Uptake and Lysosomal Delivery of Recombinant Human Alpha‐N‐Acetylglucosamine‐6‐Sulfatase to Mucopolysaccharidosis IIID Fibroblasts

Abstract

Sanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS), a glycosaminoglycan which plays important structural and functional roles in the brain and elsewhere. We now propose to develop an enzyme replacement treatment (ERT) for MPS IIID that will ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. The symptoms of MPS IIID are largely localized to the brain, therefore, our strategy proposes to deliver recombinant human alpha‐N‐acetylglucosamine‐6‐sulfatase (rhGNS) intrathecally to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. We have purified ~100 ug rhGNS from 1500 mL media of CHO cells (specific activity >10,000 nmol/hr/mg), demonstrated maximal enzymatic activity at pH 5.6 (low activity at neutral pH), which is closer to lysosomal pH, demonstrated good enzymatic activity at 37°C and showed it was stable for over one month at 4°C in artificial cerebrospinal fluid storage buffer. Additionally we have demonstrated intracellular enzymatic activity of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media, shown it colocalizes with lysosomal markers using confocal microscopy, and confirmed radiolabelled HS is diminished (33–65%) to WT levels in MPS IIID fibroblasts treated with rhGNS. These results demonstrate scale‐up is feasible in preparation for proof of concept studies in the MPS IIID knock‐out mouse. Our ultimate goal is bring this ERT to the clinic which will likely have a transformational effect on patients and families.Support or Funding InformationThis work was funded by Grant R41 NS089061‐01 from NIH NINDS