Dissertation/Thesis Abstract

Correction of Autophagic Accumulation in Skeletal Muscle of a Pompe Disease Mouse Model Following Gene Therapy Administration
by McCall, Angela Layne, Ph.D., University of Florida, 2017, 162; 13847478
Abstract (Summary)

Pompe Disease (PD) is a fatal metabolic disorder caused by mutations in the GAA gene leading to a deficiency in acid รก-glucosidase (GAA) and affects 1 in 40,000 births. Clinically, PD 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 to this strategy, chiefly that it is dependent upon receptor-mediated endocytosis. A low abundance of receptor at the plasma membrane in skeletal muscle and physical barriers by accumulated vesicles, results in much of the enzyme captured in amphisomes never reaching the lysosome. This issue illustrates the need for an alternative treatment. This project proposes that endogenous production of GAA enzyme mediated by recombinant adeno-associated virus (rAAV)-delivery of GAA will improve lysosomal delivery and reduce the overall dysregulation of vesicular systems.

In this study, AAV serotype 9 vectors carrying the GAA gene driven by a tissue restrictive promoter (rAAV9-DES-coGAA) at three doses (1x1011 vg/kg, 1x1013 vg/kg, and 1x10 14 vg/kg) were intravenously delivered to 12-week old Gaa-/- mice. One month after AAV or sham injection skeletal muscles, soleus, diaphragm, gastrocnemius, and tibialis anterior, were harvested for biochemical and histological analyses. GAA activity assays demonstrate that therapeutic levels of enzyme in skeletal muscle were attained only with a dose of 1x10 14 vg/kg. Vacuolization of fibers was lower in high dose treated mice compared to untreated controls. Immunofluorescence and western blot analysis of autophagy-associated proteins, LAMP1, LC3-I, LC3-II, Beclin1, and p62 showed a decrease in protein levels indicating an improvement in autophagic regulation in diaphragm, gastrocnemius, and tibialis anterior. Cross-sectional area, a measure of muscle growth was improved compared to hypotrophic fibers in untreated mice. Preliminary evaluation of tibialis anterior of mice treated with the high dose at birth or at 3 months of age and analyzed 6 months later, demonstrated the persistence of GAA expression and resolution of lysosomes and autophagosomes. Overall, this treatment is promising for PD patients and this study provides evidence of the amelioration of vacuolar accumulation.

Indexing (document details)
Advisor: Byrne, Barry
Commitee: Agbandje McKenna, Mavis, Boyr, Shannon, Byrne, Barry, Dunn, William, Ghivizzani, Steven
School: University of Florida
Department: Medicine
School Location: United States -- Florida
Source: DAI-B 80/07/(E), Dissertation Abstracts International
Subjects: Genetics, Cellular biology
Keywords: AAV, Autophagy, Gene therapy, Muscle, Pompe
Publication Number: 13847478
ISBN: 978-0-438-94733-7
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