Random shRNA Selection

描述（由申请人提供）：在小RNA疗法和生物工具开发中使用RNA干扰（RNAi）的现有范例是使用短发夹环RNA（shRNA）或短干扰RNA（siRNA）干扰单个基因的表达。我们试图挑战的这种现有的“单基因干扰”范式源自使用 shRNA 或 siRNA 作为研究工具，以深入了解感兴趣的蛋白质的可能功能。然而，最有效的内源性 microRNA (miRNA)（以 shRNA 和 siRNA 为模型）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，基于干扰单基因表达开发小RNA疗法和生物工具的努力受到“脱靶”效应的困扰，这可能会导致治疗指数不佳。此外，最近已证明 miRNA 可以直接激活以及干扰基因表达。控制这种“RNA 激活”（RNAa）的规则尚不清楚。事实上，小 RNA 靶标和作用的完整范围才刚刚开始被人们所认识。为了充分发挥小 RNA 疗法和生物工具开发的潜力，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在核苷酸水平上完全随机的 shRNA 表达文库。使用我们的文库进行的基于细胞的筛选测定在作用机制方面是公正的 - 实际上，我们让细胞告诉我们哪些小 RNA 最有效且毒性最小。使用选择分析和汇总方法可以在单个组织培养皿中筛选数十万种随机 shRNA。因为只有大约 20,000 个可能的种子序列（对于规范 RNAi），并且 shRNA 是生物活性分子，所以命中序列总是存在。通过随机诱变和重新筛选来优化初始命中序列非常简单。我们的方法使我们能够确定最有效、毒性最小的小RNA，用作治疗或生物工具。我们建议使用我们的文库来识别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作为 shRNA 从载体中表达，或者作为 siRNA 转染到细胞中，无需基因组整合即可发挥作用。 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用于心脏和 β 细胞分化的 shRNA。通过微阵列和/或蛋白质组分析进行分析将用于识别独特的靶基因集。 公共健康相关性：该提案描述了一种使用在核苷酸水平上完全随机的 shRNA 表达文库开发新型疗法和生物工具的方法。这种方法对基于干细胞和传染病疗法的发展具有影响，并且与公共卫生高度相关。

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.