RETROVIRAL MEDIATED GENE THERAPY OF DIABETIC RATS

糖尿病大鼠的逆转录病毒介导的基因治疗

• 批准号: 2451873
• 负责人: WILLIAM R OSBORNE
• 金额: $ 14.97万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2451873
• 关键词: Retroviridae; autoantigens; diabetes mellitus therapy; disease /disorder model; fibroblasts; fibronectins; gene expression; gene therapy; genetic promoter element; genetic transduction; glucose metabolism; immune tolerance /unresponsiveness; insulin; insulin dependent diabetes mellitus; laboratory rat; nonhuman therapy evaluation; phosphotransferases; prohormone convertase; proinsulin; transfection /expression vector; transforming growth factors; vascular smooth muscle

DESCRIPTION (Abstract of the application) The objective of this research is to develop novel retroviral vectors to treat diabetic rats with the long-term goal of treating patients with insulin dependent diabetes. We hypothesize that retroviral vectors encoding glucose responsive promoters driving furin expression may provide an amplified secretion of mature insulin. To achieve this we will construct novel 3-gene retroviral vectors with an insulin cleaving protease under the control of a glucose responsive promoter. We propose that protease expression will catalyze an amplified release of mature bioactive insulin in response to increased glucose concentration. Protease-mediated mature insulin release will be beyond that achieved by control of insulin alone. The vectors will be used to transduce vascular smooth muscle cells which are easily transduced with retroviral vectors, can be selected and returned to donor animals and express therapeutic genes for at least 1 year. The hypothesis that constitutive expression of secretable GAD65 will tolerize diabetic rats and prevent or significantly delay the onset of diabetes will be tested. In this aim we will use retroviral vectors containing elements of a fibronectin promoter to achieve long-term secretable GAD65 expression in transduced skin fibroblasts introduced into rats as skin equivalent grafts. In a third aim we will attempt to achieve in vivo retroviral infection/transduction of skin epidermal cells and will monitor a marker gene and vectors expressing secretable GAD65. The proposed vectors will be tested in a well characterized rat model of insulin dependent diabetes. The presence of homozygous lyp/lyp results in insulin-dependent diabetes in 100% of rats. The control litter mates, lyp/+ and +/+ are diabetes resistant. The onset of diabetes in the lyp/lyp rats is at 60-90 days of age. This well characterized diabetic BB rat therefore permits a unique model to study the efficacy of our novel vectors to a) control diabetes by hyperglycemia regulatable release of mature insulin and b) induce immune tolerance by secretion of the major autoantigen in diabetes, GAD65. These studies will test, in a well characterized rat model of diabetes, the ability to achieve a regulated supply of insulin at a clinically meaningful level, and to determine if tolerization can be achieved by secretion of an antigen involved in the pathogenesis of autoimmune diabetes. These studies of diabetic rats provide a model of regulated hormone gene therapy and have a potential application to other diseases requiring long-term delivery of therapeutic proteins such as cytokines, hormones, clotting factors and enzymes.

描述(应用程序摘要) 本研究的目的是开发新型逆转录病毒载体，以 治疗糖尿病大鼠的长期目标是治疗糖尿病患者 胰岛素依赖型糖尿病。我们假设逆转录病毒载体编码 驱动Furin表达的葡萄糖响应启动子可以提供 成熟胰岛素的分泌被放大。为了实现这一点，我们将构建 含胰岛素裂解酶的新型3基因逆转录病毒载体 控制葡萄糖反应启动子。我们建议，蛋白酶 表达将催化成熟生物活性胰岛素在体内的放大释放 对葡萄糖浓度升高的反应。蛋白酶介导的成熟 胰岛素的释放将超过单独控制胰岛素所达到的效果。 这些载体将用于转导血管平滑肌细胞，这些细胞 很容易用逆转录病毒载体转导，可以选择并返回到 捐献动物并表达治疗基因至少1年。这个 可分泌GAD65的结构性表达将耐受的假说 并能预防或明显延缓糖尿病大鼠的发病 接受测试。为了达到这个目的，我们将使用逆转录病毒载体 利用纤维连接蛋白启动子实现GAD65的长期分泌表达 将转导的皮肤成纤维细胞作为皮肤替代物引入大鼠体内 嫁接。在第三个目标中，我们将尝试在体内实现逆转录病毒 皮肤表皮细胞的感染/转导，并将监测一个标志物 表达分泌型GAD65的基因和载体。 建议的载体将在具有良好特性的大鼠模型中进行测试。 胰岛素依赖型糖尿病。纯合子LYP/LYP的存在导致 100%的大鼠出现胰岛素依赖型糖尿病。对照窝仔，LYP/+ 和+/+是抗糖尿病的。LYP/LYP大鼠糖尿病的发生 是60-90天龄。这很好地描述了糖尿病BB大鼠的特征 允许一个独特的模型来研究我们的新载体对a)的有效性) 通过高血糖调节成熟胰岛素的释放和控制糖尿病 B)通过分泌主要自身抗原诱导免疫耐受 糖尿病，GAD65。 这些研究将在一个具有良好特征的糖尿病大鼠模型中测试 在有临床意义的情况下实现调节的胰岛素供应的能力 水平，并确定是否可以通过分泌一种 参与自身免疫性糖尿病发病机制的抗原。这些研究 为糖尿病大鼠提供了一种调节激素基因治疗的模型 可能应用于其他需要长期给药的疾病 治疗性蛋白质，如细胞因子、激素、凝血因子和 酵素。