Gene therapy of dystrophic epidermolysis bullosa by introduction of the type VII collagen gene to keratinocytes

Dystrophic epidermolysis bullosa (EB) is an inherited disorder caused by mutations in COL7A1. Introduction of this gene to keratinocytes is now expected for potential therapy of dystrophic EB. In this study, we first constructed the full-length cDNA from cDNA clones of COL7A1. After sequence analysis, we subcloned the full-length cDNA into pCY4B expression vector and transfected it to mouse keratinocytes (Pam cells). Expression of transgenic protein was confirmed by antibodies against the NC1 and NC2 domains of the protein. Furthermore, we subcloned the cDNA to a vector of green fluorescent protein (GFP) to produce type VII collagen-GFP fusion protein. Transfection of this construct to Pam cells found that localization of the fusion protein in the cells was similar to that of type VII collagen of normal keratinocytes. Next, we deleted the cDNA from the 3 ' -end and constructed several mutant-GFP proteins, resulting in change of localization of the fusion proteins. Finally, we introduced the full-length cDNA into rat keratinocytes in vivo using the naked DNA method and found expression of the protein. This study will provide further investigation of this protein and much contribution to fundamental study of gene therapy for dystrophic EB.