Transport of gene silencing between cells during RNA interference

描述（申请人提供）：RNA干扰（RNA interference, RNAi）是一种双链RNA特异性灭活序列匹配基因的保守机制。RNAi从一个在简单蠕虫秀丽隐杆线虫中获得诺贝尔奖的发现，迅速发展成为一种治疗方法，可以沉默缺乏常规药物的人类致病基因。然而，RNAi的基本方面仍不清楚，需要了解它们以确保安全有效的RNAi治疗。模型生物秀丽隐杆线虫研究的持续相关性和重要性被以下事实所强调：在RNAi期间将RNA导入秀丽隐杆线虫细胞的RNA通道的人类对应体是将基于RNAi的药物导入人类细胞所必需的。候选人提出了5年的职业发展计划，旨在利用秀丽隐杆线虫获得RNA在RNA干扰过程中细胞间转运的基本见解，同时在研究型大学建立独立的学术生涯。该候选人将在遗传学和生物化学的坚实基础上，在动物细胞间RNA转运研究的先驱和领导者Craig Hunter博士的指导下，发展成为一名RNA转运的独立研究员。该计划将在现代生物学的领先机构哈佛大学分子生物系进行。在导师实验室的研究中，发现了保守的RNA通道SID-1，这是rnai介导的沉默信号的输入和组织内细胞间信号运输所必需的。在最近发表的一篇文章中，该候选人报告了秀丽隐杆线虫组织中RNA的输出是通过受调节的SID-1独立机制发生的。在指导阶段，候选人将：1)使用先进的显微镜解剖组织内细胞间依赖SID-1的RNA运输，并检查sid-3如何介导组织中独立于SID-1的RNA输出，这是这种输出所需的基因；2)检查细胞内RNAi途径蛋白在产生从该细胞转运的rna中的作用。除了导师的实验室外，Aim1的高级显微镜将在候选人的合作者庄晓伟博士的实验室进行，庄晓伟博士是超分辨率显微镜的先驱。在独立阶段，候选人将分析性别-1和性别-2的作用，这是候选人发现的组织间输出所需的另外两个基因，并将使用在指导阶段获得的方法和技术来确定输出的基本分子途径。在执行上述研究计划的同时，生化方法将使候选人具备作为独立研究者建立多方面和丰富的研究计划的能力。此外，拟议的研究将揭示秀丽隐杆线虫RNAi过程中RNA转运的基本方面，这将影响治疗人类疾病的RNAi方法的设计。

DESCRIPTION (provided by applicant): RNA interference (RNAi) is a conserved mechanism by which double-stranded RNA can specifically inactivate genes of matching sequence. RNAi has rapidly developed from a Nobel-prize winning discovery in the simple worm C. elegans to a therapeutic approach to silence human disease-causing genes that lack conventional medicines. However, fundamental aspects of RNAi remain unclear and they need to be understood to ensure safe and efficacious RNAi therapy. The continued relevance and significance of studies in the model organism C. elegans is underscored by the fact that the human counterpart of an RNA channel that imports RNA into C. elegans cells during RNAi is required for the import of RNAi-based drugs into human cells. The candidate presents a 5-year career development plan that aims to use C. elegans to gain fundamental insights into the transport of RNA between cells during RNA interference, while establishing an independent academic career at a research university. The candidate will build on his strong foundation in genetics and biochemistry to develop into an independent researcher in RNA transport under the mentorship of Dr. Craig Hunter, a pioneer and leader in the study of RNA transport between animal cells. The plan will be carried out in the Department of Molecular Biology at Harvard University, a leading institution in modern biology. Research in the mentor's lab led to the discovery of the conserved RNA channel SID-1 that is required for the import of RNAi-mediated silencing signals and the transport of signals between cells within a tissue. In a recent publication, the candidate reported the discovery that export of RNA from C. elegans tissues occurs through a regulated SID-1 independent mechanism. During the mentored phase, the candidate will: 1) Dissect the SID-1 dependent transport of RNA between cells within a tissue using advanced microscopy and examine how the SID-1 independent export of RNA from tissues is mediated by sid-3, a gene required for such export; and 2) examine the role of RNAi pathway proteins within a cell in generating RNAs transported from that cell. In addition to the mentor's laboratory, advanced microscopy for Aim1 will be carried out in the lab of the candidate's collaborator Dr. Xiaowei Zhuang, who is a pioneer in super-resolution microscopy. During the independent phase of the award, the candidate will analyze the roles of sexd-1 and sexd-2, two other genes discovered by the candidate that are required for inter-tissue export and will define a basic molecular pathway for export using the approaches and techniques acquired during the mentored phase Training in the complementary cell biological, genetic, and biochemical approaches while executing the above research plan will equip the candidate to establish a multi-faceted and rich research program as an independent investigator. Further, the proposed studies will reveal fundamental aspects of RNA transport during RNAi in C. elegans, which will impact the design of therapeutic RNAi approaches to human diseases. Public Health Relevance: Relevance RNAi-based drugs can specifically target disease-causing genes for which no conventional medicines are currently available. But, delivery of the drugs specifically and efficiently into diseased cells and tissues is a major barrier to RNA therapy. Understanding the mechanisms that control export of RNAi-mediated silencing signals from cells and the effect of such export on RNAi within these cells in C. elegans will provide valuable insights to overcome these barriers to therapeutic RNAi in humans.