Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme produced in pichia pastoris as an alternative for Morquio syndrome type A ERT

Mucopolysaccharidosis type IV A (Morquio A disease) is a lysosomal disease produced by mutations in GALNS enzyme. Previously, we had reported the production of the recombinant GALNS in the Pichia pastoris yeast. Here, we present the results of the in-vitro characterization, biodistribution and Immunogenicity of the recombinant GALNS enzyme. The cell up-take and intracellular traffic was evaluated in HEK293 cells, Morquio A fibroblasts and wild-type mouse chondrocytes. Reduction of Keratan Sulfate (KS), was evaluated in Morquio A fibroblasts by using HPLC-MS/MS. In-vivo biodistribution was studied in wild-type C57Bl/6 mice and indirect ELISA was used for detection of GALNS antibodies in serum. The cell uptake results showed that internalization process is mediated by both, mannose and mannose-6-phosphate receptors. Cell uptake assays using Morquio A fibroblasts, showed the enzyme was taken up reaching intracellular enzyme activity levels similar to those observed in fibroblast from healthy volunteers; while the intracellular traffic assay, showed that the enzyme was targeted to the lysosome. It is noteworthy that recombinant GALNS allowed the reduction of KS in Morquio A fibroblasts. For biodistribution assay, an intravenous doses of 5 mg/kg were administered and tissue samples were taken at 12 and 24 h. At 12 h post-injection, it was observed the enzyme was primarily recovered in the liver, spleen, kidney and heart; while at 24 h the enzyme was only detected in liver and spleen. The production of the specific antibodies was assay in wild type mice during one month. At 15 days post-infusion minimum quantity of antibodies was observed, however at 30 days a seven-fold increase in the antibodies production in plasm was detected. In conclusion, these results show the potential of this enzyme towards the development of an alternative ERT for Morquio A disease.