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

本研究的目的是建立治疗A组着色性干皮病的临床基因治疗方法。由于我们的目的是对A组着色性干皮病患者进行基因治疗，在临床试验前必须使用XPA模型小鼠，因此我们首先在XPA小鼠细胞中证实人XPA基因是否有效。将含有人XPA基因的两个质粒pCAGGS-XPA或pcHA-XPA通过电穿孔转染，并在UV照射后24或48小时测量UV诱导的UDS。pCAGGS-XPA和pcHA-XPA在XPA小鼠细胞中均恢复了修复效率。转染pcHA-XPA的细胞比转染pCAGGS-XPA的细胞修复效率更完全，因此，pcHA-XPA用于体内研究。在体内研究中，HVJ（日本血凝病毒）-脂质体用于将pcHA-XPA引入小鼠皮肤，因为HVJ-脂质体已经被接受为高效引入基因的有用方法。在UVB照射前24小时皮内注射含有pcHA-XPA的HVJ-脂质体。用4kJ/m^2 UVB照射小鼠背部皮肤，测定UV诱导的表皮细胞核UDS。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）光产物的单克隆抗体对小鼠皮肤进行化学染色，证实了XPA-HVJ脂质体在体内对UV诱导的DNA损伤的恢复。苏木精-伊红染色显示，注射XPA-HVJ脂质体的小鼠皮肤受UVB照射的损伤程度低于对照（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.