RNA Interference using gutless adenoviral vectors

描述（由申请人提供）： 非胰岛素依赖型或2型糖尿病是一种代谢疾病，其特征在于血糖浓度升高，这是由于胰岛素敏感组织中胰岛素作用不足和胰岛素分泌异常所致。在美国，欧洲和大多数西方国家，2型糖尿病影响约6%的人口。据估计，到2010年，全世界将有超过2.2亿人受到这种疾病的影响，肥胖症发病率的急剧增加和久坐不动的生活方式加剧了这种疾病。为了实现葡萄糖控制和预防糖尿病并发症（视网膜病变、心血管疾病和肾病等），患者目前接受疗效有限且与副作用相关的治疗剂。因此，需要开发更有效的治疗剂。我们的长期目标是确定可以用药物靶向的基因，以改善2型糖尿病的治疗。目前验证基因在疾病中的作用的技术涉及开发具有靶基因的无效等位基因的动物模型。在某些情况下，基因的缺失与正常发育不相容，导致子宫内死亡。此外，某些研究需要敲除成年动物的基因功能。本发明的目的是开发一种下调肝脏中基因表达的系统。我们将靶向参与脂肪酸产生的基因Srebp-1，其表达在2型糖尿病中上调。我们的目标将通过施用表达小干扰RNA（siRNA）以降低mRNA水平的重组无肠腺病毒来实现。siRNA技术是通过诱导转录后基因沉默来研究基因功能的新工具。我们期望通过追求以下具体目标来实现本提案的目的：（i）建立在肝脏中实现完全Srebp-1基因沉默的最佳条件;（ii）确定在成年动物的肝脏中表达siRNA分子多个月的可行性;以及（iii）验证siRNA表达系统作为在疾病动物模型中研究基因功能的工具。本申请中提出的工作是有意义的，因为它将提供一种新的工具来研究基因沉默在肝脏中的作用。在完成我们的研究后，该系统可以用于大量的研究应用，旨在研究该器官以及许多其他组织中的基因功能，可以通过腺病毒载体转导。

DESCRIPTION (provided by applicant): Non-insulin-dependent or type 2 diabetes mellitus is a metabolic disease characterized by an elevated blood glucose concentration that results from inadequate insulin action in insulin-sensitive tissues and from abnormal insulin secretion. In the USA, Europe and most westernized countries, type 2 diabetes affects approximately 6% of the population. It is estimated that by 2010, >220 million people worldwide will be affected by the disease, promoted by a dramatic increase in the incidence of obesity and a sedentary lifestyle. To achieve glucose control and prevent diabetic complications (retinopathy, cardiovascular disease, and nephropathy, among others), patients currently receive therapeutic agents that have limited efficacy and are associated with side effects. Thus, there is a need to develop more efficient therapeutic agents. Our long-term goal is to identify genes that can be targeted with drugs to improve the treatment of type 2 diabetes. Current technologies to validate the role of genes in disease involve the development of animal models with null alleles for the target gene. In some instances, absence of a gene is incompatible with normal development, leading to death in utero. In addition, certain studies need to knockdown gene function in an adult animal. The objective of the present proposal is to develop a system to downregulate gene expression in liver. We will target a gene involved in fatty acid production, Srebp-1, expression of which is upregulated in type 2 diabetes. Our goals will be accomplished by administration of a recombinant gutless adenovirus expressing a small interfering RNA (siRNA) to reduce mRNA levels. The siRNA technology has emerged as a new tool to study gene function by inducing posttranscriptional gene silencing. We expect that the objective of this proposal will be achieved by pursuing the following specific aims: (i) Establish the optimal conditions to achieve complete Srebp-1 gene silencing in liver (ii) Determine the feasibility of expressing a siRNA molecule in the liver of an adult animal for multiple months and (iii) validate the siRNA expression system as a tool to study gene function in an animal model of disease. The work proposed in this application is significant because it will provide a novel tool to study the effects of gene silencing in liver. Upon completion of our studies, the system could be used in a large number of research applications directed at studying gene function in this organ as well as many other tissues that can be transduced by adenoviral vectors.