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Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp−/−Upp1−/− mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2–3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp−/−Upp1−/− mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

One of the most common mitochondrial DNA (mtDNA) mutations, the A to G transition at base pair 3243, has been linked to changes in the brain, in addition to commonly observed hearing problems, diabetes and myopathy. However, a detailed quantitative description of m.3243A>G patients' brains has not been provided so far. In this study, ultra-high field MRI at 7T and volume- and surface-based data analyses approaches were used to highlight morphology (i.e. atrophy)-, microstructure (i.e. myelin and iron concentration)- and metabolism (i.e. cerebral blood flow)-related differences between patients (N = 22) and healthy controls (N = 15). The use of quantitative MRI at 7T allowed us to detect subtle changes of biophysical processes in the brain with high accuracy and sensitivity, in addition to typically assessed lesions and atrophy. Furthermore, the effect of m.3243A>G mutation load in blood and urine epithelial cells on these MRI measures was assessed within the patient population and revealed that blood levels were most indicative of the brain's state and disease severity, based on MRI as well as on neuropsychological data. Morphometry MRI data showed a wide-spread reduction of cortical, subcortical and cerebellar gray matter volume, in addition to significantly enlarged ventricles. Moreover, surface-based analyses revealed brain area-specific changes in cortical thickness (e.g. of the auditory cortex), and in T1, T2* and cerebral blood flow as a function of mutation load, which can be linked to typically m.3243A>G-related clinical symptoms (e.g. hearing impairment). In addition, several regions linked to attentional control (e.g. middle frontal gyrus), the sensorimotor network (e.g. banks of central sulcus) and the default mode network (e.g. precuneus) were characterized by alterations in cortical thickness, T1, T2* and/or cerebral blood flow, which has not been described in previous MRI studies. Finally, several hypotheses, based either on vascular, metabolic or astroglial implications of the m.3243A>G mutation, are discussed that potentially explain the underlying pathobiology. To conclude, this is the first 7T and also the largest MRI study on this patient population that provides macroscopic brain correlates of the m.3243A>G mutation indicating potential MRI biomarkers of mitochondrial diseases and might guide future (longitudinal) studies to extensively track neuropathological and clinical changes. Keywords: 7T MRI, Brain, Quantitative, m.3243A>G, Mitochondrial