Development and characterization of a recombinant truncated type VII collagen "minigene" - Implication for gene therapy of dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous diso rder of skin caused by mutations in the type VII collagen gene. The lack of therapy for DEB provides an impetus to develop gene therapy strategies. Ho wever, the full-length 9-kilobase type VII collagen cDNA exceeds the clonin g capacity of current viral delivery vectors. In this study, we produced a recombinant type VII minicollagen containing the intact noncollagenous doma ins, NC1 and NC2, and part of the central collagenous domain using stably t ransfected human 293 cell clones and purified large quantities of the recom binant minicollagen VII from culture media. Minicollagen VII was secreted a s correctly-folded, disulfide-bonded, helical trimers resistant to protease degradation. Purified minicollagen VII bound to fibronectin, laminin-5, ty pe I collagen, and type TV collagen. Furthermore, retroviral-mediated trans duction of the minigene construct into DEB keratinocytes tin which type VII collagen was absent) resulted in persistent synthesis and secretion of a 2 30-kDa recombinant minicollagen VII. In comparison with parent DEB keratino cytes, the gene-corrected DEB keratinocytes demonstrated enhanced cell-subs tratum adhesion, increased proliferative potential, and reduced cell motili ty, features that reversed the DEB phenotype toward normal. We conclude tha t the use of the minicollagen VII may provide a strategy to correct the cel lular manifestations of gene defects in DEB.