Basic Research for Ex Vivo Gene Tharapy using Gene Targeting Method

使用基因打靶方法进行离体基因治疗的基础研究

• 批准号: 06670771
• 负责人: NARITAKA Shinichi
• 金额: $ 1.22万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670771/
• 关键词: gene targeting; ex vivo gene therapy; in vivo gene therapy; Wilson disease; LEC rats; primary culture of hepatocytes; recombinant adenovirus vector; Lac Z gene of E.coli; 遺伝子治療

The aim of this study was to establish the method of ex vivo gene therapy.wilson's disase is a genetic disease and this disease gene-copper transporting ATPase (ATP-7B) has been cloning recently.Long Evans Cinnamon (LEC) rat is the model of Wilson's disease.Genetically, the disease gene (Atp-7b) of LEC rat is 70% homology of Wilson's disease.Successful gene therapy of LEC rat is the beginning of gene correction of Wilson's disease.We performed partial hepatectomy and the isolation of hepatocytes using Wister rats.Partial hepatectomy was successful, if we avoided bleeding from resected region.The isolation and primary cuture of hepatocytes was complicated and needed for special technique such as very careful handling and optical culture condition.The LacZ gene of E.coli was inserted into normal hepatocytes by using recombinant adenovirus as vector.The expression of this gene was confirmed on the cultured hepatocytes.However, in vivo introduction of this gene into rats was not successful.This phenomenon suggested that either technical problem or essential problem like inserted gene or vector itself have related to the failure.Several problems still remains unknown.We needed to resolve these problems in future.

本研究的目的是建立离体基因治疗方法。威尔逊氏病是一种遗传性疾病，该病基因铜转运ATP酶(ATP-7B)最近已被克隆。模型以Long Evans Cinnamon (LEC)大鼠为模型。从遗传学角度来看，LEC大鼠的疾病基因（Atp-7b）与Wilson病有70%同源性。LEC大鼠基因治疗成功是Wilson病基因矫正的开始。我们用Wister大鼠进行了部分肝切除和肝细胞分离。如果我们避免切除区域出血，部分肝切除是成功的。肝细胞的分离和原代培养比较复杂，需要特殊技术以重组腺病毒为载体，将大肠杆菌的LacZ基因插入正常肝细胞中，并在培养物上证实了该基因的表达。然而，将该基因导入大鼠体内并不成功。这种现象表明，要么是技术问题，要么是插入的基因或载体本身等本质问题与失败有关。还有几个问题仍然未知。我们需要解决这些问题未来。