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

在这项研究中，我们开发了侵入性更小、更有效的体内基因传递的新方法，并将其应用于大疱性营养不良表皮松解症(DEB)的基因治疗研究。通过局部应用葡萄球菌剥离毒素A(ETA)，我们成功地以较小的侵入性去除了小鼠皮肤的局限性上表皮，这种毒素A专门消化桥粒钙粘附素1(DSG1)，桥粒钙粘附素的功能是维持表皮角质形成细胞的细胞-细胞接触。这项新技术被证明允许我们引入通常不能穿透皮肤的高分子分子，如双链DNA和蛋白质。通过在DEB小鼠水疱液中直接导入VII型胶原表达载体，我们成功地建立了在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。这种裸质粒在水泡内的导入为基底膜…提供了VII型胶原DEB小鼠的更多区(BMZ)，在皮肤BMZ处缺乏III型胶原。我们研制了靶向角质形成细胞的日本血凝素病毒包膜载体(HVJ-E)，该载体是通过病毒基因组灭活和去核而产生的。将融合蛋白(F)与抗小鼠桥粒钙粘蛋白dsg3单链抗体(ScFv)进行生物融合，表达于皮肤上皮细胞的基底层角质形成细胞。然后将该dsg3-scFv-F-HVJ-E接种VII型胶原表达载体并注射到DEB小鼠的水泡中，导致VII型胶原在DEB小鼠皮肤的基底层角质形成细胞中特异而高效地表达。结合这些新技术，提供了一种独特的基因治疗系统，即所谓的基因浴系统，该系统允许EB患者对严重且难治的遗传性水疱性皮肤病进行侵入性更小且有效的基因治疗，通过将他们的皮肤浸泡在含有上述VII型胶原表达载体的液体中来缓解他们的皮肤疼痛军团。较少

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