Hepatic expression of small hairpin RNA 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性疾病，其特征是胰岛素敏感组织中胰岛素作用不足和胰岛素分泌异常导致血糖浓度升高。在美国、欧洲和大多数西方化国家，2型糖尿病影响了约6%的人口。据估计，到2010年，由于肥胖发病率的急剧增加和久坐不动的生活方式，全世界将有1.2.2亿人受到这种疾病的影响。为了实现血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），患者目前接受的治疗药物疗效有限，且伴有副作用。因此，有必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包括开发靶基因无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标的首选工具，因为它允许在成年动物中进行研究，使研究更与人类状况相关。它还显著缩短了功能研究和靶标验证之间的时间。无胆腺病毒载体是将基因传递到肝脏的最佳工具，因为它们导致转基因的长期表达，并且在高剂量使用时毒性可以忽略不计。我们开发了一种在肝脏中表达小发夹RNA （shRNA）的无胆腺病毒载体系统。可以实现75%以上的基因沉默，使该系统成为研究该组织的重要工具。然而，目前使用RNAi的限制是存在毒性作用，这是将其用作研究工具的一个问题。目前提议的目标是产生使用低转录活性启动子和诱导系统的系统，以便shRNA的水平可以通过药物管理来调节。我们期望通过追求以下具体目标来实现本提案的目标：(i)确定使用组织特异性启动子可以实现的基因下调水平；鉴定受体内高水平shRNA表达影响的细胞通路；（三）发展合成shRNA的调控转录系统。我们期望所提出的研究将证明腺病毒介导的shRNA递送是一种产生长期和可诱导的基因沉默的可行策略。该系统可以作为多种应用的研究工具，不仅包括糖尿病，还包括心血管疾病、肝炎、癌症等肝脏相关疾病的研究。

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.