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

在本研究中，我们开发了一种新的方法，用于微创，更有效的体内基因递送，并应用于营养不良性大疱性表皮细胞瘤（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）成功地微创去除了小鼠皮肤的局灶性上表皮，该毒素A特异性消化桥粒芯糖蛋白1（dsg 1），一种桥粒钙粘蛋白，其功能是维持表皮角质形成细胞的细胞-细胞接触。这种新技术被证明允许我们引入具有相当高分子量的分子，这些分子通常不能穿透皮肤，例如双链DNA和蛋白质。我们成功地开发了一种新的体内方法，通过将VII型胶原表达质粒直接导入DEB小鼠的水疱液中，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。这种裸质粒的泡内引入为基底膜提供了VII型胶原 关于我们 在皮肤BMZ处缺乏VII型胶原的DEB小鼠的BMZ区。我们开发了基底角质细胞靶向HVJ（日本血凝病毒）包膜载体（HVJ-E），该载体通过病毒基因组的灭活和去核产生。HVJ的膜融合蛋白（F）与抗小鼠桥粒钙粘蛋白dsg 3的单链抗体（scFv）生物融合，所述单链抗体在皮肤上皮的基底角质形成细胞中表达。将此dsg 3-scFv-F-HVJ-E与VII型胶原表达质粒接种并注射到DEB小鼠的水疱中，导致VII型胶原在DEB小鼠皮肤的基底角质形成细胞中的特异性和有效表达。这使得EB患者可以通过将他们的皮肤浸泡在含有VII型胶原蛋白表达载体的液体中，上面描述少

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