AAV-mediated gene therapy for galactosialidosis: A long-term safety and efficacy study.

AAV-mediated gene therapy holds promise for the treatment of lysosomal storage diseases (LSDs), some of which are already in clinical trials. Yet, ultra-rare subtypes of LSDs, such as some glycoproteinoses, have lagged. Here, we report on a long-term safety and efficacy preclinical study conducted in the murine model of galactosialidosis, a glycoproteinosis caused by a deficiency of protective protein/cathepsin A (PPCA). One-month-old Ctsa -/- mice were injected intravenously with a high dose of a self-complementary AAV2/8 vector expressing human CTSA in the liver. Treated mice, examined up to 12 months post injection, appeared grossly indistinguishable from their wild-type littermates. Sustained expression of scAAV2/8-CTSA in the liver resulted in the release of the therapeutic precursor protein in circulation and its widespread uptake by cells in visceral organs and the brain. Increased cathepsin A activity resolved lysosomal vacuolation throughout the affected organs and sialyl-oligosacchariduria. No signs of hyperplasia or inflammation were detected in the liver up to a year of age. Clinical chemistry panels, blood cell counts, and T cell immune responses were normal in all treated animals. These results warrant a close consideration of this gene therapy approach for the treatment of galactosialidosis, an orphan disease with no cure in sight.

Galactosialidosis: preclinical enzyme replacement therapy in a mouse model of the disease, a proof of concept.

Galactosialidosis is a rare lysosomal storage disease caused by a congenital defect of protective protein/cathepsin A (PPCA) and secondary deficiency of neuraminidase-1 and ß-galactosidase. PPCA is a lysosomal serine carboxypeptidase that functions as a chaperone for neuraminidase-1 and ß-galactosidase within a lysosomal multi-protein complex. Combined deficiency of the three enzymes leads to accumulation of sialylated glycoproteins and oligosaccharides in tissues and body fluids and manifests in a systemic disease pathology with severity mostly correlating with the type of mutation(s) and age of onset of the symptoms. Here, we describe a proof-of-concept, preclinical study toward the development of enzyme replacement therapy for galactosialidosis, using a recombinant human PPCA. We show that the recombinant enzyme, taken up by patient-derived fibroblasts, restored cathepsin A, neuraminidase-1, and ß-galactosidase activities. Long-term, bi-weekly injection of the recombinant enzyme in a cohort of mice with null mutation at the PPCA (CTSA) locus (PPCA -/- ), a faithful model of the disease, demonstrated a dose-dependent, systemic internalization of the enzyme by cells of various organs, including the brain. This resulted in restoration/normalization of the three enzyme activities, resolution of histopathology, and reduction of sialyloligosacchariduria. These positive results underscore the benefits of a PPCA-mediated enzyme replacement therapy for the treatment of galactosialidosis.

Conventional and Unconventional Therapeutic Strategies for Sialidosis Type I.

Congenital deficiency of the lysosomal sialidase neuraminidase 1 (NEU1) causes the lysosomal storage disease, sialidosis, characterized by impaired processing/degradation of sialo-glycoproteins and sialo-oligosaccharides, and accumulation of sialylated metabolites in tissues and body fluids. Sialidosis is considered an ultra-rare clinical condition and falls into the category of the so-called orphan diseases, for which no therapy is currently available. In this study we aimed to identify potential therapeutic modalities, targeting primarily patients affected by type I sialidosis, the attenuated form of the disease. We tested the beneficial effects of a recombinant protective protein/cathepsin A (PPCA), the natural chaperone of NEU1, as well as pharmacological and dietary compounds on the residual activity of mutant NEU1 in a cohort of patients' primary fibroblasts. We observed a small, but consistent increase in NEU1 activity, following administration of all therapeutic agents in most of the fibroblasts tested. Interestingly, dietary supplementation of betaine, a natural amino acid derivative, in mouse models with residual NEU1 activity mimicking type I sialidosis, increased the levels of mutant NEU1 and resolved the oligosacchariduria. Overall these findings suggest that carefully balanced, unconventional dietary compounds in combination with conventional therapeutic approaches may prove to be beneficial for the treatment of sialidosis type I.

MYC competes with MiT/TFE in regulating lysosomal biogenesis and autophagy through an epigenetic rheostat.

Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.

Regulated lysosomal exocytosis mediates cancer progression.

Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf (-/-) mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance.