Hepatic Insulin Gene Therapy in Swine

描述(由申请人提供)： 项目摘要/摘要：现有的糖尿病治疗方法不足。胰岛素基因治疗有可能改善糖尿病患者的长期血糖控制。在啮齿类动物中，携带胰岛素转基因的腺病毒由代谢反应灵敏的肝脏特异性启动子控制，可产生接近正常血糖。肝脏胰岛素基因疗法(HIGT)治疗的大鼠血糖在腹膜内葡萄糖负荷后两小时内恢复正常，HIGT治疗的大鼠持续长时间的饮食剥夺(24小时)，没有致命性低血糖。然而，啮齿动物生理学可能无法预测人类的反应，HIGT对非啮齿动物糖尿病相关性高血糖的影响仍是一个悬而未决的问题。猪比啮齿动物更能准确地反映人类对糖尿病的反应。和人类一样，猪是杂食性动物，会患上与年龄相关的动脉粥样硬化，糖尿病会加速这种疾病。我们建议在猪身上证明HIGT的疗效，并通过核磁共振波谱(NMRS)检测HIGT对全身碳水化合物代谢的影响。在目标1中，将创建人类衍生的启动子序列，并测试其在大鼠、猪、灵长类动物和人类肝细胞中的代谢反应。这一转基因优化将为将研究扩展到灵长类动物提供初步数据。在目标2中，我们将通过耗尽组织巨噬细胞和在腺病毒之前应用扩张剂来建立猪的肝脏基因转移技术。除了评估细胞因子和造脂血管介体外，有创性血管监测将指导其他干预措施以提高耐受性。基因标记研究将确定获得~25%肝细胞转导所需的腺病毒剂量。在AIM 3中，腺病毒将被用于向糖尿病猪传递代谢响应性胰岛素转基因，并将使用静脉内葡萄糖耐量测试和禁食研究来测试HIGT后的代谢反应性。在目标4中，将通过系列13-C NMRS研究来评估HIGT对肝脏葡萄糖代谢的影响，为猪的全身碳水化合物代谢提供新的评估，并量化HIGT在猪中的应用。这些研究的成功完成将为人类糖尿病研究提供一个重要的模型系统，并可能推动肝脏胰岛素基因治疗向人类研究迈进。 公共卫生相关性： 项目简介：与退伍军人健康相关糖尿病在退伍军人人群中的疾病负担是巨大的，每年花费退伍军人健康系统15亿美元118。大约20%的退伍军人患有糖尿病，70%的退伍军人超重，这使他们容易患上2型糖尿病。大多数患有糖尿病的退伍军人会死于心脑血管疾病。HIGT抑制大鼠的内皮功能障碍，这是动脉粥样硬化性血管疾病的早期标志。然而，HIGT是否能控制血糖并在大型哺乳动物中引起类似的血管效应仍存在争议。这项建议利用了STZ-糖尿病猪，这是一种与1型和2型糖尿病相似的疾病模型。此外，HIGT有效地降低了高血糖Zucker大鼠的血糖(未发表的数据)，这是一种公认的2型DM模型。这项提案的完成将决定HIGT在猪身上的疗效，并推动HIGT进入人体试验，并可能治疗数万名退伍军人。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Available treatments for diabetes mellitus are insufficient. Insulin gene therapy has the potential to improve long-term glycemic control in patients with diabetes. In rodents, delivery of an adenovirus containing an insulin transgene controlled by a metabolically responsive, liver specific promoter produces near normoglycemia. Blood sugars in hepatic insulin gene therapy (HIGT) treated rats normalize within two hours of an intra- peritoneal glucose load, and HIGT treated rats sustain prolonged periods of chow deprivation (24hrs) without lethal hypoglycemia. However, rodent physiology may not predict responses in humans, and the capacity of HIGT to impact diabetes associated hyperglycemia in non-rodent animals remains an open question. Swine are a more accurate model of human responses to diabetes mellitus than rodents. Like people, swine are omnivores and develop age related atherosclerosis that is accelerated by diabetes. We propose to demonstrate HIGT efficacy in pigs, and examine HIGT effects on whole body carbohydrate metabolism by nuclear magentic resonance spectroscopy (NMRS). In Aim 1 human derived promoter sequences will be created, and tested for metabolic responsiveness in rat, pig, primate, and human hepatocytes. This transgene optimization will provide preliminary data for extension of studies to primates. In Aim 2 we will establish hepatic gene transfer techniques in swine by depleting tissue macrophages and administering festral dilating agents prior to adenovirus. Invasive vascular monitoring, in addition to assessment of cytokine and lipogenic vascular mediators will guide additional interventions to improve tolerability. Gene marking studies will determine requisite doses of adenovirus to obtain ~25% hepatocyte transduction. In Aim 3 adenovirus will be used to deliver a metabolically responsive insulin transgene to diabetic pigs, and metabolic responsiveness following HIGT will be tested using intavenous glucose tolerance testing and fasting studies. In Aim 4 the effect of HIGT on hepatic glucose fluxes will be evaluated by serial 13-C NMRS studies, providing novel assessment of whole body carbohydrate metabolism in pigs, and quantifiying the utility of HIGT in pigs. Successful completion of these studies will provide an important model system for human diabetes, and may advance hepatic insulin gene therapy toward human studies. PUBLIC HEALTH RELEVANCE: Project Narrative: RELEVANCE TO VETERANS HEALTH The disease burden of diabetes mellitus among the veteran population is tremendous, costing the VA health system $1.5 billion each year 118. Approximately 20% of veterans have diabetes, and 70% are overweight, predisposing them to developing type 2 diabetes. The majority of veterans with diabetes will succumb to cardio- or cerebral vascular disease. HIGT inhibits endothelial dysfunction, an early marker of atherosclerotic vascular disease, in rats. However, whether HIGT can control glycemia and induce similar vascular effects in large mammals is disputed. This proposal utilizes STZ-diabetic pigs, a disease model with similarities to both type 1 and type 2 DM. Moreover, HIGT is effective in lowering BG in hyperglycemic Zucker rats (unpublished data), an accepted model of type 2 DM. Completion of this proposal will determine the efficacy of HIGT in pigs, and propel HIGT toward human trials, and potentially the treatment of tens of thousands of veterans.