Random shRNA Selection

随机 shRNA 选择

• 批准号: 7937761
• 负责人: ROBERT B WILSON
• 金额: $ 38.98万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 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/7937761
• 关键词: Beta Cell; Biological Assay; Cardiac; Cell Differentiation Induction; Cell Differentiation process; Cells; Communicable Diseases; Development; Gene Expression; Gene Targeting; Genes; Genomics; Libraries; MicroRNAs; Mind; Modeling; Mutagenesis; Nucleotides; Plague; Proteomics; Public Health; RNA; RNA Interference; RNA Sequences; Reporter; Research; Screening procedure; Seeds; Small RNA; Stem Cell Development; Stem cells; Therapeutic; Therapeutic Index; base; design; insight; interest; novel therapeutics; protein function; public health relevance; tissue culture; tool; vector

DESCRIPTION (provided by applicant): The existing paradigm for the use of RNA interference (RNAi) in the development of small-RNA therapeutics and biologic tools is to interfere with the expression of a single gene using a short-hairpin-loop RNA (shRNA) or short-interfering RNA (siRNA). This existing "single-gene-interference" paradigm, which we seek to challenge, derives from the use of shRNAs or siRNAs as research tools to gain insights into the possible functions of proteins of interest. However, the most potent endogenous microRNAs (miRNAs), on which shRNAs and siRNAs are modeled, target hundreds of mRNAs simultaneously through "seed-sequence" matches of 7-8 nucleotides. Not surprisingly, therefore, efforts to develop small-RNA therapeutics and biologic tools based on interfering with the expression of single genes are plagued by "off-target" effects, which are likely to result in poor therapeutic indices. In addition, miRNAs have recently been shown to directly activate, as well as interfere with, gene expression. The rules governing this "RNA activation" (RNAa) are unknown. Indeed, the complete range of small-RNA targets and effects is only beginning to be appreciated. To harness the full potential for the development of small-RNA therapeutics and biologic tools, including multi-gene targeting and RNA activation, a fundamentally different approach is needed. With this in mind, we designed and synthesized the first shRNA-expressing library that is completely random at the nucleotide level. Cell-based screening assays using our library are unbiased with respect to mechanism(s) of action - in effect, we let the cells tell us which small RNAs are the most effective and least toxic. Hundreds of thousands of random shRNAs can be screened in a single tissue- culture dish using selection assays and a pooled approach. Because there are only approximately 20,000 possible seed sequences (for canonical RNAi), and because shRNAs are bio-active molecules, hit sequences are invariably present. Optimization of initial hit sequences, by random mutagenesis and re-screening, is straightforward. Our approach allows us to identify the most effective, and least toxic, small RNAs to be used as therapeutics or biologic tools. We propose to use our library to identify and optimize shRNA sequences for stem-cell induction and for cell differentiation. Library sequences that we identify and optimize could be expressed from vectors as shRNAs, or transfected into cells as siRNAs, which exert their effects without genomic integration. Reporter constructs for Nanog and Oct4 will be used to identify shRNAs for stem-cell induction, and reporter constructs for NKX2-5 and Ngn3 will be used to identify shRNAs for cardiac and beta-cell differentiation, respectively. Profiling by microarray and/or proteomic analysis will be used to identify unique target gene sets. PUBLIC HEALTH RELEVANCE: This proposal describes an approach to develop novel therapeutics and biologic tools using an shRNA- expressing library that is completely random at the nucleotide level. This approach has implications for the development of stem-cell-based and infectious-disease therapeutics, and is highly relevant to public health.

描述（由申请人提供）：使用RNA干扰（RNAI）在开发小RNA疗法和生物学工具中的现有范例是，使用短发环蛋白 - 环RNA（SHRNA）或短速率rna（SIRNA）干扰单个基因的表达。我们寻求挑战的现有的“单基因干扰”范式源自将shrnas或siRNA作为研究工具的使用，以了解感兴趣的蛋白质的可能功能。然而，对shRNA和siRNA进行建模的最有效的内源性microRNA（miRNA），通过7-8个核苷酸的“种子序列”匹配，同时靶向数百个mRNA。因此，毫不奇怪，基于干扰单个基因表达的小型RNA治疗剂和生物学工具的努力受到“脱靶”效应的困扰，这些效应可能导致治疗指标差。另外，最近已显示miRNA直接激活并干扰基因表达。管理此“ RNA激活”（RNAA）的规则未知。实际上，小型RNA靶标和效果的完整范围才开始被理解。为了利用小型RNA治疗剂和生物学工具的全部潜力，包括多基因靶向和RNA激活，需要一种根本不同的方法。考虑到这一点，我们设计和合成了第一个表达shRNA的文库，该文库在核苷酸水平上是完全随机的。基于细胞的筛选测定使用我们的文库对作用机制无偏见 - 实际上，我们让细胞告诉我们哪种小RNA是最有效且毒性最少的小型RNA。可以使用选择分析和合并方法在单个组织培养皿中筛选成千上万的随机shRNA。由于只有大约20,000个可能的种子序列（对于典型的RNAi），并且SHRNA是生物活性分子，因此始终存在HIT序列。通过随机的诱变和重新筛选对初始命中序列的优化是直接的。我们的方法使我们能够识别最有效，最有毒的小RNA用作治疗剂或生物学工具。我们建议使用我们的库来识别和优化shRNA序列，以进行干细胞诱导和细胞分化。我们识别和优化的库序列可以从向量中作为shRNA表示，也可以像siRNA一样转染到细胞中，它们在没有基因组整合的情况下发挥其效果。 Nanog和Oct4的报告构建体将用于识别用于干细胞诱导的shRNA，而NKX2-5和NGN3的记者构造将分别用于识别用于心脏和β细胞分化的SHRNA。通过微阵列和/或蛋白质组学分析进行分析将用于识别唯一的靶基因集。 公共卫生相关性：该提案描述了一种使用shrna表达文库开发新型治疗和生物学工具的方法，该图书馆在核苷酸水平上是完全随机的。这种方法对基于干细胞和感染性疾病的疗法的发展具有影响，并且与公共卫生高度相关。