Alpha-mannosidosis (AMD) is an autosomal recessive lysosomal storage disorder secondary to mutations in the alpha-mannosidase (MANB) gene. The deficiency in MANB enzymatic activity results in accumulation of oligomannosidic- N-glycans (Manα1-3Manβ1-4GlcNAc) in the secondary lysosomes leading to cell swelling and dysfunction.¹ AMD usually occurs in childhood and predominantly affects the central nervous system. Cats with AMD exhibit similar clinical, biochemical, and neuropathological phenotypes as human patients and therefore are considered as homologues of human disease.¹ Previous in vitro² and in vivo³ proton MR spectroscopy (¹H MRS) studies have demonstrated increases in resonance at 1.8-2.2ppm region due to the N-CH₃ protons of Glc-NAc group from N-acetylated sugars and a broad resonance at 3.5-4.0ppm region corresponding to mannose-rich oligosaccharides (OG) in AMD affected feline brains suggesting that these peaks may be useful biomarkers for AMD diagnosis. Similar to the feline study, a case study of a patient with AMD demonstrated a broad resonance at 3.7ppm from basal ganglia.⁴ Following bone marrow transplant, a decline in this peak was observed signifying the clinical potential of ¹H MRS in monitoring treatment response in AMD. Adeno-associated virus (AAV) vector gene therapy has been shown to be a promising strategy in the feline AMD model.^5,6 We evaluated in vivo ¹H MRS in cats treated AAV infusion into cisterna-magna in AMD cats in comparison to untreated AMD and normal cats. Age-matched cats (n=9) raised in the animal colony of the veterinary school were used in this study. The cats were genotyped at birth. Of these 9 animals, 3 cats had no evidence of AMD and were considered as normal controls and 3 cats were affected with AMD, were not treated, and served as untreated controls. The remaining three AMD affected cats were treated with gene therapy using a single dose of AAV-1 expressing feline MANB infused through cisterna magna. Prior to imaging, all cats were anesthetized and were subjected to anatomical MR imaging and single-slice multi-voxel 2D-¹H MRS on a 3T MR system equipped with a single-channel transmit-receive birdcage coil. Sequence parameters for ¹H MRS included: TR/TE=2500/30ms, number of excitations=16, slice thickness=7mm, in-plane resolution=3.43x3.43mm². ¹H MRS data were analyzed using the Siemens Leonardo workstation. Peak areas of different metabolites [N-acetyalaspartate (NAA)+Glc-NAc, creatine (Cr), choline (Cho) and OG] were determined from each voxel encompassing cerebral cortex, and thalamus. Peak areas were normalized with respect to ipsilateral unsuppressed water for each voxel. Differences in metabolite ratios among different groups were assessed by one way ANOVA and p value < 0.05 was considered significant. Representative spectra from the thalamic region of normal control, untreated AMD cats and cats in the AAV treatment group are shown in Fig. 1. There were significant elevations in OG/water in cortical (p=0.01) and thalamic regions (p=0.004) of untreated cats compared to normal controls (Table. 1). A non-significant (p>0.05) decrease in OG/water and NAA(Glc-NAc)/Water ratio was observed in AAV treated AMD cats compared to the untreated group suggesting partial treatment response. No other metabolites demonstrated significant difference between groups. Histopathological findings revealed reduced number of lysosomal storage lesions in cats treated with gene therapy. These preliminary results indicate that OG and Glc-NAc resonances may be used as non-invasive biomarkers for evaluating treatment response to gene therapy in AMD. A single dose of AAV-fMANB into cisterna magna results in resolution of lysosomal storage lesions throughout the brain along with improved clinical symptoms and extended lifespan⁶. The decreases in OG and Glc-NAc levels in the treatment group suggesting partial response to gene therapy. We plan to determine the correlation between the in vivo and in vitro ¹H MRS changes of OG on an expanded cohort. Future multimodality imaging studies involving ¹H MRS and apparent diffusion coefficient (ADC)⁷ may also improve the ability to monitor changes during therapy non-invasively.