Autoantigen delivery to induce tolerance in diabetic ra*

描述（由申请人提供） 糖尿病是一种常见疾病，患病率为 4-5%， 分为三种主要形式： 1 型糖尿病通常称为免疫性糖尿病 介导的糖尿病、2 型糖尿病或非胰岛素依赖型糖尿病，以及 由于控制 β 细胞功能的基因突变而导致糖尿病，或 新陈代谢。年龄影响显着，1 型糖尿病在人群中占主导地位 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)、胰岛素或两者兼而有之。这项研究将检验以下假设： 可分泌型 GAD65 或胰岛素原的组成型表达将耐受 糖尿病易感大鼠并预防或显着延缓糖尿病的发作。 作为应急计划，是否应单独施用自身抗原 有效诱导耐受性，我们将共表达 GAD65 和胰岛素原 来自双顺反子向量。具有 lyp/lyp 基因型的 BB 大鼠可预测发育 60-90 日龄之间的糖尿病允许胰岛分娩 疾病发作前研究自身抗原以研究耐受诱导和保护 来自糖尿病。通过给年轻人注射可预防或延缓糖尿病 具有这两种自身抗原之一的易患糖尿病的 NOD 小鼠，我们有 显示 BB 大鼠中注射 GAD65 后 T 细胞细胞因子的偏差。 我们相信利用基因治疗来组成型表达 自身抗原比连续给药有显着改善 注射自身抗原。我们建议调查 2 个具体目标。 在目标 1 中，我们将构建编码可分泌 GAD65 的新型逆转录病毒载体 和胰岛素原在纤连蛋白启动子的控制下转导皮肤 作为皮肤等同物引入的细胞中用于长期表达的成纤维细胞 移植到对照和 BB 糖尿病大鼠。自身抗原传递的调节将 通过控制植入的成纤维细胞的数量来实现。当宽容 已实现的皮肤移植将被移除以监测其保存情况 在缺乏自身抗原的情况下改变免疫状态。 在目标 2 中，我们将生成表达胰岛素原的基于 HIV-1 的慢病毒载体 或可分泌的大鼠 GAD65 元件，用于直接肌内注射 糖尿病前期 BB 大鼠。递送的自身抗原的水平将由以下因素控制 施用的慢病毒颗粒数量的变化。 该应用程序的总体目标是开发基因疗法 适用于 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性疾病 疾病。

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.