac, M. (2020). Gene replacement therapy provides benefit in an adult mouse model of Leigh syndrome. Brain : A Journal of Neurology, 143(6), 1686–1696. https://doi.org/10.1093/brain/awaa105
37. Khazal, F. al, Holte, M. N., Bolon, B., White, T. A., LeBrasseur, N., & Iii, L. J. M. (2019). A conditional mouse model of complex II deficiency manifesting as Leigh-like syndrome. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 33(12), 13189–13201. https://doi.org/10.1096/fj.201802655RR
38. Pulliam, D. A., Deepa, S. S., Liu, Y., Hill, S., Lin, A. L., Bhattacharya, A., Shi, Y., Sloane, L., Viscomi, C., Zeviani, M., & van Remmen, H. (2014). Complex IV-deficient Surf1-/- mice initiate mitochondrial stress responses. Biochemical Journal, 462(2), 359–371. https://doi.org/10.1042/BJ20140291
39. El-Desouky, S., Taalab, Y. M., El-Gamal, M., Mohamed, W., & Salama, M. (2019). Animal Model for Leigh Syndrome. Methods in Molecular Biology (Clifton, N.J.), 2011, 451–464. https://doi.org/10.1007/978-1-4939-9554-7_27
40. Jain, I. H., Zazzeron, L., Goldberger, O., Marutani, E., Wojtkiewicz, G. R., Ast, T., Wang, H., Schleifer, G., Stepanova, A., Brepoels, K., Schoonjans, L., Carmeliet, P., Galkin, A., Ichinose, F., Zapol, W. M., & Mootha, V. K. (2019). Leigh Syndrome Mouse Model Can Be Rescued by Interventions that Normalize Brain Hyperoxia, but Not HIF Activation. Cell Metabolism, 30(4), 824-832.e3. https://doi.org/10.1016/j.cmet.2019.07.006
41. Yuan, H., Webster, K. A., Bhatti, M. T., Hauswirth, W. W., Lewin, A. S., & Guy, J. (2022). Amelioration of Leigh syndrome induced by mouse blastocyst complementation with a mutant human mitochondrial ATP synthase 6. Clinical and Translational Discovery, 2(3). https://doi.org/10.1002/ctd2.114
42. Grace, H. E., Galdun, P., Lesnefsky, E. J., West, F. D., & Iyer, S. (2019). mRNA Reprogramming of T8993G Leigh’s Syndrome Fibroblast Cells to Create Induced Pluripotent Stem Cell Models for Mitochondrial Disorders. Stem Cells and Development, 28(13), 846–859. https://doi.org/10.1089/scd.2019.0045
43. Hattori, T., Hamazaki, T., Kudo, S., & Shintaku, H. (2016). Metabolic Signature of MELAS/Leigh Overlap Syndrome in Patient-specific Induced Pluripotent Stem Cells Model. Osaka City Medical Journal, 62(2), 69–76.
44. Lickfett, S., Menacho, C., Zink, A., Telugu, N. S., Beller, M., Diecke, S., Cambridge, S., & Prigione, A. (2022). High-content analysis of neuronal morphology in human iPSC-derived neurons. STAR Protocols, 3(3), 101567. https://doi.org/10.1016/j.xpro.2022.101567
45. Zink, A., Haferkamp, U., Wittich, A., Beller, M., Pless, O., & Prigione, A. (2022). High-content screening of mitochondrial polarization in neural cells derived from human pluripotent stem cells. STAR Protocols, 3(3), 101602. https://doi.org/10.1016/j.xpro.2022.101602
46. Aguer, C., Gambarotta, D., Mailloux, R. J., Moffat, C., Dent, R., McPherson, R., & Harper, M.-E. (2011). Galactose enhances oxidative metabolism and reveals mitochondrial dysfunction in human primary muscle cells. PloS One, 6(12), e28536. https://doi.org/10.1371/journal.pone.0028536
47. Aguilar, K., Comes, G., Canal, C., Quintana, A., Sanz, E., & Hidalgo, J. (2022). Microglial response promotes neurodegeneration in the Ndufs4 KO mouse model of Leigh syndrome. Glia, 70(11), 2032–2044. https://doi.org/10.1002/glia.24234
48. Fang, H., Pengal, R. A., Cao, X., Ganesan, L. P., Wewers, M. D., Marsh, C. B., & Tridandapani, S. (2004). Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP. Journal of Immunology (Baltimore, Md. : 1950), 173(1), 360–366. https://doi.org/10.4049/jimmunol.173.1.360
49. Orecchioni, M., Ghosheh, Y., Pramod, A. B., & Ley, K. (2019). Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS-) vs. Alternatively Activated Macrophages. Frontiers in Immunology, 10, 1084. https://doi.org/10.3389/fimmu.2019.01084
50. Moore, T., Yanes, R. E., Calton, M. A., Vollrath, D., Enns, G. M., & Cowan, T. M. (2020). AMP-independent activator of AMPK for treatment of mitochondrial disorders. PLoS ONE, 15(10 October). https://doi.org/10.1371/journal.pone.0240517
51. Wu, S.-B., & Wei, Y.-H. (2012). AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases. Biochimica et Biophysica Acta, 1822(2), 233–247. https://doi.org/10.1016/j.bbadis.2011.09.014
52. Hu, Y., Lu, W., Chen, G., Wang, P., Chen, Z., Zhou, Y., Ogasawara, M., Trachootham, D., Feng, L., Pelicano, H., Chiao, P. J., Keating, M. J., Garcia-Manero, G., & Huang, P. (2012). K-ras(G12V) transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis. Cell Research, 22(2), 399–412. https://doi.org/10.1038/cr.2011.145
53. Johnson, S. C., Yanos, M. E., Kayser, E.-B., Quintana, A., Sangesland, M., Castanza, A., Uhde, L., Hui, J., Wall, V. Z., Gagnidze, A., Oh, K., Wasko, B. M., Ramos, F. J., Palmiter, R. D., Rabinovitch, P. S., Morgan, P. G., Sedensky, M. M., & Kaeberlein, M. (2013). mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome. Science (New York, N.Y.), 342(6165), 1524–1528. https://doi.org/10.1126/science.1244360
54. Zheng, X., Boyer, L., Jin, M., Kim, Y., Fan, W., Bardy, C., Berggren, T., Evans, R. M., Gage, F. H., & Hunter, T. (2016). Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration. ELife, 5. https://doi.org/10.7554/eLife.13378
55. Mattiazzi, M., Vijayvergiya, C., Gajewski, C. D., DeVivo, D. C., Lenaz, G., Wiedmann, M., & Manfredi, G. (2004). The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants. Human Molecular Genetics, 13(8), 869–879. https://doi.org/10.1093/hmg/ddh103
56. Sage-Schwaede, A., Engelstad, K., Salazar, R., Curcio, A., Khandji, A., Garvin, J. H., & de Vivo, D. C. (2019). Exploring mTOR inhibition as treatment for mitochondrial disease. Annals of Clinical and Translational Neurology, 6(9), 1877–1881. https://doi.org/10.1002/acn3.50846
57. Johnson, S. C., Martinez, F., Bitto, A., Gonzalez, B., Tazaerslan, C., Cohen, C., Delaval, L., Timsit, J., Knebelmann, B., Terzi, F., Mahal, T., Zhu, Y., Morgan, P. G., Sedensky, M. M., Kaeberlein, M., Legendre, C., Suh, Y., & Canaud, G. (2019). mTOR inhibitors may benefit kidney transplant recipients with mitochondrial diseases. Kidney International, 95(2), 455–466. https://doi.org/10.1016/j.kint.2018.08.038
58. Jain, I. H., Zazzeron, L., Goli, R., Alexa, K., Schatzman-Bone, S., Dhillon, H., Goldberger, O., Peng, J., Shalem, O., Sanjana, N. E., Zhang, F., Goessling, W., Zapol, W. M., & Mootha, V. K. (2016). Hypoxia as a therapy for mitochondrial disease. Science (New York, N.Y.), 352(6281), 54–61. https://doi.org/10.1126/science.aad9642
59. Grange, R. M. H., Sharma, R., Shah, H., Reinstadler, B., Goldberger, O., Cooper, M. K., Nakagawa, A., Miyazaki, Y., Hindle, A. G., Batten, A. J., Wojtkiewicz, G. R., Schleifer, G., Bagchi, A., Marutani, E., Malhotra, R., Bloch, D. B., Ichinose, F., Mootha, V. K., & Zapol, W. M. (2021). Hypoxia ameliorates brain hyperoxia and NAD+ deficiency in a murine model of Leigh syndrome. Molecular Genetics and Metabolism, 133(1), 83–93. https://doi.org/10.1016/j.ymgme.2021.03.005
60. Nguyen, F., Starosta, A. L., Arenz, S., Sohmen, D., Dönhöfer, A., & Wilson, D. N. (2014). Tetracycline antibiotics and resistance mechanisms. Biological Chemistry, 395(5), 559–575. https://doi.org/10.1515/hsz-2013-0292
61. Perry, E. A., Bennett, C. F., Luo, C., Balsa, E., Jedrychowski, M., O’Malley, K. E., Latorre-Muro, P., Ladley, R. P., Reda, K., Wright, P. M., Gygi, S. P., Myers, A. G., & Puigserver, P. (2021). Tetracyclines promote survival and fitness in mitochondrial disease models. Nature Metabolism, 3(1), 33–42. https://doi.org/10.1038/s42255-020-00334-y
62. Jacoby, E., ben Yakir-Blumkin, M., Blumenfeld-Kan, S., Brody, Y., Meir, A., Melamed-Book, N., Napso, T., Pozner, G., Saadi, E., Shabtay-Orbach, A., Yivgi-Ohana, N., Sher, N., & Toren, A. (2021). Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit. NPJ Regenerative Medicine, 6(1), 58. https://doi.org/10.1038/s41536-021-00167-7
63. Minovia Therapeutics Ltd. (n.d.). A Study to Evaluate the Safety and Therapeutic Effects of Transplantation of MNV-BM-BLD in Pediatric Patients With Pearson Syndrome. ClinicalTrials.gov Identifier: NCT03384420. Last Update Posted: August 31, 2021. https://clinicaltrials.gov/ct2/show/NCT03384420?term=NCT03384420&draw=2&rank=1
64. Minovia Therapeutics Ltd. (n.d.). A First in Human Study to Evaluate the Safety of Infusion of MNV-BM-PLC (Autologous CD34+ Cells Enriched With Placenta Derived Allogeneic Mitochondria) in Patients With Primary Mitochondrial Diseases Associated With Mitochondrial DNA Mutation or Deletion. ClinicalTrials.gov Identifier: NCT04548843. Last Update Posted: August 31, 2021. Retrieved January 1, 2023, from https://clinicaltrials.gov/ct2/show/NCT04548843
65. Manfredi, G. (n.d.). Expert Interview. 28 Nov 2022.
66. Manfredi, G., Fu, J., Ojaimi, J., Sadlock, J. E., Kwong, J. Q., Guy, J., & Schon, E. A. (2002). Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nature Genetics, 30(4), 394–399. https://doi.org/10.1038/ng851
67. Bonnet, C., Kaltimbacher, V., Ellouze, S., Augustin, S., Bénit, P., Forster, V., Rustin, P., Sahel, J. A., & Corral-Debrinski, M. (2007). Allotopic mRNA localization to the mitochondrial surface rescues respiratory chain defects in fibroblasts harboring mitochondrial DNA mutations affecting complex I or V subunits. Rejuvenation Research, 10(2), 127–143. https://doi.org/10.1089/rej.2006.0526
68. Oca-Cossio, J., Kenyon, L., Hao, H., & Moraes, C. T. (2003). Limitations of allotopic expression of mitochondrial genes in mammalian cells. Genetics, 165(2), 707–720. https://doi.org/10.1093/genetics/165.2.707
69. Yu-Wai-Man, P., Newman, N. J., Carelli, V., Moster, M. L., Biousse, V., Sadun, A. A., Klopstock, T., Vignal-Clermont, C., Sergott, R. C., Rudolph, G., la Morgia, C., Karanjia, R., Taiel, M., Blouin, L., Burguière, P., Smits, G., Chevalier, C., Masonson, H., Salermo, Y., … Sahel, J.-A. (2020). Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy. Science Translational Medicine, 12(573). https://doi.org/10.1126/scitranslmed.aaz7423
70. Chi, S. C., Cheng, H. C., & Wang, A. G. (2022). Leber Hereditary Optic Neuropathy: Molecular Pathophysiology and Updates on Gene Therapy. In Biomedicines (Vol. 10, Issue 8). MDPI. https://doi.org/10.3390/biomedicines10081930
71. GenSight Biologics. (n.d.). Efficacy & Safety Study of Bilateral IVT Injection of GS010 in LHON Subjects Due to the ND4 Mutation for up to 1 Year (REFLECT).ClinicalTrials.gov Identifier: NCT03293524. Last Update Posted: August 3, 2022.
72. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, N.Y.), 337(6096), 816–821. https://doi.org/10.1126/science.1225829
73. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281–2308. https://doi.org/10.1038/nprot.2013.143
74. Peeva, V., Blei, D., Trombly, G., Corsi, S., Szukszto, M. J., Rebelo-Guiomar, P., Gammage, P. A., Kudin, A. P., Becker, C., Altmüller, J., Minczuk, M., Zsurka, G., & Kunz, W. S. (2018). Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nature Communications, 9(1), 1727. https://doi.org/10.1038/s41467-018-04131-w
75. Bacman, S. R., Kauppila, J. H. K., Pereira, C. v, Nissanka, N., Miranda, M., Pinto, M., Williams, S. L., Larsson, N.-G., Stewart, J. B., & Moraes, C. T. (2018). MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation. Nature Medicine, 24(11), 1696–1700. https://doi.org/10.1038/s41591-018-0166-8
76. Rees, H. A., & Liu, D. R. (2018). Base editing: precision chemistry on the genome and transcriptome of living cells. Nature Reviews. Genetics, 19(12), 770–788. https://doi.org/10.1038/s41576-018-0059-1
77. Cho, S.-I., Lee, S., Mok, Y. G., Lim, K., Lee, J., Lee, J. M., Chung, E., & Kim, J.-S. (2022). Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell, 185(10), 1764-1776.e12. https://doi.org/10.1016/j.cell.2022.03.039
78. Mok, B. Y., de Moraes, M. H., Zeng, J., Bosch, D. E., Kotrys, A. v, Raguram, A., Hsu, F., Radey, M. C., Peterson, S. B., Mootha, V. K., Mougous, J. D., & Liu, D. R. (2020). A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 583(7817), 631–637. https://doi.org/10.1038/s41586-020-2477-4
79. Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017). Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 551(7681), 464–471. https://doi.org/10.1038/nature24644
80. Willis, J. C. W., Silva-Pinheiro, P., Widdup, L., Minczuk, M., & Liu, D. R. (2022). Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-34784-7
81. Lee, S., Lee, H., Baek, G., & Kim, J. S. (2022). Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors. Nature Biotechnology. https://doi.org/10.1038/s41587-022-01486-w
82. Lake, N. J., Bird, M. J., Isohanni, P., & Paetau, A. (2015). Leigh syndrome: neuropathology and pathogenesis. Journal of Neuropathology and Experimental Neurology, 74(6), 482–492. https://doi.org/10.1097/NEN.0000000000000195
83. Hubens, W. H. G., Vallbona-Garcia, A., de Coo, I. F. M., van Tienen, F. H. J., Webers, C. A. B., Smeets, H. J. M., & Gorgels, T. G. M. F. (2022). Blood biomarkers for assessment of mitochondrial dysfunction: An expert review. Mitochondrion, 62, 187–204. https://doi.org/10.1016/j.mito.2021.10.008
84. Sofou, K., de Coo, I. F. M., Isohanni, P., Ostergaard, E., Naess, K., de Meirleir, L., Tzoulis, C., Uusimaa, J., de Angst, I. B., Lönnqvist, T., Pihko, H., Mankinen, K., Bindoff, L. A., Tulinius, M., & Darin, N. (2014). A multicenter study on Leigh syndrome: disease course and predictors of survival. Orphanet Journal of Rare Diseases, 9, 52. https://doi.org/10.1186/1750-1172-9-52
85. Okorie, O. N., & Dellinger, P. (2011). Lactate: biomarker and potential therapeutic target. Critical Care Clinics, 27(2), 299–326. https://doi.org/10.1016/j.ccc.2010.12.013
86. Tanaka, R., Takeguchi, R., Kuroda, M., Suzuki, N., Makita, Y., Yanagi, K., Kaname, T., & Takahashi, S. (2022). Novel NARS2 variant causing leigh syndrome with normal lactate levels. Human Genome Variation, 9(1). https://doi.org/10.1038/s41439-022-00191-z
87. Peretz, R. H., Ah Mew, N., Vernon, H. J., & Ganetzky, R. D. (2021). Prospective diagnosis of MT-ATP6-related mitochondrial disease by newborn screening. Molecular Genetics and Metabolism, 134(1–2), 37–42. https://doi.org/10.1016/j.ymgme.2021.06.007
88. Dominguez-Gonzalez, C., Badosa, C., Madruga-Garrido, M., Martí, I., Paradas, C., Ortez, C., Diaz-Manera, J., Berardo, A., Alonso-Pérez, J., Trifunov, S., Cuadras, D., Kalko, S. G., Blázquez-Bermejo, C., Cámara, Y., Martí, R., Mavillard, F., Martin, M. A., Montoya, J., Ruiz-Pesini, E., … Jimenez-Mallebrera, C. (2020). Growth Differentiation Factor 15 is a potential biomarker of therapeutic response for TK2 deficient myopathy. Scientific Reports, 10(1), 10111. https://doi.org/10.1038/s41598-020-66940-8
89. Hirano, M. (n.d.). Expert Interview. 28 Feb 2022.
90. Li, Y., Li, S., Qiu, Y., Zhou, M., Chen, M., Hu, Y., Hong, S., Jiang, L., & Guo, Y. (2022). Circulating FGF21 and GDF15 as Biomarkers for Screening, Diagnosis, and Severity Assessment of Primary Mitochondrial Disorders in Children. Frontiers in Pediatrics, 10, 851534. https://doi.org/10.3389/fped.2022.851534
91. Lewis, Samantha. (n.d.). Expert Interview. 7 Mar 2022.
92. Horizon Pharma USA, Inc. (n.d.). Open-Label, Dose-Escalating Study Assessing Safety, Tolerability, Efficacy, of RP103 in Mitochondrial Disease (MITO-001). ClinicalTrials.gov Identifier: NCT02023866. May 2014-October 2016.
93. Jacoby, E., Bar-Yosef, O., Gruber, N., Lahav, E., Varda-Bloom, N., Bolkier, Y., Bar, D., Blumkin, M. B.-Y., Barak, S., Eisenstein, E., Ahonniska-Assa, J., Silberg, T., Krasovsky, T., Bar, O., Erez, N., Bielorai, B., Golan, H., Dekel, B., Besser, M. J., … Toren, A. (2022). Mitochondrial augmentation of hematopoietic stem cells in children with single large-scale mitochondrial DNA deletion syndromes. Science Translational Medicine, 14(676), eabo3724. https://doi.org/10.1126/scitranslmed.abo3724
94. Tribouillard-Tanvier, D., Dautant, A., Godard, F., Charles, C., Panja, C., di Rago, J.-P., & Kucharczyk, R. (2022). Creation of Yeast Models for Evaluating the Pathogenicity of Mutations in the Human Mitochondrial Gene MT-ATP6 and Discovering Therapeutic Molecules. Methods in Molecular Biology (Clifton, N.J.), 2497, 221–242. https://doi.org/10.1007/978-1-0716-2309-1_14