Lysosomal Diseases

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Mucolipidosis type IV (MLIV) is an autosomal recessive disorder characterized by acute psychomotor delays, achlorydria, and visual abnormalities including retinal degeneration, corneal clouding, optic atrophy, and strabismus. Lysosomal inclusions are found in most tissues in MLIV patients. The composition of the storage material is heterogeneous and includes lipids and mucopolysaccharides forming characteristic multiconcentric lamellae, as well as soluble, granulated proteins. MLIV is caused by mutations in mucolipin-1 (MCOLN1, also known as TRPML1), an endo-lysosomal cation channel belonging to the transient receptor potential (TRP) superfamily of ion channels. Whole cell patch clamp, as well as recording of native endolysosomal membranes, suggest that MCOLN1 functions as an inwardly (from lumen to cytoplasm) rectifying channel permeable to Ca2+, Na+, K+ and Fe2+/ Mn2+ whose activity is potentiated by low pH. To better understand the pathology of this disease, we used genome editing to knockout the two mcoln1 genes present in Dario rerio (zebrafish). Our model successfully reproduced the retinal and neuromuscular defects observed in MLIV patients, indicating that this model is suitable for studying the disease pathogenesis. Importantly, our model revealed novel insights into the origins and progression of the MLIV pathology, including the contribution of autophagosome accumulation to muscle dystrophy and the role of mcoln1 in embryonic development, hair cell viability and cellular maintenance. The generation of a MLIV model in zebrafish is particularly relevant given the suitability of this organism for large-scale in vivo drug screening, thus providing novel opportunities for therapeutic discovery. 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.