This project is designed to answer the critical question of whether replacement of a wild type gene using ex vivo gene therapy is necessary and sufficient to correct the abnormal would repair phenotype observed in EB tissues. Skin fragility in EB appears to derive from heritable defects in EB candidate genes coding for structural components of the basement membrane zone. Since the majority of these BMZ components have only recently been discovered it is unclear how their absence or dysfunction affects would healing and matrix remodeling in the skin. We hypothesize that mutant EB candidate gene products are proteolyticlaly unstable and when expressed their degradation products serve to includence the balance of connective tissue synthesis and degradation via soluble and solid state factors which have yet to be defined. Our preliminary studies support these ideas. We propose to systematically analyze EB skin for the presence and location of matrix metalloproteinases, serine proteinases, inhibitors and matrix products known to modulate tissue remodeling. The results of this work will be used in experiments designed to compare the proteolytic stability of native EB candidate gene products with mutant products produced by EB cells and engineered by recombinant DNA technology. The effects of modulating protolytic activities with inhibitors and antisense constructs will aid in the design of our gene therapy protocols. Finally, we utilize the EB cell lines that we have produced to test the effects of wild type candidate gene replacement on matrix metabolism in defined and novel dual cellular reaction system. In this way, we will elucidate the mechanisms responsible for the cellular """"""""wound remodeling"""""""" phenotype exhibited by EB-derived skin cells.
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