Lysosomal Diseases

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Pompe disease is a severe muscle wasting disorder caused by a deficiency of acid alphaglucosidase (GAA), the enzyme that breaks down glycogen to glucose within lysosomes. Absence of the enzyme leads to a rapidly fatal cardiomyopathy in infants, while low levels of residual enzyme activity are associated with childhood and adult-onset progressive skeletal muscle myopathy, usually without cardiac involvement. The only available therapy for Pompe disease is enzyme replacement therapy (ERT) with human recombinant GAA (rhGAA alglucosidase alpha). This therapy restores cardiac function, but its effect in skeletal muscle is much less robust, mainly due to the massive autophagic buildup that negatively affects the trafficking and lysosomal delivery of the recombinant enzyme The recent view of lysosomes as critical regulators of energy homeostasis and response to stress suggests that defects in lysosomal signaling may contribute greatly to the pathology of Lysosomal Storage Disorders (LSDs). Accordingly, we observed a profound dysregulation of mTORC1 signaling in PD. Reactivation of mTORC1 in muscle of Pompe mice by TSC knockdown or arginine supplementation resulted in the reversal of atrophy and a striking removal of autophagic buildup. We next evaluated whether ERT may work more efficiently when autophagic buildup is removed or diminished. For this, we used a dual approach in which restoration of mTORC1 activity by TSC depletion was combined with ERT. Notably, this approach resulted in increased muscle mass and reduced glycogen accumulation, suggesting reversal of the lysosomal pathology. Therefore, we proposed that a combination of TSC-mediated activation of mTORC1 with ERT may have the potential to address multiple aspects of the disease pathology. Additional contributors to the poor skeletal muscle response in PD are the inherently poor glycosylation of the rhGAA alglucosidase alpha, which negatively impact its entry into cells, and the instability of the enzyme in the bloodstream. To overcome these impediments, we collaborated with Amicus Therapeutics to assess the effectiveness of a new drug developed by (ATB200 or cipaglucosidase alpha) in comparison to alglucosidase alpha (FDA approved and the current standard of care). This two-component therapy consists of a modified rhGAA containing higher content of mannose 6-phosphate (M6P) residues to improve skeletal muscle targeting plus an enzyme stabilizer (miglustat). Short-term preclinical studies revealed that ATB200 internalization and activity was superior to alglucosidase, resulting in improved reduction in glycogen levels and lysosomal size, as well as more efficient elimination of autophagic defects. Furthermore, long-term preclinical studies showed an almost complete reversal in muscle lysosomal glycogen accumulation, elimination of autophagic buildup in most muscle fibers, and significant restoration of mTORC1/AMPK signaling, muscle proteostasis, and metabolic abnormalities. The efficiency of ATB200 is further demonstrated by the fact that the therapy was initiated in 3–4-month-old animals that already showed fully developed muscle alterations, suggesting that the treatment not only prevents but potentially reverses the muscle pathology. Overall, our results clearly underscore the potential therapeutic benefit of ATB200. The treatment significantly improved or reversed multiple aspects of the disease pathogenesis in pre-clinical studies, thus offering clear advantage over the current standard of care. Despite the improved efficiency of the new-generation ERT, development of gene therapy for PD, which would provide a continuous supply of the therapeutic enzyme with a single intervention, is an approach of potentially great significance to the field. In pursuit of this goal, we recently evaluated the effect of gene therapy in GAA-KO mice. We used a newly developed adeno-associated virus vector (AVV9) encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues We demonstrated that (AAV9)-mediated systemic gene transfer fully reversed glycogen storage in all key therapeutic targets - skeletal and cardiac muscles, the diaphragm, and the central nervous system (CNS) - in both young and severely affected old Gaa knockout mice. Furthermore, the therapy reversed secondary cellular abnormalities in skeletal muscle, such as autophagy and mTORC1/AMPK signaling. These results lay the groundwork for future clinical development strategy in Pompe disease. Preclinical testing of new therapies is a lengthy undertaking that requires a large number of animals and involves an array of techniques to analyze samples ex vivo. For this reason, we also investigated the use of high‐resolution intravital microscopy (IVM) to visualize and quantify the effectiveness of new treatments in animals within the natural tissue context. By imaging the tongue muscle of GFP-LC3 Gaa-KO live animals, we were able to observe the disease progression and response to therapeutic intervention through a non-invasive methodology. We propose that IVM may represent a promising tool for assessing emerging therapies for PD.