Gene Silencing with U1 Adaptor Oligonucleotides

使用 U1 接头寡核苷酸进行基因沉默

• 批准号: 7899508
• 负责人: Mark Aaron Behlke
• 金额: $ 9.98万
• 依托单位: INTEGRATED DNA TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-17 至 2010-02-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7899508
• 关键词: 3' Untranslated Regions; 5' Splice Site; Anabolism; Antisense Oligonucleotides; Antisense Technology; Area; Base Pairing; Binding; Biochemical Pathway; Biological Phenomena; Biology; Catalytic RNA; Cell Nucleus; Cells; Chemicals; Chemistry; Classification; Cleaved cell; Communities; Complex; Cytoplasm; DNA; Data; Development; Disease Progression; Down-Regulation; Effectiveness; Evaluation; Excision; Exons; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Goals; Human; Indium; Introns; Mediating; Messenger RNA; Methods; MicroRNAs; Modification; Mutate; Mutation; Nucleic Acids; Oligonucleotides; Pathway interactions; Plasmids; Polyadenylation; Post-Transcriptional Regulation; Process; Proteins; RNA; RNA Degradation; RNA Interference; RNA Splicing; Reagent; Refractory; Regulation; Regulator Genes; Research; Ribonuclease H; Role; Scientist; Site; Small Interfering RNA; Specificity; Tail; Techniques; Technology; Testing; Translations; U1 Small Nuclear Ribonucleoprotein; U1 small nuclear RNA; Untranslated Regions; Vertebrates; Work; base; cell type; combinatorial; commercialization; design; improved; mRNA Precursor; new technology; programs; public health relevance; success; synthetic nucleic acid

DESCRIPTION (provided by applicant): Our understanding of complex biological phenomena and disease progression has led to the realization that changes in the expression of genes underlie many of these processes. Developing reagents that can selectively alter the expression level of any desired gene has been a goal of both scientists and clinicians for years. Historically, the most common approach was based on antisense oligonucleotides (ASOs) that encompass a broad variety of mechanisms that have in common an oligonucleotide designed to base pair with its complementary target mRNA leading to either degradation or impaired function of the mRNA. Classically, ASOs were designed to interfere with translation of the target mRNA or induce its degradation via RNase H or more recently by ribozyme activity. Current excitement has focused on RNAi that uses a distinct mechanism where oligonucleotides trigger an endogenous pre-existing gene suppression pathway that is fundamental to cellular gene regulatory networks. In spite of its general success, some mRNAs are only modestly downregulated (2-fold) by RNAi and others are refractory. Further, certain off-target effects can arise leading to unexpected consequences, underscoring the need for additional methods. The rapid rise of the RNAi field has led to an increased appreciation, of direct relevance to the present proposal, that regulatory sequence elements in mRNA 3' ends (eg. 3'UTRs) control the expression of that gene. Here we present preliminary data on the development of a new gene silencing technology that uses oligonucleotides annealing to specific sequence regions within the 3'UTR to inhibit pre-mRNA processing. We plan to improve this new technology by systematically analyzing modified bases to increase inhibitory activity. We will also determine how robust the technology is by testing its effectiveness in a variety of human cell types as well as cells from other vertebrates and by silencing several endogenous human genes as a proof-of-principle. Finally, we will also determine whether enhanced inhibition is seen when this new technology is used in conjunction with other gene silencing technologies such as RNAi where enhancement is expected because these different methods utilize fundamentally distinct mechanisms. We believe this new technology will make a significant addition to our gene silencing toolkit and may even aid emerging oligonucleotide-based therapies, although that is beyond the scope of this proposal. PUBLIC HEALTH RELEVANCE: The commercialization of this new U1 Adaptor mediated gene silencing technology will be a significant addition to the scientific research community's "gene silencing toolkit". Because this method exploits a distinctly different mechanism compared to more common gene silencing approaches, it has the potential of enhancing these traditional technologies when used in combination with them via synergistic effects. This may aid in the development of emerging oligonucleotide-based gene silencing therapies by improving sensitivity and efficacy.

描述（由申请人提供）：我们对复杂的生物学现象和疾病进展的理解已导致认识到基因表达的变化是许多此类过程的基础。多年来，开发可以选择性改变任何所需基因表达水平的试剂一直是科学家和临床医生的目标。从历史上看，最常见的方法是基于反义寡核苷酸（ASO）的基础，这些寡核苷酸（ASO）涵盖了各种各样的机制，这些机制具有共同的寡核苷酸，旨在将其与其互补靶mRNA相对基础，从而导致mRNA降解或功能受损。通常，ASO被设计为干扰靶mRNA的翻译或最近通过RNase H或通过核酶活性诱导其降解。当前的兴奋集中在RNAi上，它使用了一种不同的机制，在这种机制中，寡核苷酸触发了内源性的先前存在的基因抑制途径，该途径是细胞基因调节网络至关重要的。尽管取得了一般成功，但RNAi和其他mRNA仅由RNAi和其他mRNA被适度下调（2倍）。此外，可能会出现某些脱离目标效应，从而导致意外后果，从而强调对其他方法的需求。 RNAi场的迅速上升导致了与本提案直接相关的欣赏，即mRNA 3'末端（例如3'UTRS）控制该基因的表达。在这里，我们提供了有关开发新基因沉默技术的初步数据，该数据使用将寡核苷酸退火到3'UTR内的特定序列区域来抑制前MRNA处理。我们计划通过系统地分析改良基础以增加抑制活性来改善这项新技术。我们还将通过测试其在各种人类细胞类型以及其他脊椎动物的细胞中测试其有效性，并使几个内源性人类基因作为原则证明，通过测试其有效性，以确定该技术的鲁棒性。最后，我们还将确定当将这种新技术与其他基因沉默技术（例如RNAi）结合使用时，是否可以看到增强的抑制作用，因为这些不同的方法利用了从根本上不同的机制。我们认为，这项新技术将为我们的基因沉默工具包提供重要的补充，甚至可以帮助新兴的基于寡核苷酸的疗法，尽管这超出了该提案的范围。 公共卫生相关性：这种新的U1适配器介导的基因沉默技术的商业化将是科学研究界的“基因沉默工具包”的重要补充。由于该方法与更常见的基因沉默方法相比利用了明显不同的机制，因此当通过协同作用与它们结合使用时，它具有增强这些传统技术的潜力。这可能有助于通过提高敏感性和功效来发展基于新兴的寡核苷酸基因沉默疗法。