Hepatic expression of small hairpin RNA using gutless adenoviral vectors

使用无肠腺病毒载体肝脏表达小发夹 RNA

• 批准号: 8467899
• 负责人: NURIA MORRAL
• 金额: $ 6.06万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8467899
• 关键词: Address; Adenovirus Vector; Adenoviruses; Adipose tissue; Adverse effects; Affect; Alleles; Animal Model; Animals; Anti-Progestin; Binding; Biology; Blood Glucose; Bypass; Cardiovascular Diseases; Communicable Diseases; Complications of Diabetes Mellitus; Country; Data; Development; Diabetes Mellitus; Disease; Dose; Down-Regulation; Drug Delivery Systems; Dyslipidemias; Europe; Fatty Acids; Functional disorder; Funding; Gene Delivery; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Goals; HIV; Hepatic; Hepatitis; Hepatocyte; Homeostasis; Human; Incidence; Injury; Insulin; Insulin Resistance; Interferons; Kidney Diseases; Knock-out; Lead; Life Style; Lipids; Liver; Longitudinal Studies; Malignant Neoplasms; Mediating; Messenger RNA; Metabolic Diseases; Metabolic syndrome; Methods; MicroRNAs; Mifepristone; Modeling; Molecular; Mus; Muscle; Non-Insulin-Dependent Diabetes Mellitus; Nonesterified Fatty Acids; Obesity; Pathway interactions; Patients; Pharmaceutical Preparations; Population; Positioning Attribute; Process; Production; Proteins; RNA Interference; RNA chemical synthesis; Recombinants; Research; Retinal Diseases; Role; Small Interfering RNA; Study Section; System; Technology; Therapeutic Agents; Time; Tissues; Toxic effect; Transgenes; Validation; Viral Genes; Work; animal model development; blood glucose regulation; cost; drug development; effective therapy; experience; gene delivery system; gene function; gutless adenoviral vector; improved; in vivo; innovation; instrument; insulin secretion; knock-down; lipid metabolism; macrovascular disease; mature animal; mortality; nonhuman primate; novel; prevent; promoter; public health relevance; research study; response; sedentary; small hairpin RNA; tool; transgene expression; vector

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.

描述(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、欧洲和大多数西化国家，2型糖尿病影响了大约6%的人口。据估计，到2010年，由于肥胖症发病率的急剧增加和久坐不动的生活方式，全球将有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，且与副作用相关。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作用的技术包括建立目标基因零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身上进行研究，使研究与人类状况更相关。它还显著减少了功能研究和靶点验证之间的时间。无胆腺病毒载体是向肝脏传递基因的最佳工具，因为它们导致转基因的长期表达，并且在高剂量给药时毒性可以忽略不计。我们建立了一种在肝脏中表达小发夹RNA(ShRNA)的无胆腺病毒载体系统。超过75%的基因沉默可以实现，使该系统成为研究该组织的非凡工具。然而，目前使用RNAi的局限性是存在毒性效应，这是将其用作研究工具的一个令人担忧的问题。本提案的目的是建立使用低转录活性启动子和可诱导系统的系统，以便可以通过给药来调节shRNA的水平。我们期望这项建议的目标将通过追求以下具体目标来实现：(I)建立可利用组织特异性启动子实现的基因下调水平；(Ii)确定在体内受高水平shRNA表达影响的细胞途径；以及(Iii)开发用于合成shRNA的受调控的转录系统。我们期望这项拟议的研究将提供概念证明，即腺病毒介导的shRNA传递是产生长期和可诱导的基因沉默的可行策略。该系统可用作各种应用的研究工具，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：本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，2型糖尿病是一种影响大约6%美国人口的疾病。产生的数据将提供有关siRNA生物学基本方面的信息，siRNA生物学是治疗糖尿病、肥胖症、传染病和癌症等疾病的一种有前途的治疗剂。

项目成果

Enhancing hepatic mitochondrial fatty acid oxidation stimulates eating in food-deprived mice.

增强肝线粒体脂肪酸氧化可刺激食物匮乏小鼠的进食。

• DOI: 10.1152/ajpregu.00279.2014
• 发表时间: 2015
• 期刊: American journal of physiology. Regulatory, integrative and comparative physiology
• 作者: Mansouri,Abdelhak;Pacheco-López,Gustavo;Ramachandran,Deepti;Arnold,Myrtha;Leitner,Claudia;Prip-Buus,Carina;Langhans,Wolfgang;Morral,Núria
• 通讯作者: Morral,Núria

Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies.

• DOI: 10.1186/1756-0500-4-8
• 发表时间: 2011-01-18
• 期刊: BMC research notes
• 影响因子: 1.8
• 作者: Park, Jae-Seung;Surendran, Sneha;Morral, Nuria
• 通讯作者: Morral, Nuria