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: 8130855
• 负责人: CRAIG Patrick HUNTER
• 金额: $ 36.7万
• 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/8130855
• 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; 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）：在C. elegans是它与细胞间RNA转运途径的关联。这种连接调动dsRNA沉默信号，并使沉默能够从起始位点传播到整个动物和后代。这种现象被称为系统性RNAi，是许多多细胞生物中的保守过程。通过遗传分析，我们分离了系统性RNAi缺陷突变体（sid），并鉴定了相应的蛋白质（SID）。SID-1是一个广泛保守的dsRNA通道，它选择性地和特异性地将dsRNA转运到细胞中，并且是系统性RNAi所必需的。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，表明dsRNA转运是该通道蛋白家族的保守功能。SID-2是一种推定的dsRNA受体，其仅表达并定位于肠的腔膜。SID-2将摄取的dsRNA穿过肠上皮转运到动物中以触发RNAi。这种响应于环境遇到的dsRNA的序列特异性基因沉默的过程，称为环境RNAi，在整个自然界中广泛存在，包括哺乳动物。 基于RNAi的药物的开发使得能够靶向先前“不可治疗”的疾病相关基因，并且具有令人兴奋的治疗潜力。然而，RNAi作为基因沉默疗法的有效性和安全性需要了解治疗性dsRNA如何进入细胞以获得沉默机制。此外，我们必须合理地确定如何修饰这些dsRNA分子，以更有效地递送和靶向选择的组织和细胞。我们对dsRNA通过SID-1通道转运的分析表明，即使是微小的修饰，例如从核糖中去除一个氧原子，也可以阻止dsRNA的转运。因此，了解这些蛋白在实验上易处理的线虫C。elegans的研究将为基于RNAi的药物作为人类新型治疗剂的发展提供有价值的见解。 该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，本提案的具体目标是： 1)表征SID-1 dsRNA通道的特异性和调节; 2)描述环境RNAi中摄入dsRNA的摄取机制; 3)表征细胞外dsRNA转运途径;以及 4)分离和鉴定C.优雅的 这些目标解决，通过遗传，生物化学和生物物理方法的组合，在最近发现的细胞间RNA运输领域的前沿问题，并进一步探索细胞外RNA分子在多细胞生物体中的信号转导的新手段的可能性。值得注意的是，这一过程在10年前还不为人知，现在已具有直接的临床意义。 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多种人类疾病带来了巨大的希望。尽管几种基于RNAi的药物的临床试验已经在进行中，但对dsRNA转运和摄取的机制知之甚少。了解dsRNA如何穿过细胞膜触发RNAi将导致靶向和递送效率的改善，并将限制这种令人兴奋的治疗策略的潜在有害副作用。