382. Correction of Autophagic Dysregulation in Pompe Disease Following Gene Therapy

Abstract

Pompe Disease (PD) is a fatal metabolic disorder caused by mutations in the GAA gene leading to a deficiency in acid alpha-glucosidase (GAA) and affects 1 in 40,000 births. Clinically, this lysosomal storage disorder presents with cardiomegaly and skeletal muscle weakness, leading to cardiorespiratory failure. GAA is responsible for the breakdown of glycogen in the lysosome, an important energy source for striated muscle and neurons. Currently, the only approved treatment for PD is enzyme replacement therapy (ERT). While ERT has increased patient survival, there are several limitations including treatment cost and the inability of the enzyme to cross the blood-brain barrier and breakdown lysosomal glycogen deposition within the central nervous system. ERT therapy is dependent upon receptor-mediated endocytosis of the exogenous enzyme; much of which remains in amphisomes resulting in insufficiently trafficking of GAA to the lysosome. These issues illustrate the need for an alternative treatment. We propose that endogenous production of GAA enzyme mediated by adeno-associated virus (AAV)-delivery of the GAA gene will improve targeting of GAA to the lysosome and reduce the overall dysregulation of vesicular systems. In this study, we performed intravenous delivery of AAV9-DES-coGAA to 12-week old Gaa−/− animals at three doses (1×1011 vg/kg, 1×1013 vg/kg, and 1×1014vg/kg) and compared to AAV9-CMV-GFP (1×1013 vg/kg) and vehicle-control groups. Previous research has revealed that cellular dysregulation due to lysosomal storage of glycogen is amassed by twelve-weeks of age prior to onset of the physiological phenotype of PD in the Gaa−/− murine model. One month after AAV or sham injection, cardiac and skeletal muscles were harvested for biochemical and histological analyses. GAA activity assays demonstrate that therapeutic levels of enzyme activity are not attained with doses 1×1011 vg/kg and 1×1013 vg/kg, but were when a dose of 1×1014 vg/kg is administered. Western Blot analysis of autophagy-associated proteins, such as Beclin, LAMP1, LC3-I, and LC3-II show a decrease in protein levels following gene therapy indicating an improvement in autophagic regulation. Hematoxalin & Eosin staining of tibialis anterior and gastrocnemius muscle sections show an inverse relationship between level of vacuolarization of muscle fibers and dose of AAV9-DES-coGAA vector. Additionally, preliminary data suggest increased endocytosis of AAVs without reaching the therapeutic threshold may activate autophagy. Further analysis by RT-qPCR and immunofluorescence will elucidate the mechanism by which treatment modifies autophagic flux.