The broad goal of this research is to continue studies of receptor- mediated transport of lysosomal enzymes and its relevance to enzyme replacement and gene therapy using beta-glucuronidase as a model lysosomal enzyme, and mucopolysaccharidosis type VII (Sly disease) as a model lysosomal storage disease. Previous studies focused on the biochemistry of the enzyme, the cell biology of its targeting and transport, the molecular genetics underlying the disease, and the mouse model of MPS VII which provided unique opportunities to study enzyme replacement therapy and gene therapy. We seek continued support to extend these studies. We have six specific aims: 1) Produce monoclonal antibodies to murine beta-glucuronidase in MPS VII mice treated from birth with the human enzyme. 2) Define the active site residues of human beta-glucuronidase by site-directed mutagenesis. 3) Use active site mutations in the human gus transgene and targeted active site mutations in the endogenous mouse gus gene to produce tolerant MPS VII mice models for enzyme and gene therapy. 4) Compare the responses to Man 6-P targeted and mannose-targeted human beta-glucuronidase in tolerant MPS VII mice. 5) Produce PEG-modified, Man 6-P-targeted stealth human beta-glucuronidase that is shielded from the immune response in non-tolerant MPS VII mice but still corrects the lysosomal storage. 6) Test the effects of transgene-conferred tolerance on persistence of expression and response to gene therapy in MPS VII mice. We will use a variety of biochemical, cell biological, immunological, and molecular genetic approaches. We will also use transgenic mouse and mouse gene knockout technology as well as sophisticated histochemistry and histopathology. The answers sought have fundamental significance, and should provide information leading to novel therapeutic approaches to enzyme replacement and gene therapy for lysosomal storage diseases.
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