High throughput RNAi based functional genomics in primary cells and in vivo

描述(申请人提供)：HGP(人类基因组计划)的完成开启了生物医学研究的“基因组时代”，并使各种基因元素的功能基因组分析成为可能。ENCODE项目的长期目标是识别和确定所有基于序列的基因组元件的功能特征。在ENCODE中研究的功能元件包括转录序列、转录调控因子和RNA转录产物本身的调控因子。超过50%的转录基因序列的功能未知。其中大多数在专门的细胞中表达，因此它们的功能只能在生物学相关的背景下(即体内的原代细胞)进行研究。在这个提案中，我们计划开发一个新的RNAi平台并将其商业化，该平台能够在生物相关的背景下进行基因研究，长期目标是构建人类基因组的功能图谱。通过转染剂将小干扰RNA(SiRNAs)导入细胞，可导致有效的基因沉默。基于siRNA的功能基因组学在体外建立的细胞系中得到了广泛的应用，但由于缺乏高效无毒的递送系统，其在原代细胞和体内靶向验证方面的适用性一直受到限制。在与RXi制药公司的合作中，我们开发了一类新的RNAi化合物-自我递送RNAs。这些小的、不对称的、疏水修饰的RNA化合物无需递送配方即可进入细胞和组织，并在体外和体内有效地沉默基因，使原代细胞、胚胎细胞、组织、体外和体内的功能基因组学研究成为可能。阻碍科学界广泛使用这一平台的主要技术障碍是复杂而昂贵的化合物鉴定、合成和验证过程。这个快速通道方案的重点是优化化合物发现过程(第一阶段)和开发一个由200-1000个功能验证的sdRNAs组成的小组(第二阶段)，该方案的完成将建立一个商业可用的产品平台，将彻底改变生物相关系统(如初级细胞、干细胞、组织和器官模型)以及最终在体内的功能基因组研究。

DESCRIPTION (provided by applicant): Completion of the HGP (human genome program) has opened the 'Genomic Era' in biomedical research and enables functional genomics analysis of various gene elements. The long-term goal of the ENCODE Project is to identify and functionally characterize all of the sequence-based genomic elements. Functional elements that have been studied in ENCODE include transcribed sequences, regulators of transcription, and regulators of RNA transcripts themselves. The function of more than 50% of transcribed gene sequences is unknown. Most of them are expressed in specialized cells and thus their functions can only be studied in a biologically relevant context (i.e. primary cells, in vivo). In this propoal we plan to develop and commercialize a new RNAi platform that enables gene studies in a biologically relevant context with a long term goal of building a functional map of human genome. Introduction of small interfering RNAs (siRNAs) into cells with transfection reagents results in efficient gene silencing. siRNA-based functional genomics is widely used in established cell lines in vitro, but its applicability to primary cells and in vivo target validatin has been limited because of lack of efficient and non-toxic delivery systems. In collaboration with RXi pharmaceuticals we have developed a novel class of RNAi compounds - self deliverable RNAs. These small, asymmetric, hydrophobically modified RNA compounds enter cells and tissues without requirement for delivery formulation and efficiently silence genes in vitro and in vivo, enabling functional genomics studies in primary cells, embryonic cells, tissues, ex vivo, and in vivo. The major technical hurdle, which impedes wide spread use of this platform by scientific community is the complex and costly process of compound identification, synthesis and validation. The focus of this fast-track proposal is optimization of compound discovery process (Phase I) and development of a panel of 200-1000 functionally validated sdRNAs (Phase II), Completion of this proposal will build a commercially available product platform that will revolutionize functional genomics studies in biologically relevant systems such as primary cells, stem cells, tissue and organ models and eventually in vivo.