Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal–epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes

A Phenotypic Analysis of Involucrin-Membrane-Bound Ovalbumin Mice after Adoptive Transfer of Ovalbumin-Specific CD8⁺ T Cells

To investigate the mechanism of autoimmunity and peripheral tolerance in the skin, several transgenic mouse strains expressing membrane-bound ovalbumin (mOVA) as an epidermal self-antigen under the control of keratinocyte-specific promotors, such as keratin 5 and keratin 14, were employed in combination with adoptive transfer of CD8⁺ T cells from OT-I mice (OT-I T cells) that recognize an ovalbumin-derived peptide. However, these strains showed bodyweight loss and required additional inflammatory stimuli, such as γ-irradiation and tape-stripping, to induce skin inflammation. In this study, we generated a mouse strain expressing mOVA under the control of human involucrin promoter (involucrin-mOVA mice). In contrast to previous strains, involucrin-mOVA mice spontaneously developed skin inflammation after the transfer of OT-I T cells in the absence of external stimuli without significant bodyweight loss. We focused on the skin infiltration process of OT-I T cells and found that transferred OT-I T cells accumulated around the hair follicles in the early phase of skin inflammation, and in the later phase, the skin inflammation spontaneously resolved despite the remaining OT-I T cells in the skin. Our involucrin-mOVA mice will provide a promising tool to investigate the pathogenesis and the tolerance mechanisms of cytotoxic skin autoimmunity