Pathological Features in Paediatric Patients with TK2 Deficiency.

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.

Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit.

Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance.

Dysfunctional mitochondrial translation and combined oxidative phosphorylation deficiency in a mouse model of hepatoencephalopathy due to Gfm1 mutations.

Hepatoencephalopathy due to combined oxidative phosphorylation deficiency type 1 (COXPD1) is a recessive mitochondrial translation disorder caused by mutations in GFM1, a nuclear gene encoding mitochondrial elongation factor G1 (EFG1). Patients with COXPD1 typically present hepatoencephalopathy early after birth with rapid disease progression, and usually die within the first few weeks or years of life. We have generated two different mouse models: a Gfm1 knock-in (KI) harboring the p.R671C missense mutation, found in at least 10 patients who survived more than 1 year, and a Gfm1 knock-out (KO) model. Homozygous KO mice (Gfm1-/- ) were embryonically lethal, whereas homozygous KI (Gfm1R671C/R671C ) mice were viable and showed normal growth. R671C mutation in Gfm1 caused drastic reductions in the mitochondrial EFG1 protein content in different organs. Six- to eight-week-old Gfm1R671C/R671C mice showed partial reductions of in organello mitochondrial translation and respiratory complex IV enzyme activity in the liver. Compound heterozygous Gfm1R671C/- showed a more pronounced decrease of EFG1 protein in liver and brain mitochondria, as compared with Gfm1R671C/R671C mice. At 8 weeks of age, their mitochondrial translation rates were significantly reduced in both tissues. Additionally, Gfm1R671C/- mice showed combined oxidative phosphorylation deficiency (reduced complex I and IV enzyme activities in liver and brain), and blue native polyacrylamide gel electrophoresis analysis revealed lower amounts of both affected complexes. We conclude that the compound heterozygous Gfm1R671C/- mouse presents a clear dysfunctional molecular phenotype, showing impaired mitochondrial translation and combined respiratory chain dysfunction, making it a suitable animal model for the study of COXPD1.

Therapy Prospects for Mitochondrial DNA Maintenance Disorders.

Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.

Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides.

BACKGROUND: Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches. METHODS: dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals. FINDINGS: Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy. INTERPRETATION: Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype. FUNDING: This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Growth Differentiation Factor 15 is a potential biomarker of therapeutic response for TK2 deficient myopathy.

GDF-15 is a biomarker for mitochondrial diseases. We investigated the application of GDF-15 as biomarker of disease severity and response to deoxynucleoside treatment in patients with thymidine kinase 2 (TK2) deficiency and compared it to FGF-21. GDF-15 and FGF-21 were measured in serum from 24 patients with TK2 deficiency treated 1-49 months with oral deoxynucleosides. Patients were grouped according to age at treatment and biomarkers were analyzed at baseline and various time points after treatment initiation. GDF-15 was elevated on average 30-fold in children and 6-fold in adults before the start of treatment. There was a significant correlation between basal GDF-15 and severity based on pretreatment distance walked (6MWT) and weight (BMI). During treatment, GDF-15 significantly declined, and the decrease was accompanied by relevant clinical improvements. The decline was greater in the paediatric group, which included the most severe patients and showed the greatest clinical benefit, than in the adult patients. The decline of FGF-21 was less prominent and consistent. GDF-15 is a potential biomarker of severity and of therapeutic response for patients with TK2 deficiency. In addition, we show evidence of clinical benefit of deoxynucleoside treatment, especially when treatment is initiated at an early age.

Age-related metabolic changes limit efficacy of deoxynucleoside-based therapy in thymidine kinase 2-deficient mice.

BACKGROUND: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. METHODS: We daily administered TK2 knockout mice (Tk2KO) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. FINDINGS: dThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. INTERPRETATION: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. FUND: This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Identification and Characterization of New RNASEH1 Mutations Associated With PEO Syndrome and Multiple Mitochondrial DNA Deletions.

Mitochondrial DNA (mtDNA) depletion and deletion syndrome encompasses a group of disorders caused by mutations in genes involved in mtDNA replication and maintenance. The clinical phenotype ranges from fatal infantile hepatocerebral forms to mild adult onset progressive external ophthalmoplegia (PEO). We report the case of a patient with PEO and multiple mtDNA deletions, with two new homozygous mutations in RNASEH1. The first mutation (c.487T>C) is located in the same catalytic domain as the four previously reported mutations, and the second (c.258_260del) is located in the connection domain, where no mutations have been reported. In silico study of the mutations predicted only the first mutation as pathogenic, but functional studies showed that both mutations cause loss of ribonuclease H1 activity. mtDNA replication dysfunction was demonstrated in patient fibroblasts, which were unable to recover normal mtDNA copy number after ethidium bromide-induced mtDNA depletion. Our results demonstrate the pathogenicity of two new RNASEH1 variants found in a patient with PEO syndrome, multiple deletions, and mild mitochondrial myopathy.

Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Adipocyte MTERF4 regulates non-shivering adaptive thermogenesis and sympathetic-dependent glucose homeostasis.

In humans, low brown adipose tissue (BAT) mass and activity have been associated with increased adiposity and fasting glucose levels, suggesting that defective BAT-dependent thermogenesis could contribute to the development of obesity and/or type 2 diabetes. The thermogenic function of BAT relies on a vast network of mitochondria exclusively equipped with UCP1. Mitochondrial biogenesis is exquisitely regulated by a well-defined network of transcription factors that coordinate the expression of nuclear genes required for the formation of functional mitochondria. However, less is known about the mitochondrial factors that control the expression of the genes encoded by the mitochondrial genome. Here, we have studied the role of mitochondrial transcription termination factor-4 (MTERF4) in BAT by using a new mouse model devoid of MTERF4 specifically in adipocytes (MTERF4-FAT-KO mice). Lack of MTERF4 in BAT leads to reduced OxPhos mitochondrial protein levels and impaired assembly of OxPhos complexes I, III and IV due to deficient translation of mtDNA-encoded proteins. As a result, brown adipocytes lacking MTERF4 exhibit impaired respiratory capacity. MTERF4-FAT-KO mice show a blunted thermogenic response and are unable to maintain body temperature when exposed to cold. Despite impaired BAT function, MTERF4-FAT-KO mice do not develop obesity or insulin resistance. Still, MTERF4-FAT-KO mice became resistant to the insulin-sensitizing effects of ß3-specific adrenergic receptor agonists. Our results demonstrate that MTERF4 regulates mitochondrial protein translation and is essential for proper BAT thermogenic activity. Our study also supports the notion that pharmacological activation of BAT is a plausible therapeutic target for the treatment of insulin resistance.

Retrospective natural history of thymidine kinase 2 deficiency.

BACKGROUND: Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. OBJECTIVE: To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. METHODS: The study was conducted by 42 investigators across 31 academic medical centres. RESULTS: We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. CONCLUSIONS: In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder.

Cardiomyocyte hypertrophy induced by Endonuclease G deficiency requires reactive oxygen radicals accumulation and is inhibitable by the micropeptide humanin.

The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3ß and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency.

Long-Term Sustained Effect of Liver-Targeted Adeno-Associated Virus Gene Therapy for Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in TYMP, the gene encoding the enzyme thymidine phosphorylase (TP). TP dysfunction results in systemic accumulation of the noxious TP substrates thymidine and deoxyuridine. Gene therapy using either a lentiviral vector or adeno-associated vector (AAV) has proven to be a feasible strategy, as both vectors restore biochemical homeostasis in a murine model of the disease. This study shows that the effect of an AAV containing the TYMP coding sequence transcriptionally targeted to the liver persists long term in mice. Although the vector copy number was diluted and AAV-mediated liver TP activity eventually reduced or lost after 21 months at the lowest vector doses, the effect was sustained (with a negligible decrease in TP activity) and fully effective on nucleoside homeostasis for at least 21 months at a dose of 2 × 1012 vg/kg. Macroscopic visual inspection of the animals' organs at completion of the study showed no adverse effects associated with the treatment. These results further support the feasibility of gene therapy for MNGIE.

Identification and characterization of the novel point mutation m.3634A>G in the mitochondrial MT-ND1 gene associated with LHON syndrome.

Leber's hereditary optic neuropathy (LHON) is a mitochondrial genetic disease characterized by bilateral acute or subacute progressive central visual loss. Most cases of LHON syndrome are caused by point mutations in the MT-ND1, MT-ND4, and MT-ND6 genes. Here, we report a novel homoplasmic mutation in the MT-ND1 gene (m.3634A>G, p.Ser110Gly) in a patient with the classical clinical features of LHON syndrome. Several observations support the idea that the mutation is pathogenic and involved in the clinical phenotype of the patient: 1) The mutation affected a highly conserved amino acid, 2) A pathogenic mutation in the same amino acid (m.3635G>A, p.Ser110Asn) was previously reported in a patient with LHON syndrome, 3) The mutation is not recorded in the Mitomap or Human Mitochondrial Genome Database, 4) In silico predictors classified the mutation as "probably damaging", and 5) Cybrids carrying the mutation showed decreased Complex I enzyme activity, lower cell proliferation, and decreased mitochondrial membrane potential relative to control cybrids.

Long-Term Restoration of Thymidine Phosphorylase Function and Nucleoside Homeostasis Using Hematopoietic Gene Therapy in a Murine Model of Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a metabolic disorder caused by mutations in TYMP, encoding thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction produces systemic thymidine and deoxyuridine accumulation, which ultimately impairs mitochondrial DNA replication and results in mitochondrial dysfunction. To date, only allogeneic hematopoietic stem cell transplantation has demonstrated long-term clinical efficacy, but high morbidity and mortality associated with this procedure necessitate the search for safer alternatives. In a previous study, we demonstrated that hematopoietic stem cell gene therapy using a lentiviral vector containing the coding sequence of TYMP restored the biochemical homeostasis in an animal model of MNGIE. In the present follow-up study, we show that ectopic expression of TP in the hematopoietic system restores normal nucleoside levels in plasma, as well as in tissues affected in MNGIE such as small intestine, skeletal muscle, brain, and liver. Mitochondrial dNTP pool imbalances observed in liver of the animal model were also corrected by the treatment. The biochemical effects were maintained at least 20 months even with low levels of chimerism. No alterations in the blood cell counts or other toxic effects were observed in association with the lentiviral transduction or TP overexpression. These results further support the notion that gene therapy is a feasible treatment option for MNGIE.

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria.

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients' quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.

Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.