The broad goal of this research is to continue studies of receptor-mediated transport of lysosomal enzymes and its relevance to enzyme replacement (ERT) and gene therapy using beta-glucuronidase (GUSB) as a model enzyme and mucopolysaccharidosis type VII (Sly disease) as a model lysosomal storage disease. Previous studies focused on the active site mutations in the human and murine disease, the cell biology of enzyme targeting and transport, the immune response in treated MPS VII mice, and therapeutic responses to ERT with murine GUSB. MPS VII mice showed remarkable benefit from ERT, though enzyme did not cross the blood-brain barrier after 2 weeks of age. We seek support to extend these studies and use novel approaches to improve results of ERT and achieve correction in brain. We have four specific aims: 1) Determine to what extent the species of overproducing mammalian cell line influences the receptor targeting and effectiveness of beta-glucuronidase for enzyme replacement therapy for MPS VII. 2) Introduce the mannose receptor knockout allele onto the MPS VII background to determine how Man6-Ptargeted tissue distribution and correction differ in the absence of mannose-mediated clearance. 3) Use human beta-glucuronidase-vector fusion proteins to target receptors that normally mediate transcytosis of their ligands across the blood-brain barrier to achieve correction of storage in brain in MPS VII mice. 4) Identify chemical chaperones, which correct defective enzyme folding, transport, or stability in cells from MPS VII patients and mouse models with missense mutations. We will use a variety of biochemical, cell biological, immunological, and molecular genetic approaches. We take advantage of novel mouse models of MPS VII produced in our laboratory by transgenic and mouse knockout technology. We combine histochemistry, histopathology, and immunoelectron microscopy to measure enzyme delivery to brain. The answers sought have fundamental significance, and should provide information leading to novel therapeutic approaches to enzyme replacement for lysosomal storage diseases involving the central nervous system.
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