Development of less-invasive in vivo gene delivery system and application to gene therapy for dystrophic epidermolysis bullosa

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In this study, we have developed novel methods for less invasive, more efficient gene delivery in vivo, and applied to the study for gene therapy of dystrophic epidermolysis bullosa (DEB).1. We succeeded in less-invasive removal of focal upper epidermis of the mouse skin by topical application of staphylococcal exfoliative toxin A (ETA), which specifically digest desmoglein 1 (dsg1), a desmosomal cadherin functioning to maintain cell-cell contact of the epidermal keratinocytes. This novel technique was shown to allow us to introduce molecules with rather high molecular weight which usually are not able to penetrate in the skin, such as double strand DNA and proteins.2. We succeeded to develop novel in vivo method to express type VII collagen in the DEB mouse skin keratinocytes and fibroblasts by introducing type VII collagen expression plasmid directly in the blister fluid of DEB mouse. This intra-blister introduction of naked plasmid provided type VII collagen to the basement membrane … More zone (BMZ) of the DEB mouse which lacks type VII collagen at cutaneous BMZ.3. We developed basal keratinocyte-targeting HVJ (hemoagglutinating virus of Japan) envelope vector (HVJ-E) which was generated by inactivation and enucleation of the viral genome. Membrane fusion protein (F) of HVJ was biogenetically fused with single chain antibody (scFv) against mouse desmosomal cadherin dsg3, which is expressed in basal keratinocytes of the cutaneous epithelia. This dsg3-scFv-F-HVJ-E was then inoculated with type VII collagen expression plasmids and injected into the blister of DEB mouse, resulted in specific and efficient expression of type VII collagen in the basal keratinocytes of the DEB mouse skin.Combination of those novel techniques provide an unique gene therapy system, so called gene bath system, which allow EB patients to have a less invasive and efficient gene therapy for the severe and intractable genetic blistering skin disease to relieve them from those painful skin legions just by soaking their skin in the liquid containing type VII collagen expression vectors described above. Less