GENE THERAPY TRIAL FOR XERODERMA PIGMENTOSUM GROUP using xeroderma pigmentosum model mice USING LIPOSMESE.

本研究的目的是建立A组着色性干皮病的临床基因治疗方法。为此，我们首先确定人XPA基因在XPA小鼠细胞中是否有效，因为我们的目标是对XPA患者进行基因治疗，并且在临床试验之前必须使用XPA模型小鼠。电穿孔转染人XPA基因真核表达载体pCAGGS-XPA或pcHA-XPA，分别于紫外线照射后24或48小时检测紫外线诱导的UDS。PCAGGS-xPA和pcHA-xPA均恢复了XPA小鼠细胞的修复效率。与pCAGGS-XPA相比，pcHA-XPA能更好地恢复细胞的修复能力。在体内研究中，由于hHJ-脂质体已被认为是一种高效导入基因的有效方法，因此采用hHJ-脂质体将pcHA-XPA导入小鼠皮肤。在UVB照射前24小时皮下注射含有pcHA-XPA的HVJ-脂质体。用4kJ/m~2中波紫外线照射小鼠背部皮肤，观察紫外线对小鼠表皮细胞核UDS的影响。注射XPA-HVJ脂质体的小鼠UDS高于对照脂质体。用抗嘧啶二聚体和(6-4)光产物的单抗对小鼠皮肤进行免疫组织化学染色，证实XPA-HVJ脂质体对紫外线所致DNA损伤的体内修复作用。苏木精-伊红染色显示，注射XPA-HVJ脂质体的小鼠皮肤受到UVB照射的损伤轻于对照(空)脂质体。

The purpose of this study is to establish the clinical gene therapy for xeroderma pigmentosum group A.For this purpose, firstly we confirmed if human XPA gene is effective in XPA mice cells, because our goal is gene therapy for XPA patients and we must use XPA model mice before clinical trial. Two plasmids containing human XPA gene, pCAGGS-XPA or pcHA-XPA was transfected by electroporation and the UV induced UDS was measured 24 or 48 hr after UV irradiation. Both pCAGGS-XPA and pcHA-XPA recovered the repair efficiency in XPA mice cells. Cells transfected with pcHA-XPA recovered repair efficiency more completely than those with pCAGGS-XPA.Consequently, pcHA-XPA was used for in vivo study. In in vivo study HVJ (Hemagglutinating Virus of Japan)-liposomes was used for introducing the pcHA-XPA into mice skin, because HVJ-liposomes was already accepted as a useful method for introducing genes with high efficiency. HVJ-liposomes containing pcHA-XPA was injected intradermaly 24 hr before UVB irradiation. 4kJ/m^2 UVB was irradiated at back skin of the mice and UV induced UDS in the epidermal nuclei was measured. UDS of mice injected XPA-HVJ liposmes was higher than that of control liposomes. Recovery of UV induced DNA damage by XPA-HVJ liposomes in vivo was also confirmed by staining the mice skin immunohistochemically using monoclonal antibodies againt pyrimidine dimer and (6-4) photoproduct. Hematozylin-eosin staininig revealed that mice skin injected XPA-HVJ liposomes was less severely damaged by UVB irradiation than that of a control (empty) liposomes.