Autoantigen delivery to induce tolerance in diabetic ra*

输送自身抗原以诱导糖尿病患者耐受*

• 批准号: 6534383
• 负责人: WILLIAM R OSBORNE
• 金额: $ 15.16万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534383
• 关键词: autoantigens; biotechnology; cell transplantation; disease /disorder prevention /control; gene therapy; genetic strain; glutamate decarboxylase; immune tolerance /unresponsiveness; immunotherapy; insulin dependent diabetes mellitus; isozymes; laboratory rat; prediabetic state; proinsulin; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant) Diabetes mellitus is a common disorder with a prevalence of 4-5 percent and is classified into three major forms: type 1 diabetes often referred to as immune mediated diabetes, type 2 diabetes or non-insulin dependent diabetes, and diabetes due to mutations in genes controlling beta cell function or metabolism. The effect of age is marked and type 1 diabetes dominates among children and teenagers. The etiology and pathogenesis of type 1 diabetes is strongly associated with autoreactivity to glutamic acid decarboxylase 65 (GAD65), insulin or both. This research will test the hypothesis that constitutive expression of secretable GAD65 or proinsulin will tolerize diabetes prone rats and prevent or significantly delay the onset of diabetes. As a contingency plan, should single administration of autoantigen not be effective in inducing tolerance, we will co-express both GAD65 and proinsulin from bicistronic vectors. BB rats with the lyp/lyp genotype predictably develop diabetes between 60-90 days of age permitting the delivery of islet autoantigens before disease onset to study tolerance induction and protection from diabetes. Diabetes is prevented or delayed by injecting young diabetes-prone NOD mice with either one of these two autoantigens and we have shown T cell cytokine deviation in response to GAD65 injections in the BB rat. We believe that utilizing gene therapy for the constitutive expression of autoantigens presents a significant improvement over serial administration of autoantigen by injection. We propose to investigate 2 Specific Aims. In aim 1 we will construct novel retroviral vectors encoding secretable GAD65 and proinsulin under the control of a fibronectin promoter to transduce skin fibroblasts for long-term expression from cells introduced as skin equivalent grafts to control and BB diabetic rats. Regulation of autoantigen delivery will be achieved by controlling the number of fibroblasts implanted. When tolerance has been achieved skin grafts will be removed to monitor preservation of this altered immune state in the absence of autoantigen. In aim 2 we will generate HIV-1 based lentiviral vectors expressing proinsulin or secretable rat GAD65 elements for direct intramuscular injection into prediabetic BB rats. The levels of autoantigen delivered will be controlled by variation in number of lentivirus particles administered. The overall goal of this application is the development of a gene therapy method of tolerance induction that can be applied to patients with type 1 diabetes and their high risk relatives, and may be applied to other autoimmune diseases.

描述(由申请人提供) 糖尿病是一种常见疾病，患病率为4%-5%， 主要分为三种形式：1型糖尿病，通常被称为免疫性 介导型糖尿病、2型糖尿病或非胰岛素依赖型糖尿病，以及 控制β细胞功能的基因突变导致的糖尿病或 新陈代谢。年龄的影响是显著的，1型糖尿病在 儿童和青少年。1型糖尿病的病因和发病机制是 与谷氨酸脱羧酶65的自身反应性密切相关 (GAD65)、胰岛素或两者兼有。这项研究将检验这一假设 分泌型GAD65或胰岛素原的组成性表达将耐受 对易患糖尿病的大鼠有效预防或显著推迟糖尿病的发生。 作为一种应急计划，单次注射自身抗原不应该 有效地诱导耐受，我们将同时表达GAD65和胰岛素原 来自双顺反子载体。携带LYP/LYP基因的BB大鼠可预测发育 60-90日龄允许分娩胰岛的糖尿病 发病前自身抗原研究耐受诱导和保护 得了糖尿病。糖尿病的预防或延迟是通过注射年轻的 有糖尿病倾向的NOD小鼠携带这两种自身抗原中的任何一种，我们有 在BB大鼠注射GAD65后显示T细胞细胞因子偏离。 我们认为，利用基因疗法进行结构性表达 自身抗原与连续给药相比有显著的改善 注射自身抗原。我们建议调查两个具体目标。 在目标1中，我们将构建编码可分泌的GAD65的新型逆转录病毒载体 和胰岛素原在纤维连接蛋白启动子的控制下转导皮肤 成纤维细胞作为皮肤替代物从细胞中长期表达 移植给正常大鼠和BB糖尿病大鼠。对自身抗原递送的监管将 通过控制植入的成纤维细胞的数量来实现。当容差 已经达到的皮肤移植物将被移除，以监测保存情况 在没有自身抗原的情况下免疫状态的改变。 在目标2中，我们将构建基于HIV-1的表达胰岛素原的慢病毒载体 或可分泌的大鼠GAD65元件直接肌肉注射到 糖尿病前期BB大鼠。交付的自身抗原水平将受到以下因素的控制 给药的慢病毒颗粒数量的变化。 这项应用的总体目标是开发一种基因疗法。 可应用于1型患者的耐受诱导方法 糖尿病及其高危亲属，并可应用于其他自身免疫 疾病。