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

微创体内基因传递系统的开发及其在营养不良性大疱性表皮松解症基因治疗中的应用

基本信息

批准号：
16390317
负责人：
TAMAI Katsuto
金额：
$ 9.22万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

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

在本研究中，我们开发了一种侵入性更小、更高效的体内基因传递的新方法，并应用于营养不良性大疱性表皮松解症（DEB）的基因治疗研究。1．我们通过局部应用葡萄球菌剥脱性毒素 A (ETA) 成功地以微创方式去除了小鼠皮肤的局灶性上表皮，该毒素专门消化桥粒芯糖蛋白 1 (dsg1)，桥粒钙粘蛋白的作用是维持表皮角质形成细胞的细胞间接触。这项新技术被证明允许我们引入通常无法渗透到皮肤中的分子量相当高的分子，例如双链 DNA 和蛋白质。 2．我们成功开发了一种新的体内方法，通过将 VII 型胶原蛋白表达质粒直接引入 DEB 小鼠的泡液中，在 DEB 小鼠皮肤角质形成细胞和成纤维细胞中表达 VII 型胶原蛋白。这种泡罩内引入裸露质粒向 DEB 小鼠的基底膜区 (BMZ) 提供了 VII 型胶原蛋白，该小鼠的皮肤 BMZ.3 缺乏 VII 型胶原蛋白。我们开发了靶向基底角质形成细胞的 HVJ（日本血凝病毒）包膜载体（HVJ-E），该载体是通过病毒基因组失活和去核而产生的。 HVJ 的膜融合蛋白 (F) 与针对小鼠桥粒钙粘蛋白 dsg3 的单链抗体 (scFv) 进行生物遗传融合，该抗体在皮肤上皮的基底角质形成细胞中表达。然后将这种dsg3-scFv-F-HVJ-E接种VII型胶原蛋白表达质粒并注射到DEB小鼠的水泡中，从而在DEB小鼠皮肤的基底角质形成细胞中特异性高效地表达VII型胶原蛋白。这些新技术的结合提供了一种独特的基因治疗系统，即所谓的基因浴系统，使EB患者能够获得微创且高效的治疗。针对严重且顽固的遗传性起疱性皮肤病的基因疗法，只需将他们的皮肤浸泡在含有上述 VII 型胶原蛋白表达载体的液体中即可减轻他们的皮肤军团的痛苦。较少的

项目成果

期刊论文数量（45）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Dominant dystrophic epidermolysis bullosa caused by a novel G2037R mutation and by a known G2028R mutation in the type VII collagen gene(COL7A1).

由 VII 型胶原基因 (COL7A1) 中的新 G2037R 突变和已知 G2028R 突变引起的显性营养不良性大疱性表皮松解症。

DOI：
发表时间：
2006
期刊：
J Dermatol. 33(8)
影响因子：
0
作者：
Iwata T;Nakano H;Nakano A;Toyomaki Y;Tamai K;Tomita Y.
通讯作者：
Tomita Y.

Targeted chemotherapy against intraperitoneally disseminated colon carcinoma using a cationized gelatin-conjugated HVJ envelope vector

DOI：
10.1158/1535-7163.mct-05-0352
发表时间：
2006-04-01
期刊：
MOLECULAR CANCER THERAPEUTICS
影响因子：
5.7
作者：
Mima, H;Yamamoto, S;Kaneda, Y
通讯作者：
Kaneda, Y

Interferon-gamma down-regulates expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1) in epidermal keratinocytes via novel chimeric sequences of ISRE and GAS

干扰素-γ 通过 ISRE 和 GAS 的新型嵌合序列下调表皮角质形成细胞中 230 kDa 大疱性类天疱疮抗原基因 (BPAG1) 的表达

DOI：
发表时间：
2006
期刊：
Exp Dermatol 15
影响因子：
0
作者：
Kakizaki I;Takahashi R;Ibori N;Kojima K;Takahashi T;Yamaguchi M;Kon A;Takagaki K;Kondo N;Yamaguchi M;Morohashi H;Kaneko T
通讯作者：
Kaneko T

Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells