Hepatic expression of small hairpin RNA using gutless adenoviral vectors

描述（由申请方提供）：非胰岛素依赖型或2型糖尿病是一种代谢性疾病，其特征为血糖浓度升高，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，全球将有超过2.2亿人受到这种疾病的影响，肥胖发生率的急剧增加和久坐不动的生活方式将加剧这种疾病。为了实现葡萄糖控制和预防糖尿病并发症（视网膜病、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基因靶点，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。小干扰RNA（siRNA）技术已成为研究基因功能和验证靶点的首选工具，因为它允许在成年动物中进行研究，使研究与人类状况更相关。它还显著缩短了功能研究和目标验证之间的时间。无肠腺病毒载体是基因递送至肝脏的最佳工具，因为它们导致转基因的长期表达，并且当以高剂量施用时毒性可忽略不计。我们建立了一个无肠腺病毒载体系统，在肝脏中表达小发夹RNA（shRNA）。可以实现高于75%的基因沉默，使该系统成为该组织研究的显着工具。然而，目前使用RNAi的限制是存在毒性作用，这是其作为研究工具的一个问题。本发明的目的是产生使用具有低转录活性的启动子和诱导型系统的系统，使得可以通过施用药物来调节shRNA的水平。我们希望通过追求以下具体目标来实现本提案的目标：（i）建立可以使用组织特异性启动子实现的基因下调水平;（ii）识别受体内高水平shRNA表达影响的细胞途径;以及（iii）开发用于合成shRNA的调节转录系统。我们希望，拟议的研究将提供概念证明，腺病毒介导的shRNA递送是一种可行的策略，以产生长期和诱导的基因沉默。该系统可用作各种应用的研究工具，其中不仅包括糖尿病，还包括与肝脏相关的疾病，如心血管疾病，肝炎和癌症的研究。 公共卫生相关性：这项申请中提出的工作将提供新的工具来确定2型糖尿病的药物靶点，这种疾病影响了大约6%的美国人口。所产生的数据将提供关于siRNA生物学的基本方面的信息，siRNA生物学是用于治疗糖尿病、肥胖症、传染病和癌症等疾病的有前景的治疗剂。

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 ~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 gene targets 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. The small interfering RNA (siRNA) technology has become the tool of choice to study gene function and validate targets because it allows carrying out studies in adult animals, making the study more relevant to the human condition. It also significantly reduces the time between functional studies and target validation. Gutless adenoviral vectors are optimal tools for gene delivery to the liver because they result in long-term expression of transgenes and negligible toxicity when administered at high doses. We have developed a gutless adenoviral vector system to express small hairpin RNA (shRNA) in liver. Higher than 75% gene silencing can be achieved, making this system a remarkable instrument for studies in this tissue. However, current limitations to using RNAi are the presence of toxic effects, which is a concern for its use as a research tool. The objective of the present proposal is to generate systems that use promoters with low transcriptional activity and inducible systems, so that the level of shRNA can be regulated by administration of a drug. We expect that the objective of this proposal will be achieved by pursuing the following specific aims: (i) Establish the level of gene downregulation that can be achieved using tissue-specific promoters; (ii) Identify cellular pathways affected by high-level shRNA expression in vivo; and (iii) Develop regulated transcription systems for synthesis of shRNA. We expect that the proposed research will provide the proof of concept that adenovirus-mediated shRNA delivery is a feasible strategy to generate long-term and inducible gene silencing. The system could be used as research tool for a variety of applications, which include not only diabetes, but also studies on liver-related diseases such as cardiovascular disease, hepatitis, and cancer. Public Health Relevance: The work proposed in this application will provide novel tools to identify drug targets for type 2 diabetes, a disease that affects approximately 6% of the US population. The data generated will provide information on basic aspects of siRNA biology, a promising therapeutic agent for the treatment of diseases such as diabetes, obesity, infectious diseases, and cancer.