Molecular Genetic Analysis of Extracellular RNAs in C. elegans

描述(申请人提供)：线虫中RNA干扰(RNAi)的一个显著特征是它与细胞间RNA运输途径有关。这种连锁动员dsRNA沉默信号，使沉默能够从起始点传播到整个动物和后代。这种现象被称为系统RNAi，在许多多细胞生物体中是一个保守的过程。通过遗传分析，我们分离到了系统性RNAi缺陷突变体(SID)，并鉴定了相应的蛋白质(SID)。SID-1是一种广泛保守的dsRNA通道，它选择性和特异性地将dsRNA转运到细胞内，是系统RNAi所必需的。哺乳动物的SID-1同源基因对于修饰的siRNAs的细胞质传递至关重要，这表明dsRNA运输是这一家族通道蛋白的一种保守功能。SID-2是一种可能的dsRNA受体，仅在肠腔膜上表达和定位。SID-2将摄入的dsRNA通过肠道上皮运输到动物体内，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，广泛存在于自然界，包括哺乳动物中。 以RNAi为基础的药物的开发使以前“无法治愈”的疾病相关基因成为靶点，并具有令人兴奋的治疗潜力。然而，RNAi作为基因沉默治疗的有效性和安全性需要了解治疗性dsRNA是如何进入细胞以获得沉默机制的。此外，我们必须合理地确定如何修饰这些dsRNA分子，以便更有效地输送和靶向选择组织和细胞。我们对SID-1通道中dsRNA运输的分析表明，即使是微小的修饰，如从核糖中移除一个氧原子，也可以阻止dsRNA的运输。因此，了解这些蛋白在实验上可驯化的线虫中的调节和功能将为基于RNAi的药物作为一类新的人类治疗剂的发展提供有价值的见解。 这项拟议研究的长期目标是了解动物细胞间RNA运输的生理重要性和机制。为此，这项建议的具体目标是： 1)研究SID-1 dsRNA通道的特异性及其调控； 2)研究环境RNAi中摄取dsRNA的机制； 3)研究细胞外dsRNA转运途径； 4)分离和鉴定线虫内源性胞外RNA。 这些目的是通过结合遗传、生化和生物物理方法，解决最近发现的细胞间RNA运输领域的前沿问题，并进一步探索细胞外RNA分子是多细胞生物体中一种新的信号传递手段的可能性。值得注意的是，这一10年前还不为人所知的过程，现在具有直接的临床意义。 与公共卫生相关：基于dsRNA的基因沉默的特异性和有效性为包括癌症在内的多种人类疾病的治疗带来了巨大的希望。尽管几种基于RNAi的药物的临床试验已经在进行中，但dsRNA转运和摄取的机制尚不清楚。了解dsRNA是如何穿过细胞膜触发RNAi的，将有助于提高靶向和递送效率，并将限制这一令人兴奋的治疗策略的潜在有害副作用。

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.