Longterm gene therapy of xeroderma pigmentosum group A mice using HVJ-liposomes.

着色性干皮病（Xeroderma pigmentosum，XP）是一种常染色体隐性遗传性疾病，其核苷酸切除修复缺陷，导致患者易发生日光暴露区皮肤癌。在XP互补组中，XP互补组A（XPA）显示出最常见的神经系统异常临床表型，XPA是日本XPA患者中最常见的类型。本项目的目的是通过以HVJ-脂质体的形式处理人XPA基因来挑战XPA模型小鼠的体内基因治疗，因为我们的研究目标之一是建立XPA患者的基因治疗。将含有人XPA基因的质粒pCAGGS-XPA和以血凝素基因为标签的质粒pcHA-XPA分别转染细胞，用程序外DNA合成法检测UVC照射后24和48小时细胞的修复能力。两者都恢复了XPA小鼠细胞的修复效率。在体内研究中，使用HVJ（日本血凝病毒）-脂质体将含有人XPA基因的质粒导入小鼠皮肤。在4kJ/m^2 UVB照射前24小时皮内注射HVJ-脂质体-XPA，或在UVB照射前48小时局部涂抹。注射XPA-HVJ脂质体的小鼠表皮细胞核的程序外DNA合成高于对照脂质体。接下来，我们用500 J/m^2的UVB每周照射小鼠1-2次，持续3个月，在UVB照射前24或48小时处理或不处理HVJ-XPA-脂质体。HVJ-脂质体包封的pcHA-XPA和pCAGGS-XPA均能缩小肿瘤体积、减少肿瘤数量、延长肿瘤存活时间。然而，两种治疗都没有完全抑制肿瘤形成。肿瘤的组织学为鳞状细胞癌。总之，HVJ-XPA脂质体的治疗对减少XPA模型小鼠中UV诱导的肿瘤具有作用。

Xeroderma pigmentosum (XP) is an autosomalrecessibely inheritated disorder in which nucleotide excision repair is deficient, thus the patients with XP cause skin cancer of sun-exposed area in high frequency. Among XP complementation groups, XP complementation group A (XPA) reveals the severest clinical phenotype with neurological abnormalities and XPA is the most common type among Japanese XPA patients. The aim of this project is to challenge the gene therapy of XPA model mice in vivo with treating human XPA gene in the form of HVJ-liposomes, since one of our research goal is to establish the gene therapy for XPA patients. Two plasmids containing human XPA gene, pCAGGS-XPA or pcHA-XPA, which contains hemagultinin gene as a tag, was transfected and the repair ability was measured by unscheduled DNA syntshesis at post 24 or 48 hr UVC irradiation. Both recovered the repair efficiency in XPA mice cells. In in vivo study HVJ (Hemagluutinating Virus of Japan)-liposomes was used for introducing the plasmid containing human XPA gene into mice skin. HVJ-liposomes-XPA was injected intradermaly 24 hr before 4 kJ/m^2 of UVB irradiation or topically painted 48 hr before UVB irradiation. Unscheduled DNA synthesis of epidermal nuclei in mice injected XPA-HVJ liposmes was higher than that of control liposomes. Next we irradiated the mice with 500 J/m^2 of UVB 1-2 times per week for 3 months with or without treating HVJ-XPA-liposmes 24 or 48 hr prior to UVB irradiation. Both pcHA-XPA and pCAGGS-XPA encapsulated in HVJ-liposmes reduced the size, the number of the UV-induced tumors and elongate the survival duration. However, both treatment did not completely inhibit the tumor formation. The histology of the tumor was squamous cell carcinoma. In conclusion, the treatment of HVJ-XPA liposomes has effect on reducing the UV-induced tumors in XPA model mice.