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.

这项研究的目的是建立针对静脉的临床基因治疗。临床试验前的小鼠模型。紫外线辐照后24或48小时测量了两个含有人XPA基因的质粒，pCAGGS-XPA或PCHA-XPA被转染，并测量UV诱导的UDS。 PCAGGS-XPA和PCHA-XPA都恢复了XPA小鼠细胞中的修复效率。与PCAGGS-XPA相比，用PCHA-XPA转染的细胞更完全恢复了修复效率。结果，PCHA-XPA用于体内研究。在体内研究中，使用HVJ（日本的血凝病毒） - 脂肪体用于将PCHA-XPA引入小鼠皮肤，因为HVJ-脂质体已经被接受为引入高效率的基因的有用方法。 UVB辐照前24小时注射含有PCHA-XPA的HVJ脂质体。在小鼠的背部皮肤上照射4KJ/m^2 UVB，并测量表皮核中紫外线诱导的UDS。注射XPA-HVJ脂质的小鼠UDS高于对照脂质体。还通过使用单克隆抗体再次染色小鼠皮肤免疫组织化学染色的XPA-HVJ脂质体在体内恢复了紫外线诱导的DNA损伤。血红蛋白 - 欧洲蛋白斑点表明，与对照（空）脂质体的小鼠皮肤注入的XPA-HVJ脂质体受到UVB辐射的严重破坏相比，XPA-HVJ脂质体受到严重的伤害。