Optimization of U1 Adaptor Technology

U1适配器技术优化

• 批准号: 7804100
• 负责人: Mark Aaron Behlke
• 金额: $ 46.09万
• 依托单位: INTEGRATED DNA TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7804100
• 关键词: 3' Untranslated Regions; Adoption; Affect; Algorithms; Alternative Splicing; Antisense Oligonucleotides; Base Pairing; Biological Phenomena; Biology; Chemicals; Chemistry; Communities; Complex; Cultured Cells; DNA Microarray Chip; Data Set; Development; Disease Progression; Exons; Fingerprint; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genome; Goals; Human; Indium; Introns; Investigation; Length; Measures; Mediating; Messenger RNA; Methodology; Methods; Microarray Analysis; Modification; Oligonucleotides; Pathway interactions; Pattern; Phase; Polyadenylation; Positioning Attribute; Precursor RNA; Process; Progress Reports; Publications; Publishing; RNA Interference; Reagent; Refractory; Regulator Genes; Research; Resistance; Ribonuclease H; Scientist; Selection Criteria; Site; Small Business Innovation Research Grant; Small Interfering RNA; Specificity; Tail; Techniques; Technology; Testing; To specify; Transcript; Transfection; Translations; U1 small nuclear RNA; Untranslated Regions; Walking; Work; base; commercialization; design; genome wide association study; genome-wide; genome-wide analysis; high throughput analysis; improved; knock-down; mRNA Precursor; new technology; novel; novel therapeutics; potency testing; programs; public health relevance; research study; response; success; tool

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. Currently, greater excitement has focused on RNAi which 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 may be 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 (i.e., 3'UTRs) control the expression of that gene. Here we present a progress report from a Phase 1 SBIR on the development of a new gene silencing technology which we call "U1 Adaptors," that uses oligonucleotides annealing to specific sequence regions within the 3'UTR to inhibit pre-mRNA processing. In this Phase 2 proposal, we plan to continue the optimization work begun in Phase 1 and expand to perform high throughput analysis of U1 Adaptors to establish site selection criteria and create an algorithm and design tool to assist with application of this technology to new gene targets with ease. We further propose to test the specificity of the method using whole genome microarray analysis. We intend to focus on use of this technology in genes which appear to be difficult to suppress using RNAi methods. Finally, we propose to study use of U1 Adaptors in complex genes having more than one polyadenylation site or alternative splicing involving the terminal exon. 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 additive 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。从经典上讲，ASO被设计为干扰靶mRNA的翻译或通过RNase H诱导其降解。目前，更大的兴奋集中在RNAi上，它使用了一种独特的机制，其中寡核苷酸触发了内源性预先存在的基因抑制途径，该途径是细胞基因调节网络基础的基础。尽管取得了一般成功，但RNAi和其他mRNA仅由RNAi和其他mRNA被适度地下调（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（即3'Utrs）中的调节序列要素控制该基因的表达。在这里，我们介绍了1阶段SBIR的进度报告，涉及我们称为“ U1适配器”的新基因沉默技术的开发，该技术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。在此第2阶段的建议中，我们计划继续在第1阶段开始的优化工作，并扩展以对U1适配器进行高吞吐量分析，以建立站点选择标准并创建算法和设计工具，以帮助将该技术轻松地应用于新的基因目标。我们进一步建议使用整个基因组微阵列分析测试该方法的特异性。我们打算专注于在基因中使用该技术，这些基因似乎很难使用RNAi方法抑制。最后，我们建议研究在具有多个多腺苷酸化位点或涉及末端外显子的替代剪接的复杂基因中使用U1适配器的使用。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，因此当通过添加效应与它们结合使用时，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。