Pompe disease is an autosomal recessive disorder caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The disease is characterized by lysosomal glycogen storage in heart and muscles, and manifests as a fatal cardiomyopathy in infantile form. Cardiac correction by enzyme replacement therapy (ERT) has recently prolonged the lifespan of these patients, revealing a new natural history. The emergent neurologic phenotype and the poor correction of skeletal muscles in survivors are currently partly attributed to central nervous system (CNS) glycogen storage, uncorrected by ERT. A gene therapy strategy using AAV vectors delivered to cerebrospinal fluid has been set up to restore GAA activity into the CNS. We demonstrate the use of Infrared Micro spectroscopy with synchrotron light as an innovative tool to map glycogen at the subcellular level in motor neurons and cardiac fibers. Principal Component Analysis (PCA) of infrared spectral data from motor neurons and cardiac fibers show that both treated and wild-type animals are merged in the same cluster whereas infrared spectra obtained from untreated Pompe mice are characterized by increase of the bands assigned to the carbohydrates of glycogen.

This new analytical approach that allows an highly sensitive and resolutive direct probing of tissue glycogen is required to explore early biochemical change at a subcellular level and therefore to assess therapeutic efficiency for Pompe disease.

Acknowledgments : We thank the vector core of the Atlantic Gene Therapies Institute (AGT) in Nantes for the preparation of the rAAV vectors, Véronique Blouin and Philippe Moullier (INSERM UMR1089) for vector production and the technical staff of Oniris rodent facility for animal care. We acknowledge assistance from SOLEIL SMIS beamline staff for his help. This work was supported by a  grant from “Investissement d’Avenir – ANR-11-INBS-0011”  – NeurATRIS :  A Translational Research Infrastructure for Biotherapies in Neurosciences

Figures:

Figure 1 : A, Periodic acid-Schiff (PAS) luxol-fast blue staining used to detect glycogen cardiac muscle. B, Glycogen mapping by infrared microspectroscopic imaging in cardiac muscle. Wild type mice (Wt-PBS), non treated Pompe mice (nt-PBS), AAV10 treated Pompe mice (pt-/- AAV10), AAV9 treated Pompe mice (pt-/- AAV9).

Figure 2 : Principal component analysis (PCA) scores plot showing the unique chemical composition of the cardiac fibers of wild type mice (Awtmap), non treated Pompe mice (Antmap) and AAV treated Pompe mice (A09map for AAV9, A10map for AAV10) visualized as distinct clusters along PC1 versus PC2. AAV9 treated Pompe mice (A09map) and wild type animals (Awtmap) were merged in the same cluster.

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EMC Abstracts - https://emc-proceedings.com/abstract/synchrotron-infrared-microspectroscopy-an-innovative-approach-to-investigate-tissue-chemical-changes-in-mouse-model-of-pompe-of-disease-glycogenosis-type-ii-and-to-assess-efficiency-in-gene-therapy/