Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease caused by deficiency of α-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans ¹. Manifestations of the disease include multi-systemic disorders, and in the severe form of the disease (Hurler syndrome) can lead to death by age ten if left untreated. Currently used treatments, such as enzyme replacement therapy and hematopoietic stem cell transplantation, appear to be inefficient for CNS treatment. In this study we have used intrathecal delivery of an adeno-associated virus serotype 9 vector transducing the IDUA gene (AAV9-IDUA) to the CNS in a knock-out mouse model of MPS I ^2,3. The purpose of this study was to assess the ability of the AAV-mediated gene therapy to prevent the pathological neurochemical changes associated with the MPS I disease.C57BL/6 knock-out mice deficient for IDUA were used as a well-established model of Hurler syndrome. AAV9-miniCags-IDUA vector was delivered intrathecally to MPS I mice at 12 weeks of age. Prior to AAV administration, the mice were injected with mannitol to open the blood-brain barrier and immunotolerized with laronidase to prevent anti-IDUA immune response. In vivo ¹H MR spectra were acquired from the hippocampus and cerebellum of AAV9-IDUA gene treated MPS I mice (MPS I treated, N = 11), untreated MPS I mice (MPS I, N = 12) and heterozygote littermates (control, N = 12) at 9 months of age. In vivo ¹H MRS data were acquired at 9.4T using FASTMAP shimming and ultra-short TE STEAM (TE = 2 ms) localization sequence combined with VAPOR water suppression ⁴. Metabolites were quantified using LCModel with the spectrum of fast relaxing macromolecules included in the basis set. Spontaneously breathing animals were anesthetized with 1.0 – 1.5% isoflurane.The spectral quality consistently accomplished in this study (Figs. 1 and 3) enabled reliable quantification of fifteen brain metabolites (Figs. 2 and 4). Small but significant increases in ascorbate (Asc, +0.6 µmol/g, p = 0.003) and N-acetylaspartylglutamate (NAAG, +0.3 μmol/g, p = 0.015) concentrations were observed in the hippocampus of untreated MPS I mice relative to controls (Fig. 2). In addition, a trend of increased glutathione level (GSH, +0.2 µmol/g, p = 0.054) has been observed. Differences between cerebellar neurochemical profiles of untreated MPS I mice and controls (Fig. 4) include an increase in NAAG (+0.25 µmol/g, p = 0.026) and a decrease in phosphoethanolamine (PE, -0.44 µmol/g, p = 0.04). Neurochemical profiles of MPS I mice treated with AAV9-IDUA showed remarkable similarity to those of control mice (Figs. 2 and 4). In the hippocampus of treated MPS I mice, the levels of Asc, NAAG and GSH were normalized; only lactate (Lac) showed a small difference relative to control. In the cerebellum of treated MPS I mice, PE but not NAAG level was normalized. Small, but significant differences between treated and control mice were observed for Asc, Lac taurine (Tau) and total creatine (Cr+PCr). Except Asc, changes in metabolite concentrations in treated MPS-I mice were always opposite to those observed in the untreated group. In addition, for a number of metabolites that did not show significant changes between untreated MPS I mice and controls (e.g. glucose, glutamate, NAA) it appears that metabolite levels found in treated MPS I mice were closer to controls than to untreated MPS I mice.Significantly increased concentrations of Asc and a trend for increased GSH in the hippocampus of untreated MPS I mice indicate a protective response against the oxidative stress reported in lysosomal diseases ^5,6. Whereas decreased PE in the cerebellum and increased NAAG in both brain regions of untreated MPS I may indicate demyelination ^7,8. A similar pattern of decreased PE and increased NAAG was observed in iron deficiency model where altered myelination was confirmed ⁹. The comparison of hippocampal and cerebellar neurochemical profiles of treated MPS I mice against those of untreated MPS I and control mice (Figs. 2 and 4) clearly demonstrates that direct transfer of the missing IDUA gene to the CNS using intrathecal delivery of AAV9 (at 12 weeks of age) prevented neurochemical alternations (at 9 months of age) associated with the neurodegenerative processes in this MPS I mouse model. These neurochemical results are in agreement with similar gene therapy approaches tested in the mouse model of MPS I ^3,10.Gene therapy based on direct AAV9-IDUA delivery to the CNS indicates that the oxidative stress and demyelination associate with this mouse model of MPS I can be prevented.