Random shRNA Selection

随机 shRNA 选择

• 批准号: 8329704
• 负责人: ROBERT B WILSON
• 金额: $ 38.59万
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8329704
• 关键词: Affect; Age related macular degeneration; Area; Beta Cell; Binding; Biological Assay; Cardiac; Cardiac Myocytes; Cardiology; Cell Differentiation Induction; Cell Differentiation process; Cell Line; Cells; Chemicals; Clinical Trials; Communicable Diseases; Communities; Complement; DNA Sequence; Development; Diabetes Mellitus; Gene Expression; Gene Targeting; Genes; Genome; Genomics; IL3 gene; Islet Cell; Libraries; Messenger RNA; MicroRNAs; Mind; Modeling; Mutagenesis; Neurology; Neurons; Nucleotides; Oncogenes; Pathway interactions; Plague; Proteins; Proteomics; Public Health; RNA; RNA Interference; RNA Sequences; Regenerative Medicine; Reporter; Research; Risk; Screening procedure; Seeds; Small RNA; Specificity; Stem Cell Development; Stem cells; Testing; Therapeutic; Therapeutic Index; Toll-like receptors; Tumor Suppressor Genes; Vascular Endothelial Growth Factors; Withdrawal; base; design; gene therapy; insight; interest; new technology; novel; novel therapeutics; protein function; small molecule; tissue culture; tool; vector

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.

在小RNA疗法和生物工具的开发中使用RNA干扰(RNAi)的现有范例是使用短发夹环RNA(ShRNA)或短干扰RNA(SiRNA)来干扰单个基因的表达。这种现有的“单基因干扰”范式，我们试图挑战，源于使用shRNAs或siRNAs作为研究工具，以获得对可能的 感兴趣蛋白质的功能。然而，最有效的内源microRNAs(MiRNAs)，在其上模拟shRNAs和siRNAs，通过7-8个核苷酸的“种子序列”匹配同时靶向数百个mRNAs。因此，毫不奇怪，开发基于干扰单基因表达的小RNA疗法和生物工具的努力受到“非靶标”效应的困扰，这可能导致较差的治疗指数。 此外，最近发现miRNAs可以直接激活基因表达，也可以干扰基因表达。 管理这种“RNA激活”(RNAa)的规则尚不清楚。事实上，小RNA靶标和效应的完整范围才刚刚开始受到重视。为了充分利用小RNA疗法和生物工具的发展潜力，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，我们设计并合成了第一个shRNA表达 在核苷酸水平上完全随机的文库。使用我们的文库的基于细胞的筛选分析在作用机制(S)方面是公正的--实际上，我们让细胞告诉我们哪些小RNA是最有效和毒性最低的。在一个组织培养皿中，可以使用选择分析和汇集的方法来筛选数十万个随机的shRNA。因为只有大约两万人 可能的种子序列(对于规范的RNAi)，并且因为shRNAs是生物活性分子，所以总是存在命中序列。通过随机突变和重新筛选来优化初始命中序列是简单的。我们的方法使我们能够确定最有效、毒性最低的小RNA，作为治疗或生物工具使用。 我们建议使用我们的文库来鉴定和优化用于干细胞诱导和细胞分化的shRNA序列。我们鉴定和优化的文库序列可以以shRNAs的形式从载体中表达，也可以作为siRNAs导入细胞，发挥其作用而不需要基因组整合。Nanog和Oct4的报告构建体将用于识别用于干细胞诱导的shRNA，NKX2-5和Ngn3的报告构建体将分别用于识别心脏和β细胞分化的shRNA。将使用微阵列和/或蛋白质组学分析来识别独特的目标基因集。