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

在这项研究中，我们开发了一种微创、高效的基因在体内传递的新方法，并将其应用于营养不良大疱性表皮松解症（DEB）的基因治疗研究。我们通过局部应用葡萄球菌剥脱毒素A （ETA）成功地微创切除了小鼠皮肤的局灶上表皮，该毒素能特异性地消化桥粒蛋白1 (dsg1)，一种桥粒体钙粘蛋白，其功能是维持表皮角质形成细胞的细胞间接触。这项新技术使我们能够引入通常无法穿透皮肤的高分子量分子，如双链DNA和蛋白质。我们通过将VII型胶原表达质粒直接导入DEB小鼠的水泡液中，成功地建立了在DEB小鼠皮肤角质形成细胞和成纤维细胞中表达VII型胶原的新方法。裸露质粒在水泡内的引入为DEB小鼠的基底膜提供了VII型胶原蛋白，而在皮肤的基底膜上缺乏VII型胶原蛋白。利用日本血液凝集病毒基因组灭活和去核制备了靶向基底角化细胞的HVJ包膜载体（HVJ- e）。HVJ的膜融合蛋白(F)与单链抗体（scFv）生物融合，抗小鼠桥粒钙粘蛋白dsg3（表达于皮肤上皮基底角化细胞）。将该dsg3-scFv-F-HVJ-E与VII型胶原表达质粒接种于DEB小鼠水疱内，在DEB小鼠皮肤基底角质形成细胞中特异性高效表达VII型胶原。这些新技术的结合提供了一种独特的基因治疗系统，即基因浴系统，使EB患者只需将其皮肤浸泡在含有上述VII型胶原表达载体的液体中，即可对严重难治性遗传性起泡性皮肤病进行侵入性更小、效率更高的基因治疗，使其从疼痛的皮肤军团中解脱出来。少

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