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: 8325718
• 负责人: CRAIG Patrick HUNTER
• 金额: $ 36.85万
• 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/8325718
• 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）：线虫中RNA干扰（RNAi）的一个显着特性是其与细胞间RNA转运途径的关联。这种连接调动了 dsRNA 沉默信号，并使沉默能够从起始位点传播到整个动物并传播到后代。这种现象被称为系统性 RNAi，是许多多细胞生物体中的保守过程。通过遗传分析，我们分离出了系统性RNAi缺陷突变体（sid）并鉴定了相应的蛋白（SID）。 SID-1 是一种广泛保守的 dsRNA 通道，可选择性、特异性地将 dsRNA 转运至细胞内，对于系统性 RNAi 至关重要。哺乳动物 SID-1 同源物对于修饰 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 是一种推定的 dsRNA 受体，仅表达并定位于肠腔膜。 SID-2 将摄入的 dsRNA 穿过肠上皮转运到动物体内以触发 RNAi。这种响应环境中遇到的 dsRNA 的序列特异性基因沉默过程（称为环境 RNAi）在自然界中广泛存在，包括在哺乳动物中。 基于 RNAi 的药物的开发能够靶向以前“不可成药”的疾病相关基因，并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 如何进入细胞以访问沉默机制。此外，我们必须合理确定如何修饰这些dsRNA分子，以更有效地递送和靶向选择的组织和细胞。我们对通过 SID-1 通道的 dsRNA 运输的分析表明，即使是微小的修饰，例如从核糖中去除氧原子，也可以阻止 dsRNA 运输。因此，了解这些蛋白质在实验上易处理的线虫秀丽隐杆线虫中的调节和功能将为基于 RNAi 的药物作为人类新型治疗剂的发展提供有价值的见解。 本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通道的特异性和调控； 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) 分离和表征线虫内源性细胞外RNA。 这些目标通过结合遗传、生物化学和生物物理方法，解决最近发现的细胞间 RNA 转运领域的前沿问题，并进一步探索细胞外 RNA 分子作为多细胞生物中新的信号传导手段的可能性。值得注意的是，这一过程在 10 年前还不为人知，但现在已经具有直接的临床意义。 公共健康相关性：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希望。尽管几种基于 RNAi 的药物的临床试验已经在进行中，但人们对 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 将提高靶向和递送效率，并将限制这种令人兴奋的治疗策略的潜在有害副作用。