PROJECT SUMMARY Candidate & Environment: The short and long-term goals of the candidate are to gain skills, knowledge, and experience necessary to become a successful independent investigator in gene therapy for neuromuscular diseases (NMDs) with respiratory pathology, such as Pompe disease. Duke University is the optimal location for the candidate to achieve these goals with a world-renowned Pompe disease clinical and research center, along with myology and gene therapy experts, who frequently collaborate and who host monthly research seminars. Research: Pompe disease is a fatal glycogen storage disease caused by mutations in the gene encoding acid α-glucosidase (GAA), which is responsible for hydrolyzing lysosomal glycogen. GAA deficiency results in glycogen accumulation in the lysosomes of muscles (cardiac, skeletal, smooth) and motor neurons. The only FDA approved treatment is enzyme replacement therapy (ERT) of recombinant human GAA (rhGAA). ERT effectively treats cardiac muscle glycogen accumulation but cannot completely correct skeletal muscle pathology due to disrupted autophagy. In addition, ERT cannot cross the blood-brain barrier to treat motor neurons, and therefore, respiratory failure persists. Thus, to prevent respiratory distress in Pompe patients, there is a need for therapies that can clear glycogen and repair autophagy in the respiratory muscles and motor neurons. The overall goal is to identify novel adjunctive therapies to improve respiratory muscle and motor neuron pathology in Pompe disease. We propose to administer three autophagy activators which can cross the blood- brain barrier and assess their therapeutic impact on the motor unit of Pompe mice (Gaa-/-). Acute intermittent hypoxia (AIH), rapamycin, and recombinant adeno-associated virus (rAAV) gene therapy are potential therapies to treat Pompe disease, which will be evaluated across three aims. Each therapeutic has a unique and complementary ability to address autophagy, a critical component of Pompe disease cellular therapy, in key respiratory muscles and motor neurons. (1) Hypoxia is an activator of the autophagosome initiation complex. Additionally, AIH induces neuroplasticity in respiratory motor neurons in neurogenerative disorders. (2) Rapamycin is currently administered to Pompe patients as an immune suppressor, however, rapamycin also has a direct negative impact on master metabolic regulator, mTORC1, thus activating autophagy. (3) rAAV-GAA provides the deficient enzyme, thereby reducing glycogen and improving lysosome health which is a key to restoring normal autophagy. In the final aim of this proposal, we will combine rAAV-GAA therapy with AIH and rapamycin to determine the cumulative effect. Key methods of analysis include neurophysiology to assess neural output of respiratory nerves, whole body plethysmography to assess respiration, molecular proteomics analysis for autophagy pathway proteins, and immunohistochemistry to identify cellular architecture of muscle and CNS. These novel interventions are already used in clinical trials and have the potential to quickly translate to clinical care in Pompe patients suffering from respiratory distress.

PROJECT NARRATIVE Pompe disease is a devastating disease that affects the heart, muscles, and breathing, particularly in infants. In this project we will use a Pompe disease mouse model to test how novel therapies are able to clear the accumulated glycogen which is a classic sign of this disease. Once glycogen is cleared, we will analyze the organelles responsible for nutrient recycling, which become nonfunctional and lead to breathing problems caused by neural and muscle weakness in Pompe disease.