GENE THERAPY FOR ALCOHOLIC LIVER DISEASE

酒精性肝病的基因治疗

• 批准号: 6267147
• 负责人: RONALD G THURMAN
• 金额: $ 18.05万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6267147
• 关键词: Kupffer's cell; adeno associated virus group; alcoholic fatty liver; alcoholic liver cirrhosis; antioxidants; ethanol; free radicals; gender difference; gene therapy; green fluorescent proteins; histopathology; laboratory rat; liver cells; liver disorder; liver ischemia /hypoxia; liver metabolism; nonhuman therapy evaluation; oxidative stress; polymerase chain reaction; superoxide dismutase; transfection /expression vector

Recent work from this laboratory and others has shown that alcoholic liver disease is associated with oxidative stress via mechanisms most likely involving Kupffer cells, yet effective therapeutic modalities are still lacking. This led us to hypothesize now that delivery of antioxidant genes would prevent early hepatic damage due to alcohol. The first goal of these studies is to prevent oxidative stress by gene delivery using superoxide dismutase(SOD) and catalase to small Wistar rats required for the Tsukamoto-French model. Initially, recombinant adeno associated virus (rAAV) vectors containing transgenes for the jellyfish (Aequorea victoria) green fluorescent protein (GFP) will be injected into small male and female Wistar rats to determine the time course of gene transduction and protein expression after vector injection. Gene transduction and protein expression will be quantitated using in situ PCR and fluorescence image-analysis, respectively. Next, similar experiments will be performed with rAAV containing SOD and catalase transgenes at various dose levels. The protective effect of delivery of SOD/CAT genes against oxidative stress will be assessed in a model of hypoxia/reoxygenation in perfused liver. We predict that hepatic expression of SOD/CAT will protect the liver from oxidative stress. Our second goal is to determine whether Kupffer or parenchymal cells predominate in mechanisms of oxidative stress. To do this, Kupffer and parenchymal cells will be selectively targeted with rAAV containing SOD/CAT. Initially, to determine optimal doses and times for maximal gene incorporation selectively into isolated Kupffer cells, rAAV containing GFP transgenes will be given to isolated cultured Kupffer cells for various times and gene transduction and protein expression monitored. Seeding of liver by injection of transduced Kupffer cells after elimination of native Kupffer cells with gadolinium chloride (GGdCl3) will be monitored via real-time video microscopy of GFP fluorescence in living animals using techniques developed by our team. Parenchymal cells will be selectively transduced in vivo in GdCl3-treated rats. Protection from oxidative stress will be assessed in the perfused liver with SOD/CAT exclusively in Kupffer or parenchymal cells. We expect that selectively targeting Kupffer and parenchymal cells with SOC/CAT will determine which cell-type predominates in oxidative stress associated with early liver injury due to alcohol. Our third goal will be to prevent alcoholic liver injury in vivo with gene delivery using a clinically relevant enteral alcohol administration model, the so-called gold standard. Accordingly, male and female Wistar rats will be injected with rAAV containing SOC/CAT. Animals will be placed on the Tsukamoto-French enteral alcohol delivery protocol for at least two months, and livery injury will be assessed from enzyme release and histology. Hypoxia and free radicals will be measured using a 2- nitroimidazole hypoxia marker and the spin trapping technique with electron spin resonance, respectively. We expect that oxidative stress will explain gender differences in alcoholic liver injury and that delivery of SOD/CAT genes in a rAAV vector targeting Kupffer cells will prevent this disease completely.

该实验室和其他实验室最近的工作表明，酒精性肝 疾病与氧化应激有关， 涉及枯否细胞，但有效的治疗方式仍然是 缺乏这让我们假设，现在，抗氧化基因的传递 可以预防酒精引起的早期肝损伤 这些研究的第一个目标是通过基因预防氧化应激 用超氧化物歧化酶（SOD）和过氧化氢酶向小Wistar大鼠递送 Tsukamoto-French模型所需的。最初，重组腺病毒 含有水母转基因的rAAV相关病毒载体 （Aequorea维多利亚）绿色荧光蛋白（GFP）将被注射到 小雄性和雌性Wistar大鼠，以确定基因的时间过程 转染和蛋白表达。基因 使用原位PCR定量转导和蛋白表达 和荧光图像分析。接下来，类似的实验 将用含有SOD和过氧化氢酶转基因的rAAV进行， 不同剂量水平。转SOD/CAT基因的保护作用 抗氧化应激将在以下模型中进行评估： 缺氧/复氧。我们预测肝脏 SOD/CAT的表达将保护肝脏免受氧化应激。 我们的第二个目标是确定枯否细胞或实质细胞 在氧化应激机制中占主导地位。为此，库普弗和 实质细胞将选择性地被含有rAAV的靶向， 首先，为了确定最大基因表达的最佳剂量和时间， 选择性掺入分离的Kupffer细胞，含有GFP的rAAV 将转基因给予分离培养的库普弗细胞， 时间，并监测基因转导和蛋白质表达。播种 通过在消除天然Kupffer细胞后注射转导的Kupffer细胞 将通过以下方法监测含氯化钆（GGdCl 3）的枯否细胞： 使用实时视频显微镜观察活体动物的GFP荧光， 我们团队开发的技术。实质细胞将被选择性地 在GdCl 3处理的大鼠体内转导。保护免受氧化应激 将在Kupffer中仅用SOD/CAT灌注的肝脏中进行评估 或实质细胞。我们希望有选择地针对库普弗， 具有SOC/CAT的实质细胞将决定哪种细胞类型占优势 与酒精引起的早期肝损伤相关的氧化应激。 我们的第三个目标是用基因工程技术在体内预防酒精性肝损伤。 使用临床相关的肠内酒精给药的递送 所谓的黄金标准。因此，雄性和雌性Wistar 将用含有SOC/CAT的rAAV注射大鼠。 Tsukamoto-French肠内酒精输送方案， 几个月后，将从酶释放和 组织学缺氧和自由基将使用2- 硝基咪唑缺氧标记物和自旋捕获技术 电子自旋共振。我们认为氧化应激 将解释酒精性肝损伤的性别差异， 在靶向库普弗细胞的rAAV载体中递送SOD/CAT基因将 彻底预防这种疾病。