Molecular Genetic Analysis of Extracellular RNAs in C. elegans

线虫细胞外 RNA 的分子遗传学分析

• 批准号: 7936354
• 负责人: CRAIG Patrick HUNTER
• 金额: $ 34.88万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936354
• 关键词: Address; Adverse effects; Animals; Bathing; Binding; Biochemical; Biogenesis; Biological; Body cavities; Caenorhabditis elegans; Carrier Proteins; Cell Communication; Cell membrane; Cells; Cholesterol; Clinical Trials; Complement; Coupled; Cytoplasm; Development; Disease; Double-Stranded RNA; Drosophila genus; Endocytosis; Epithelium; Family; Gene Proteins; Gene Silencing; Genes; Genetic; Genetic Screening; Genetic screening method; Genome; Homologous Gene; Human; In Vitro; Ingestion; Integral Membrane Protein; Intestines; Knowledge; Lead; Ligands; Liquid substance; Lung; Malignant Neoplasms; Mammals; Measures; Membrane; Methods; Minor; Modification; Molecular Analysis; Molecular Genetics; Movement; Nature; Nematoda; Organ; Organism; Oxygen; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Process; Property; Proteins; RNA; RNA Interference; RNA Interference Pathway; RNA Transport; Regulation; Research; Ribose; Role; Safety; Signal Transduction; Site; Small Interfering RNA; Specificity; Therapeutic; Therapeutic Agents; Tissues; base; body cavity; clinically relevant; design; extracellular; genetic analysis; human disease; in vivo; insight; intestinal epithelium; luminal membrane; mutant; novel; protein function; protein transport; public health relevance; receptor; receptor mediated endocytosis; response; sugar; targeted delivery; tool; uptake

DESCRIPTION (provided by applicant): A remarkable property of RNA interference (RNAi) in C. elegans is its association with intercellular RNA transport pathways. This linkage mobilizes dsRNA-silencing signals and enables silencing to spread from the site of initiation throughout the animal and to the progeny. This phenomenon, known as systemic RNAi, is a conserved process among many multicellular organisms. Through genetic analysis, we have isolated systemic RNAi defective mutants (sid) and have identified the corresponding proteins (SID). SID-1 is a widely conserved dsRNA channel that selectively and specifically transports dsRNA into cells and is essential for systemic RNAi. A mammalian SID-1 homolog is critical for cytoplasmic delivery of modified siRNAs, suggesting that dsRNA transport is a conserved function for this family of channel proteins. SID-2 is a putative dsRNA receptor that is expressed and localized exclusively to the luminal membrane of the intestine. SID-2 transports ingested dsRNA across the intestinal epithelium into the animal to trigger RNAi. This process of sequence-specific gene silencing in response to environmentally-encountered dsRNA, known as environmental RNAi, is widespread throughout nature, including in mammals. The development of RNAi-based drugs enables targeting of previously "undruggable" disease related genes and has exciting therapeutic potential. However, the efficacy and safety of RNAi as a gene-silencing therapy requires understanding how therapeutic dsRNAs enter cells to gain access to the silencing machinery. In addition, we must rationally determine how to modify these dsRNA molecules for more efficient delivery and targeting to select tissues and cells. Our analysis of dsRNA transport through the SID-1 channel indicates that even minor modifications, such as removing an oxygen atom from the ribose sugar, can block dsRNA transport. Thus, understanding the regulation and function of these proteins in the experimentally tractable nematode C. elegans will provide valuable insights for the advancement of RNAi-based drugs as a novel class of therapeutic agents in humans. The long-term objective of the proposed research is to understand the physiological importance and mechanism of intercellular RNA transport in animals. Towards this end, the specific aims of this proposal are: 1) To characterize the specificity and regulation of the SID-1 dsRNA channel; 2) To characterize ingested dsRNA uptake mechanism in environmental RNAi; 3) To characterize extracellular dsRNA transport pathways; and 4) To isolate and characterize endogenous extracellular RNAs in C. elegans. These aims address, through a combination of genetic, biochemical and biophysical approaches, questions at the leading edge of the recently discovered field of intercellular RNA transport and further explore the possibility that extracellular RNA molecules underlie a novel means of signaling in multicellular organisms. Remarkably, this process, which was unknown 10 years ago, now has immediate clinical relevance. PUBLIC HEALTH RELEVANCE: The specificity and potency of dsRNA-based gene silencing lends tremendous hope for the treatment of a wide range of human diseases including cancer. Although clinical trials for several RNAi-based drugs are already underway, the mechanisms underlying dsRNA transport and uptake are poorly understood. Understanding how dsRNA crosses cell membranes to trigger RNAi will lead to improvements in targeting and delivery efficiency, and will limit potential deleterious side effects of this exciting therapeutic strategy.

描述（由申请人提供）：秀丽隐杆线虫中RNA干扰（RNAi）的显着特性是其与细胞间RNA传输途径的关联。这种连锁动员了dsRNA - 沉默信号，并使沉默能够从启动部位传播到整个动物和后代。这种现象被称为全身RNAi，是许多多细胞生物中的保守过程。通过遗传分析，我们具有分离的全身RNAi缺陷突变体（SID），并鉴定了相应的蛋白质（SID）。 SID-1是一种广泛保守的DSRNA通道，有选择地，特异性地将DSRNA转运到细胞中，对于全身RNAi至关重要。哺乳动物SID-1同源物对于修饰的siRNA的细胞质递送至关重要，这表明dsRNA转运是该通道蛋白家族的保守功能。 SID-2是一种假定的DSRNA受体，该受体被表达并仅在肠道的腔膜中定位。 SID-2将摄入的DSRNA横穿肠上皮摄入动物，以触发RNAi。响应环境中的dsRNA（称为环境RNAi）的序列特异性基因沉默的过程在整个自然界中广泛存在，包括在哺乳动物中。 基于RNAi的药物的开发使以前的“不难”疾病相关基因的靶向具有令人兴奋的治疗潜力。然而，RNAi作为基因分解疗法的功效和安全性需要了解治疗性DSRNA如何进入细胞以进入沉默机制。此外，我们必须合理地确定如何修改这些DSRNA分子，以更有效地递送和靶向选择组织和细胞。我们对DSRNA通过SID-1通道传输的分析表明，即使是从核糖糖中去除氧原子等小修饰也可以阻止DSRNA的转运。因此，了解这些蛋白质在实验可牵引的线虫C.秀丽隐杆线虫中的调节和功能将为基于RNAi的药物的发展提供有价值的见解，作为人类中一种新型的治疗剂。 拟议研究的长期目标是了解动物细胞间RNA转运的生理重要性和机制。为此，该提案的具体目的是： 1）表征SID-1 DSRNA通道的特异性和调节； 2）表征环境RNAi中摄入的dsRNA摄取机制； 3）表征细胞外DSRNA转运途径；和 4）分离并表征秀丽隐杆线虫中内源性细胞外RNA。 这些目的是通过遗传，生化和生物物理方法的结合来解决在最近发现的细胞间RNA转运领域的前沿问题，并进一步探索了细胞外RNA分子的可能性，即在多细胞生物中的新型信号手段构成了一种新颖的手段。值得注意的是，这个过程是10年前未知的，现在具有立即的临床意义。 公共卫生相关性：基于DSRNA的基因沉默的特异性和效力为治疗包括癌症在内的广泛的人类疾病的治疗带来了巨大的希望。尽管已经进行了几种基于RNAi的药物的临床试验，但对DSRNA运输和摄取的机制知之甚少。了解dsRNA如何越过细胞膜以触发RNAi将导致靶向和递送效率的提高，并将限制这种令人兴奋的治疗策略的潜在有害副作用。