Lysosomal Diseases

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Pompe disease, a severe muscle wasting disorder characterized by altered lysosomal function. Profound muscle atrophy is a hallmark of Pompe disease, a rare genetic 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 and skeletal muscle myopathy in infants; low levels of residual enzyme activity are associated with childhood and adult onset progressive skeletal muscle myopathy usually without cardiac involvement. Recently, we found dysregulation of mTOR signaling in the diseased muscle cells and focused on the identification of potential sites for therapeutic intervention. Importantly, reactivation of mTOR in the whole muscle of Pompe mice by TSC knockdown or arginine supplementation resulted in the reversal of atrophy and a striking removal of autophagic buildup. 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. The massive autophagic buildup in Pompe skeletal muscle negatively affects the trafficking and lysosomal delivery of the recombinant enzyme. Since we showed that restoration of mTORC1 activity in Pompe skeletal muscle dramatically reduces autophagosome accumulation, we 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. Notable, 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 mTOR 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 initiated a collaboration to assess the effectiveness of a new drug developed by Amicus Therapeutics (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 the majority of 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. Treatment of PD patients with ERT requires large amounts of recombinant enzyme (2040 mg/kg body weight). Genetically modified CHO cells constitute the main source of rhGAA but its production requires in vitro deglycosylation of complex oligosaccharides and the financial cost is prohibitive. To address such limitations, we established a collaboration with Dr. Lai-Xi Wang (University of Maryland College Park) in which we evaluated an array of M6P-containing oxazolines as donor substrates for the synthesis of M6P-containing glycoproteins and the glycan remodeling of rhGAA. This allowed the site-selective conjugation of high affinity M6P glycan ligands, resulting in a remodeled rhGAA with 20-fold enhanced binding affinity for its receptor. The effectivity of this enzyme was evaluated in GAA-deficient multinucleated myotubules, an in vitro system for PD previously developed by Dr. Nina Raben. The remodeled rhGAA showed significantly enhance cellular uptake when compared to the commercial GAA alglucosidase and effected a much more efficient reduction in glycogen levels, lysosomal enlargement and autophagic debris accumulation. One of the main advantages of this approach is that this glycan remodeling can be performed using rhGAA produced in easily available plant or insect cells, potentially allowing production of large amounts of recombinant enzyme at a much-reduced cost. It is important to consider that even if the newly developed rhGAAs prove efficient in reversing skeletal muscle pathology in Pompe patients, some general limitations of ERT will remain. These include the development of immune response to the recombinant enzyme, the requirement for frequent (most commonly every 2 weeks) life-long i.v. injections, a significant financial burden, and the inability of the therapeutic enzyme to cross the blood brain barrier. For these reasons, 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 demonstrated that adeno-associated virus (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. We used a newly developed AAV9 vector encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues. These results lay the groundwork for future clinical development strategy in Pompe disease.