Hepatic Insulin Gene Therapy in Swine

描述（由申请人提供）： 项目总结/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，递送含有由代谢响应性肝特异性启动子控制的胰岛素转基因的腺病毒产生接近正常的胰岛素。肝胰岛素基因疗法（HIGT）处理的大鼠中的血糖在腹膜内葡萄糖负荷的两小时内正常化，并且HIGT处理的大鼠维持长时间的食物剥夺（24小时）而没有致命的低血糖。然而，啮齿动物生理学可能无法预测人类的反应，并且HIGT影响非啮齿动物中糖尿病相关高血糖症的能力仍然是一个悬而未决的问题。与啮齿类动物相比，猪是人类对糖尿病反应的更准确模型。像人一样，猪是杂食动物，并发展与糖尿病加速的年龄相关的动脉粥样硬化。我们建议在猪中证明HIGT的功效，并通过核磁共振光谱（NMRS）检查HIGT对全身碳水化合物代谢的影响。在目的1中，将创建人源启动子序列，并在大鼠、猪、灵长类动物和人肝细胞中测试代谢反应性。这种转基因优化将为灵长类动物研究的扩展提供初步数据。目的2：建立猪肝脏基因转移技术，先去除组织巨噬细胞，再用腺病毒进行肝细胞扩张。除了评估细胞因子和脂肪生成血管介质外，有创血管监测还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，腺病毒将用于向糖尿病猪递送代谢反应性胰岛素转基因，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研究评价HIGT对肝脏葡萄糖通量的影响，提供猪全身碳水化合物代谢的新评估，并量化HIGT在猪中的效用。这些研究的成功完成将为人类糖尿病提供一个重要的模型系统，并可能推动肝胰岛素基因治疗向人类研究。 公共卫生相关性： 项目叙述：糖尿病在退伍军人中的疾病负担是巨大的，每年花费VA卫生系统15亿美元。大约20%的退伍军人患有糖尿病，70%的人超重，易患2型糖尿病。大多数患有糖尿病的退伍军人将死于心血管或脑血管疾病。HIGT抑制内皮功能障碍，动脉粥样硬化性血管疾病的早期标志，在大鼠。然而，在大型哺乳动物中，HIGT是否能控制血管扩张并诱导类似的血管效应仍有争议。该建议利用STZ-糖尿病猪，一种与1型和2型DM相似的疾病模型。此外，HIGT可有效降低高血糖Zucker大鼠（未发表数据）（一种公认的2型DM模型）的BG。这项提案的完成将确定HIGT在猪身上的疗效，并推动HIGT进行人体试验，并可能治疗成千上万的退伍军人。

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.