RETROVIRAL MEDIATED GENE THERAPY OF DIABETIC RATS

说明（申请摘要） 本研究的目的是开发新型逆转录病毒载体， 治疗糖尿病大鼠的长期目标是治疗 胰岛素依赖型糖尿病我们假设逆转录病毒载体编码 驱动弗林蛋白酶表达的葡萄糖响应性启动子可以提供 成熟胰岛素分泌增加。为了实现这一目标，我们将建立 新的3-基因逆转录病毒载体，其具有胰岛素裂解蛋白酶， 葡萄糖响应启动子的控制。我们认为蛋白酶 表达将催化成熟生物活性胰岛素的放大释放， 对葡萄糖浓度增加的反应。蛋白酶介导的成熟 胰岛素释放将超过单独控制胰岛素所达到的释放。 这些载体将用于培养血管平滑肌细胞， 容易用逆转录病毒载体转导，可以选择并返回到 供体动物和表达治疗基因至少1年。的 假设分泌型GAD 65组成型表达将耐受 糖尿病大鼠和预防或显着延迟糖尿病的发病将 得到考验在这个目标中，我们将使用含有元件的逆转录病毒载体， 的纤连蛋白启动子，以实现长期分泌型GAD 65表达 在作为皮肤等同物引入大鼠的转导的皮肤成纤维细胞中， 移植物在第三个目标中，我们将尝试实现体内逆转录病毒 皮肤表皮细胞的感染/转导，并将监测标记物 基因和表达分泌型GAD 65的载体。 将在充分表征的大鼠模型中测试所提出的载体。 胰岛素依赖型糖尿病纯合子lyp/lyp的存在导致 100%的大鼠患有胰岛素依赖型糖尿病。对照窝交配，lyp/+ 和+/+是耐糖尿病的。LYP/LYP大鼠糖尿病的发病 是在60 - 90天的年龄。因此，这种良好表征的糖尿病BB大鼠 允许一个独特的模型来研究我们的新载体的功效， 通过调节成熟胰岛素的高血糖释放来控制糖尿病， B）通过分泌主要自身抗原诱导免疫耐受， 糖尿病GAD65 这些研究将在一个良好表征的糖尿病大鼠模型中测试， 能够在临床上有意义的水平上实现胰岛素的调节供应， 水平，并确定耐受化是否可以通过分泌 自身免疫性糖尿病的发病机制中所涉及的抗原。这些研究 糖尿病大鼠提供了调节激素基因治疗的模型， 可能应用于需要长期递送的其他疾病， 治疗性蛋白质，例如细胞因子、激素、凝血因子和 内切酶

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.