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AAV9/hSURF1 normalized exhaustive exercise-induced lactic acidosis in SURF1 KO mice (A) Scheme of study design. (B) Running time on treadmill until exhaustion. (C) Resting blood lactate level before treadmill running. (D) Change of lactate after running on treadmill until exhaustion. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with KO+Vehicle mice using a Tukey's multiple comparison test following two-way ANOVA. (E and F) COX activity of cerebrum (E) (n = 8-10 per group) and liver (F) (n = 9-10 per group) of mice at 18 months of age. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with WT mice using Dunn's multiple comparison method following a Kruskal-Wallis test, since KO+High mice did not pass the normality test using the ShapiroWilk's test. # p < 0.05, ### p < 0.001 compared with KO+Vehicle mice using a one-tailed Student's t test. A dashed line in (E) and (F) indicates the level of KO+Vehicle, and the percentage of improvement compared with the KO+Vehicle group from each treatment is provided at the bottom of the respective column. Each data point represents measurement from an individual animal, with bars representing the mean ± SEM.
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SURF1 (surfeit locus protein 1)-related Leigh syndrome is an early onset neurodegenerative disorder, characterized by reduction in complex IV activity, resulting in disrupted mitochondrial function. Currently, there are no treatment options available. To test our hypothesis that AAV9/hSURF1 gene replacement therapy can provide a potentially meaning...
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Context 1
... and examined blood lactate levels both at rest and after exhaustive exercise. The study scheme is shown in Figure 4A. The mice were tested at two ages, 10 weeks (6 weeks after treatment) and 10 months (9 months after treatment). ...
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... were no differences in their running time (Fig- ure 4B) or resting lactate level among groups at both ages (Fig- ure 4C). However, as shown in Figure 4D, DLactate of SURF1 KO mice was significantly higher than that of WT animals when tested at 10 months of age (p < 0.001). Both low-dose and high-dose treatments significantly reduced DLactate of KO mice compared with vehicle-treated KO mice (p < 0.01 and p < 0.001, respectively). ...
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... we collected their tissues at 18 months of age and examined COX activity of cerebrum and liver. We obtained similar trends of improvement as found in previous analysis from the 8-week-old mice ( Figures 4E and 4F). Taken together, our data suggest that both low and high doses of AAV9/hSURF1 treatment administered i.t. ...
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Background
Mutation of the m. 8993 T > G ATP6 subunit of ATP synthase causes a maternally inherited Leigh Syndrome (LS), a rapidly fatal encephalomyelopathy in childhood called and related Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) in adults. There is no cure, and relevant animal models are few. Here we describe a novel ATP6 mouse model of...
Citations
... Symptomatic treatments include manipulating cell content of mitochondria, inducing mitochondrial turnover through rapamycin, restoring NAD + levels, modulating the production of reactive oxygen species and oxidative stress, ect (Russell et al., 2020). Gene therapy includes direct editing of mitochondrial genomes, gene replacement therapy (Silva-Pinheiro et al., 2020;Ling et al., 2021), and mitochondria transfer therapy (Greenfield et al., 2017). ...
Introduction: Mitochondrial diseases caused by mtDNA have no effective cures. Recently developed DddA-derived cytosine base editors (DdCBEs) have potential therapeutic implications in rescuing the mtDNA mutations. However, the performance of DdCBEs relies on designing different targets or improving combinations of split-DddA halves and orientations, lacking knowledge of predicting the results before its application.
Methods: A series of DdCBE pairs for wide ranges of aC or tC targets was constructed, and transfected into Neuro-2a cells. The mutation rate of targets was compared to figure out the potential editing rules.
Results: It is found that DdCBEs mediated mtDNA editing is predictable: 1) aC targets have a concentrated editing window for mtDNA editing in comparison with tC targets, which at 5’C8-11 (G1333) and 5’C10-13 (G1397) for aC target, while 5’C4-13 (G1333) and 5’C5-14 (G1397) for tC target with 16bp spacer. 2) G1333 mediated C>T conversion at aC targets in DddA-half-specific manner, while G1333 and G1397 mediated C>T conversion are DddA-half-prefer separately for tC and aC targets. 3) The nucleotide adjacent to the 3’ end of aC motif affects mtDNA editing. Finally, by the guidance of these rules, a cell model harboring a pathogenic mtDNA mutation was constructed with high efficiency and no bystander effects.
Discussion: In summary, this discovery helps us conceive the optimal strategy for accurate mtDNA editing, avoiding time- and effort-consuming optimized screening jobs.
... Examples of this include the MPV17 deficiency model in which mice did not develop cirrhosis or cerebral dysfunction compared to the human phenotype, and the SURF1 deficient mouse model which did not demonstrate reduced survival. 50,71 In TK2 deficiency, 52 the model displayed encephalomyopathy and spinal cord disease that is not seen in patients and may explain why only partial improvement in the disease phenotype could be achieved. Conclusions regarding efficacy can thus be difficult to deduce when organ involvement is milder, since it is not possible to predict how the treatment would perform in humans, or if it is more severe or grossly different (e.g. if there is organ involvement that is not seen in humans), this may lead to an inability to detect a therapeutic effect. ...
... This poses an additional translational hurdle due to the route of delivery. In SURF1 deficiency, 50 an intrathecal route was used instead, although the translatability of using this route to target the brain in humans with Leigh syndrome is yet to be demonstrated. In TYMP deficiency, 69 vector dilution was seen in liver due to loss of transduction as the liver divides, despite adult gene transfer. ...
... 132,133 The main drawback is the high risk of injury to the medulla in humans. 134 An AAV9 lumbar intrathecal approach was able to ameliorate brain mitochondrial dysfunction in murine SURF1 deficiency, however biodistributional studies in NHPs yielded conflicting results, 50,132,135 therefore it is unclear whether this approach could be translated in humans. ...
In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno‐associated virus‐based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator‐like effector nucleases, zinc‐finger nucleases, and meganucleases (mitoARCUS). rAAV‐based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR‐Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR‐free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design.
... Dr. Steven Gray provided an overview of adenoassociated viruses as a vector for gene therapy [11]. He presented data showing that adeno-associated virus 9-mediated expression of human SURF1 in Surf1 knockout mice restored Surf1 expression and normalized measures of mitochondrial dysfunction, including complex IV levels and lactic acidosis [11]. ...
... Dr. Steven Gray provided an overview of adenoassociated viruses as a vector for gene therapy [11]. He presented data showing that adeno-associated virus 9-mediated expression of human SURF1 in Surf1 knockout mice restored Surf1 expression and normalized measures of mitochondrial dysfunction, including complex IV levels and lactic acidosis [11]. However, the value of the Surf1 knockout mouse model has been contested as not reflective of the symptomology observed in patients; future studies are needed to evaluate the efficacy of adeno-associated virus 9-mediated expression of the genes affected in other models of Leigh syndrome. ...
Background
Leigh syndrome, an inherited neurometabolic disorder, is estimated to be the most common pediatric manifestation of mitochondrial disease. No treatments are currently available for Leigh syndrome due to many hurdles in drug discovery efforts. Leigh syndrome causal variants span over 110 different genes and likely lead to both unique and shared biochemical alterations, often resulting in overlapping phenotypic features. The mechanisms by which pathogenic variants in mitochondrial genes alter cellular phenotype to promote disease remain poorly understood. The rarity of cases of specific causal variants creates barriers to drug discovery and adequately sized clinical trials.
Body
To address the current challenges in drug discovery and facilitate communication between researchers, healthcare providers, patients, and families, the Boston University integrative Cardiovascular Metabolism and Pathophysiology (iCAMP) Lab and Cure Mito Foundation hosted a Leigh Syndrome Symposium. This symposium brought together expert scientists and providers to highlight the current successes in drug discovery and novel models of mitochondrial disease, and to connect patients to providers and scientists to foster community and communication.
Conclusion
In this symposium review, we describe the research presented, the hurdles ahead, and strategies to better connect the Leigh syndrome community members to advance treatments for Leigh syndrome.
... However, due to genetic differences between mice and humans, creating a model that mimics human disease can sometimes be challenging, especially for rare diseases, which are generally less studied. For example, this has been a challenge in the development of a gene therapy for an early-onset neurodegenerative disease called SURF1 (Surfeit locus protein 1)-related Leigh syndrome, which has only one viable animal model available 98 . Even with complete knockout of Surf1 in mice, disease-related phenotypes are not recapitulated, including a lack of neurological dysfunction and even longer life spans than WT mice 99 . ...
... Our group has recently published a preclinical proof-of-concept study on using AAV gene therapy for SURF1-related Leigh syndrome, which is a severe early-onset neurodegenerative mitochondrial disease 98 . Other studies have demonstrated that AAV gene therapy can rescue phenotypes in Nduf3 and Ndufs4 knockout mouse models, which represent other common causes for Leigh syndrome [105][106][107][108] . ...
Recent advancements in gene supplementation therapy are expanding the options for the treatment of neurological disorders. Among the available delivery vehicles, adeno-associated virus (AAV) is often the favoured vector. However, the results have been variable, with some trials dramatically altering the course of disease whereas others have shown negligible efficacy or even unforeseen toxicity. Unlike traditional drug development with small molecules, therapeutic profiles of AAV gene therapies are dependent on both the AAV capsid and the therapeutic transgene. In this rapidly evolving field, numerous clinical trials of gene supplementation for neurological disorders are ongoing. Knowledge is growing about factors that impact the translation of preclinical studies to humans, including the administration route, timing of treatment, immune responses and limitations of available model systems. The field is also developing potential solutions to mitigate adverse effects, including AAV capsid engineering and designs to regulate transgene expression. At the same time, preclinical research is addressing new frontiers of gene supplementation for neurological disorders, with a focus on mitochondrial and neurodevelopmental disorders. In this Review, we describe the current state of AAV-mediated neurological gene supplementation therapy, including critical factors for optimizing the safety and efficacy of treatments, as well as unmet needs in this field.
... Ling et al. [58] treated 1 month old Surf1-KO mice with self-complimentary AAV9 carrying human SURF1 under control of the CB promoter. Unlike the native AAV genome, self-complementary vectors are double stranded, making synthesis of the second strand unnecessary [59]. ...
... A self-complementary vector, however, can only carry half the genetic material of a single-stranded vector (about 2.4 kb vs 4.7 kb). Ling et al. [58] chose to use self-complimentary AAV due to the relatively small size of the SURF1 cDNA and CB promoter and some evidence that self-complimentary AAV9 vectors result in a higher number of transduced cells [60]. scAVV9-CB-SURF1 was administered by intrathecal (IT) injection or both IT and IV injection. ...
... scAVV9-CB-SURF1 was administered by intrathecal (IT) injection or both IT and IV injection. Surf1-KO mice treated with the highest intrathecal dose of scAAV9-CB-hSURF1 had a statistically significant, though very modest, increase in CIV activity in the cerebrum, cerebellum, liver and muscle compared to wild-type animals [58]. A combination of IV and IT delivery of the therapeutic vector did not result in increased CIV activity compared to IT treatment alone [58]. ...
Mitochondrial diseases are a group of rare, heterogeneous diseases caused by gene mutations in both nuclear and mitochondrial genomes that result in defects in mitochondrial function. They are responsible for significant morbidity and mortality as they affect multiple organ systems and particularly those with high energy-utilizing tissues, such as the nervous system, skeletal muscle, and cardiac muscle. Virtually no effective treatments exist for these patients, despite the urgent need. As the majority of these conditions are monogenic and caused by mutations in nuclear genes, gene replacement is a highly attractive therapeutic strategy. Adeno-associated virus (AAV) is a well-characterized gene replacement vector, and its safety profile and ability to transduce quiescent cells nominates it as a potential gene therapy vehicle for several mitochondrial diseases. Indeed, AAV vector-based gene replacement is currently being explored in clinical trials for one mitochondrial disease (Leber hereditary optic neuropathy) and preclinical studies have been published investigating this strategy in other mitochondrial diseases. This review summarizes the preclinical findings of AAV vector-based gene replacement therapy for mitochondrial diseases including Leigh syndrome, Barth syndrome, ethylmalonic encephalopathy, and others.
... Ultimately, we want to further explore the possibility to adopt the proposed protocol not only for replacing the Ndufs4 gene but also other nucleus-encoded genes known to be responsible of Leigh disease, as recently shown for a Surf1 −/− model generated by us and treated elsewhere with a single intra-thecal injection at 4 weeks after birth of a scAAV9 SURF1 vector. 21 Alternatively, the direct administration of Ndufs4 RNA can be considered, given the important results recently obtained by an approach, termed selective endogenous encapsidation for cellular delivery. Selective endogenous encapsidation for cellular delivery exploits the capacity of some retroviral-like proteins to secrete its own mRNA, opening the possibility to exploit them to deliver functional mRNA cargos to mammalian cells. ...
Leigh disease, or subacute necrotizing encephalomyelopathy, a genetically heterogeneous condition consistently characterized by defective mitochondrial bioenergetics, is the most common oxidative-phosphorylation related disease in infancy. Both neurological signs and pathological lesions of Leigh disease are mimicked by the ablation of the mouse mitochondrial respiratory chain subunit Ndufs4-/-, which is part of, and crucial for, normal Complex I activity and assembly, particularly in the brains of both children and mice. We previously conveyed the human NDUFS4 gene to the mouse brain using either single-stranded adeno-associated viral 9 recombinant vectors or the PHP.B adeno-associated viral vector. Both these approaches significantly prolonged the lifespan of the Ndufs4-/- mouse model but the extension of the survival was limited to a few weeks by the former approach, whereas the latter was applicable to a limited number of mouse strains, but not to primates. Here, we exploited the recent development of new, self-complementary adeno-associated viral 9 vectors, in which the transcription rate of the recombinant gene is markedly increased compared with the single-stranded adeno-associated viral 9 and can be applied to all mammals, including humans. Either single intra-vascular or double intra-vascular and intra-cerebro-ventricular injections were performed at post-natal Day 1. The first strategy ubiquitously conveyed the human NDUFS4 gene product in Ndufs4-/- mice, doubling the lifespan from 45 to ≈100 days after birth, when the mice developed rapidly progressive neurological failure. However, the double, contemporary intra-vascular and intra-cerebroventricular administration of self-complementary-adeno-associated viral NDUFS4 prolonged healthy lifespan up to 9 months of age. These mice were well and active at euthanization, at 6, 7, 8 and 9 months of age, to investigate the brain and other organs post-mortem. Robust expression of hNDUFS4 was detected in different cerebral areas preserving normal morphology and restoring Complex I activity and assembly. Our results warrant further investigation on the translatability of self-complementary-adeno-associated viral 9 NDUFS4-based therapy in the prodromal phase of the disease in mice and eventually humans.
... An scAAV9 vector carrying hSURF1 cDNA under the control of the hybrid chickenβ-actin/CMV enhancer (CAG) promoter was delivered through an intrathecal injection (8 × 10 11 vg/mouse) to one-month-old KO mice. The treatment effectively increased the SURF1 mRNA expression in multiple relevant organs, including the brain and spinal cord, leading to a partial recovery of the COX activity in all tissues as well as the abnormal lactate acidosis induced by exhaustive exercise [116]. ...
Mitochondrial diseases (MDs) are a group of severe genetic disorders caused by mutations in the nuclear or mitochondrial genome encoding proteins involved in the oxidative phosphorylation (OXPHOS) system. MDs have a wide range of symptoms, ranging from organ-specific to multisystemic dysfunctions, with different clinical outcomes. The lack of natural history information, the limits of currently available preclinical models, and the wide range of phenotypic presentations seen in MD patients have all hampered the development of effective therapies. The growing number of pre-clinical and clinical trials over the last decade has shown that gene therapy is a viable precision medicine option for treating MD. However, several obstacles must be overcome, including vector design, targeted tissue tropism and efficient delivery, transgene expression, and immunotoxicity. This manuscript offers a comprehensive overview of the state of the art of gene therapy in MD, addressing the main challenges, the most feasible solutions, and the future perspectives of the field.
... 10 The peculiarities of mitochondrial genetics mean that replicating this for mitochondrial DNA (mtDNA) has been more challenging, but a clinical trial success in LHON was reported recently 15 and work is underway for SURF1 associated Leigh syndrome. 16 Another new development, which holds promise for new genetic therapies for mitochondrial disease, is the discovery of an effective method for base editing mtDNA. 17 Priority 4. What are the biological mechanisms that cause mitochondrial disease to get worse over time? ...
Primary mitochondrial disorders encompass a wide range of clinical presentations and a spectrum of severity. They currently lack effective disease‐modifying therapies and have a high mortality and morbidity rate. It is therefore essential to know that competitively‐funded research designed by academics meets core needs of people with mitochondrial disorders and their clinicians. The Priority Setting Partnerships are an established collaborative methodology that brings patients, carers and families, charity representatives and clinicians together to try to establish the most pressing and unanswered research priorities for a particular disease. We developed a web‐based questionnaire, requesting all patients affected by primary mitochondrial disease, their carers, and clinicians to pose their research questions. This yielded 709 questions from 147 participants. These were grouped into overarching themes including basic biology, causation, health services, clinical management, social impacts, prognosis, prevention, symptoms, treatment, and psychological impact. Following the removal of ‘answered questions’ the process resulted in a list of 42 discrete, answerable questions. This was further refined by web‐based ranking by the community to 24 questions. These were debated at a face‐to‐face workshop attended by a diverse range of patients, carers, charity representatives and clinicians to create a definitive ‘Top Ten of unanswered research questions for primary mitochondrial disorders’. These Top Ten questions related to understanding biological processes, including triggers of disease onset, mechanisms underlying progression and reasons for differential symptoms between individuals with identical genetic mutations; new treatments; biomarker discovery; psychological support; and optimal management of stroke‐like episodes and fatigue. This article is protected by copyright. All rights reserved.
... Gene therapy for spinal muscle atrophy (SMA) has recently been developed and has shown promising effects [82]. In LS with SURF1 mutations, intrathecal delivery of adeno-associated viral vector serotype 9 (AAV9)/human SURF1 (hSURF1) was studied in mice and was found effective in improving the biochemical abnormalities induced by SURF1 deficiency, thus showing potential applicability for patients with SURF1related Leigh syndrome in the future [83]. ...
SURF1 encodes the assembly factor for maintaining the antioxidant of cytochrome c oxidase (COX) stability in the human electron respiratory chain. Mutations in SURF1 can cause Leigh syndrome (LS), a subacute neurodegenerative encephalopathy, characterized by early onset (infancy), grave prognosis, and predominant symptoms presenting in the basal ganglia, thalamus, brainstem, cerebellum, and peripheral nerves. To date, more than sixty different SURF1 mutations have been found to cause SURF1-associated LS; however, the relationship between genotype and phenotype is still unclear. Most SURF1-associated LS courses present as typical LS and cause early mortality (before the age of ten years). However, 10% of the cases present with atypical courses with milder symptoms and increased life expectancy. One reason for this inconsistency may be due to specific duplications or mutations close to the C-terminus of the SURF1 protein appearing to cause less protein decay. Furthermore, the treatment for SURF1-associated LS is unsatisfactory. A ketogenic diet is most often prescribed and has proven to be effective. Supplementing with coenzyme Q and other cofactors is also a common treatment option; however, the results are inconsistent. Importantly, anti-epileptic drugs such as valproate—which cause mitochondrial dysfunction—should be avoided in patients with SURF1-associated LS presenting with seizures.
The proximity and association of cerebrospinal fluid (CSF) and the intrathecal (IT) space with deep targets in the central nervous system (CNS) parenchyma makes IT injection an attractive route of administration for brain drug delivery. However, the extent to which intrathecally administered macromolecules are effective in treating neurological diseases is a question of both clinical debate and technological interest. We present the biological, chemical, and physical properties of the intrathecal space that are relevant to drug absorption, distribution, metabolism, and elimination from CSF. We then analyze the evolution of IT drug delivery in clinical trials over the last 20 years. Our analysis revealed that the percentage of clinical trials assessing IT delivery for the delivery of biologics (i.e., macromolecules, cells) for treatment of chronic conditions (e.g., neurodegeneration, cancer, and metabolic diseases) has steadily increased. Clinical trials exploring cell or macromolecular delivery within the IT space have not evaluated engineering technologies, such as depots, particles, or other delivery systems. Recent pre-clinical studies have evaluated IT macromolecule delivery in small animals, postulating that delivery efficacy can be assisted by external medical devices, micro- or nanoparticles, bulk biomaterials, and viral vectors. Further studies are necessary to evaluate the extent to which engineering technologies and IT administration improve CNS targeting and therapeutic outcome.
