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

着色性干皮病是一种核苷酸切除修复缺陷的常染色体隐性遗传性疾病，常导致日照部位皮肤癌的高发。在XP互补组中，XP互补组A(XPA)表现出最严重的临床表型并伴有神经功能异常，XPA是日本XPA患者中最常见的类型。我们的研究目标之一是建立XPA患者的基因治疗方法，本项目的目的是通过以HVJ-脂质体的形式治疗人XPA基因来挑战XPA模型小鼠体内的基因治疗。将含有血凝素基因标签的两个含有人XPA基因的载体pCAGGS-XPA或pcHA-XPA分别在UVC照射后24小时和48小时通过非程序DNA合成法检测其修复能力。两者均恢复了XPA小鼠细胞的修复效率。在体内实验中，用日本血凝素病毒脂质体将含有人XPA基因的载体导入小鼠皮肤。在紫外线照射4kJ/m^2前24小时皮下注射HVJ-脂质体-XPA，或在紫外线照射前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500J/m^2的UVB照射小鼠，每周1-2次，连续3个月，在UVB照射前24小时或48小时使用或不使用HVJ-XPA脂质体。脂质体包裹的pcHA-xPA和pCAGGS-xPA均能明显减小紫外线诱导的肿瘤体积，减少肿瘤数目，延长生存期。然而，两种治疗方法都没有完全抑制肿瘤的形成。肿瘤组织学诊断为鳞状细胞癌。综上所述，HVJ-XPA脂质体对紫外线诱导的XPA模型小鼠肿瘤有一定的抑制作用。

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.