Gaucher disease is the most common of the lysosomal storage disorders and is a significant genetic risk for Ashkenazi Jews. The long range goals of the project are to improve the diagnosis and treatment of this group of related disorders. The current proposal builds on information gained during the previous grant period. Studies will be performed to 1) map the site in glucocerebrosidase (GC) for saposin C activation, 2) determine the physical and kinetic properties of activated GC, 3) test methods to covalently link GC with sap-C to produce a """"""""preactivated"""""""" enzyme complex, and 4) study the effect of substituents bound to the free sulfhydryl at cysteine 18 on the properties of GC. Work will be done to complete the description of the structure of GC by X-ray crystallography. The development of a viable transgenic mouse model of Gaucher disease will be studied. Strategies to accomplish this aim include rescue of the lethal """"""""knockout"""""""" phenotype by breeding carriers to transgenic mice with a milder mutation introduced onto the genetic background of the strain. A cre/lox mediated insertion of the rescuing transgene directly into the targeted mutation is also being studied. The mouse model will be used to re- examine some of the basic questions related to the pathogenesis of the disease. We will also use the model to study questions important to improving enzyme replacement techniques using both conventional and chemically altered enzymes developed in our laboratory. We will also study other therapeutic strategies involving gene transfer to myoblasts. These experiments will utilize a retroviral vector which is highly efficient in transducing primary myoblasts and has resulted in robust expression. Gene transfer studies will be conducted in mice to assess the value of muscle cell transduction and transplantation as an approach to enzyme therapy of Gaucher disease. Preliminary studies demonstrate that GC is secreted from transduced primary mouse and human muscle cells in vitro. The secreted enzyme is taken up by macrophages in culture, but not other cell types. When transduced myoblasts are transplanted into syngeneic recipients, GC can be detected in plasma more than 100 fold greater than endogenous levels, demonstrating that the enzyme is secreted in vivo. These results provide encouragement that this approach could result in an alternative therapeutic strategy. The studies in this proposal develop new therapeutic possibilities derived from basic information learned from the study of glucocerebrosidase and the gene coding it. Studies of enzyme and gene transfer in human cells and animal models complement the development and improvement of these strategies which this grant support helped bring forward.
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