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

描述（申请人提供）：人类基因组计划（HGP）的完成开启了生物医学研究的“基因组时代”，并使各种基因元件的功能基因组学分析成为可能。ENCODE项目的长期目标是鉴定和功能性表征所有基于序列的基因组元件。在ENCODE中已经研究的功能元件包括转录序列、转录调节子和RNA转录物本身的调节子。超过50%的转录基因序列的功能是未知的。它们中的大多数在特化细胞中表达，因此它们的功能只能在生物学相关的背景下（即原代细胞，体内）进行研究。在这个项目中，我们计划开发和商业化一个新的RNAi平台，使基因研究在生物学相关的背景下，建立一个人类基因组的功能图谱的长期目标。用转染试剂将小干扰RNA（siRNA）引入细胞中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被广泛使用，但由于缺乏有效且无毒的递送系统，其对原代细胞和体内靶点验证的适用性受到限制。在与RXi制药公司的合作中，我们开发了一类新型的RNAi化合物-可自我递送的RNA。这些小的、不对称的、疏水修饰的RNA化合物进入细胞和组织而不需要递送制剂，并在体外和体内有效地沉默基因，使得能够在原代细胞、胚胎细胞、组织、离体和体内进行功能基因组学研究。阻碍科学界广泛使用该平台的主要技术障碍是化合物鉴定、合成和验证的复杂和昂贵的过程。该快速通道提案的重点是优化化合物发现过程（第一阶段）和开发一组200-1000个功能验证的sdRNA（第二阶段），该提案的完成将建立一个商业化的产品平台，这将彻底改变生物相关系统（如原代细胞，干细胞，组织和器官模型）中的功能基因组学研究，并最终在体内进行。

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.