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.

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.

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.