Abstract
BACKGROUND: Mucopolysaccharidosis IVA (Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which results in the accumulation of the glycosaminoglycans (GAGs), keratan sulfate, and chondroitin-6-sulfate in the lysosomes of all tissues causing systemic dysfunction. Current treatments include enzyme replacement therapy (ERT) which can treat only certain aspects of the disease such as endurance-related biological endpoints. A key challenge in ERT is ineffective enzyme uptake in avascular tissues, which makes the treatment of the corneal, cartilage, and heart valvular tissue difficult. The aim of this study was to culture human umbilical mesenchymal stem cells (UMSC), demonstrate presence of GALNS enzyme activity within the extracellular vesicles (EVs) derived from these UMSC, and study how these secreted EVs are taken up by GALNS-deficient cells and used by the deficient cell's lysosomes. METHODS: We obtained and cultured UMSC from the umbilical cord tissue from anonymous donors from the Saint Louis Cord Blood Bank. We characterized UMSC cell surface markers to confirm phenotype by cell sorting analyses. In addition, we confirmed that UMSC secrete GALNS enzyme creating conditioned media for co-culture experiments with GALNS deficient cells. Lastly, we isolated EVs derived from UMSC by ultracentrifugation to confirm source of GALNS enzyme. RESULTS: Co-culture and confocal microscopy experiments indicated that the lysosomal content from UMSC migrated to deficient cells as evidenced by the peak signal intensity occurring at 15 min. EVs released by UMSC were characterized indicating that the EVs contained the active GALNS enzyme. Uptake of GALNS within EVs by deficient fibroblasts was not affected by mannose-6-phosphate (M6P) inhibition, suggesting that EV uptake by these fibroblasts is gradual and might be mediated by a different means than the M6P receptor. CONCLUSIONS: UMSC can deliver EVs containing functional GALNS enzyme to deficient cells. This enzyme delivery method, which was unaffected by M6P inhibition, can function as a novel technique for reducing GAG accumulation in cells in avascular tissues, thereby providing a potential treatment option for Morquio A syndrome.