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

使用脂质干皮病模型小鼠对色素性干皮病组进行基因治疗试验。

基本信息

批准号：
11670825
负责人：
NISHIGORI Chikako
金额：
$ 2.43万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670825/
关键词：
Xeroderma Pigmentosum gene therapy XPA mice UDS (unscheduled DNA Synthesis)Pyrimidine dimers マウス XPAモデルマウス pCHA pCAGGS

项目摘要

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.

本研究的目的是建立对a组色素性干皮病的临床基因治疗。为此，我们首先要确认人类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，在临床试验前必须使用XPA模型小鼠。采用电穿孔法转染含有人XPA基因的pCAGGS-XPA和pcHA-XPA两个质粒，在紫外线照射24小时和48小时后测定紫外诱导的UDS。pCAGGS-XPA和pcHA-XPA均能恢复XPA小鼠细胞的修复效率。转染pcHA-XPA的细胞修复效率比转染pCAGGS-XPA的细胞恢复得更彻底。因此，采用pcHA-XPA进行体内研究。在体内研究中，采用HVJ（日本血凝病毒）脂质体将pcHA-XPA导入小鼠皮肤，因为HVJ脂质体已被公认为是一种高效导入基因的有效方法。在UVB照射前24小时皮下注射含pcHA-XPA的hvj脂质体。用4kJ/m^2 UVB照射小鼠背部皮肤，测定紫外线诱导的表皮核内UDS。注射XPA-HVJ脂质体小鼠的UDS高于对照脂质体。用抗嘧啶二聚体和（6-4）光产物单克隆抗体对小鼠皮肤进行免疫组化染色，证实了XPA-HVJ脂质体在体内恢复紫外线诱导的DNA损伤。Hematozylin-eosin染色显示，注射XPA-HVJ脂质体的小鼠皮肤受到UVB照射的损伤程度较对照组（空脂质体）轻。