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Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders caused by mutations in the human type VII collagen gene (COL7A1). Individuals with DEB lack type VII collagen and anchoring fibrils, structures that attach epidermis and dermis. The current lack of treatment for DEB is an impetus to develop gene therapy strategies that efficiently transfer and stably express genes delivered to skin cells in vivo. In this study, we delivered and expressed full-length type VII collagen using a self-inactivating minimal lentivirus-based vector. Transduction of lentiviral vectors containing the COL7A1 transgene into recessive DEB (RDEB) keratinocytes and fibroblasts (in which type VII collagen was absent) resulted in persistent synthesis and secretion of type VII collagen. Unlike RDEB parent cells, the gene-corrected cells had normal morphology, proliferative potential, matrix attachment and motility. We used these gene-corrected cells to regenerate human skin on immune-deficient mice. Human skin regenerated by gene-corrected RDEB cells had restored expression of type VII collagen and formation of anchoring fibrils at the dermal-epidermal junction in vivo. These studies demonstrate that it is possible to restore type VII collagen gene expression in RDEB skin in vivo.

The large T protein coded by the early region of simian virus 40 (SV40) is involved in the induction and maintenance of cell transformation. It is not clear which properties of T are important in causing the transformation, since the protein is multifunctional. To clarify the action of T proteins in transformation, we have prepared several monoclonal antibodies directed against different regions of the T molecule. One of these antibodies, DL 3C4, recognizes an antigenic determinant on T that is also present on a host cell protein of molecular weight 68,000. This cross-reactive 68K protein is located within the nucleus of all mammalian cell types examined and has a characteristic granular distribution as shown by immunofluorescence. The antigenic determinant recognized by DL 3C4 is resistant to denaturation. Studies on Adeno-SV40 hybrid viruses show that the antigenic site on T is coded for by sequences located between 0.44 and 0.29 on the SV40 map.

Laminin-5 is a heterotrimer composed of ␣3, ␤3, and ␥2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of ␣3, ␤3, and ␥2 monomers, a ␤3␥2 heterodimer, and an ␣3␤3␥2 heterotrimer. The presence of the ␤3␥2 heterodimer, but not heterodimers containing an ␣3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a ␤3␥2 heterodimer to an ␣3␤3␥2 heterotrimer. We showed, by cotransfection experiments using fulllength recombinant ␤3 and ␥2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of ␣3 chain expression. In the SCC-25 cell fraction, the ␣3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24-h chase. These results are consistent with the synthesis of ␣3 being limiting for heterotrimer assembly, with rapid association of the ␣3 chain with ␤3␥2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion.Laminin-5 (kalinin/nicein) is an epithelium-specific laminin subtype and is a component of the anchoring filament of the lamina lucida in the basement membrane of the skin (1). The anchoring filament is the bridge between hemidesmosomes and the lamina densa region of the basement membrane and is believed to mediate the adhesion of the epithelium to the basement membrane. Laminin-5 is therefore one of the primary adhesion proteins holding together the epidermis and the dermis. Laminin-5 is initially synthesized in a cell-associated form, estimated to be 460 kDa, and is composed of three polypeptides: the 200-kDa (␣3), 145-kDa (␤3), and 155-kDa (␥2) chains (2). The three chains are presumed to form a cruciform structure where the chains are bound by disulfide linkages (2). The two heterotrimeric forms in keratinocyte cell-conditioned culture medium are derived from the cellular form by extracellular processing. A 440-kDa medium form results from the processing of the 200-kDa ␣3 chain to 165 kDa, while the 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155-kDa ␥2 chain to 105 kDa (2).A number of variant laminin subunits, in addition to those of laminin-5, are assembled in the rough endoplasmic reticulum. N-Linked oligosaccharide processing of some of these has been shown to occur within the Golgi apparatus prior to secretion (3, 4). These variant chains are produced in different cell types (5, 6); for exa...