A Novel Method for Efficiently and Robustly Retrieving Circulating miRNAs

一种高效、稳健地检索循环 miRNA 的新方法

• 批准号: 10013246
• 负责人: Qipan Deng
• 金额: $ 75万
• 依托单位: GLC BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-09 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10013246
• 关键词: Adsorption; Biological Assay; Biological Markers; Biological Models; Blood; Cardiovascular Diseases; Clinical; DNA; Detection; Development; Diagnosis; Disease; FDA approved; Foundations; Health; Human; Individual; Interruption; Malignant Neoplasms; Methods; MicroRNAs; Monitor; Nucleic Acids; Phase; Plasma; Protocols documentation; RNA; Sampling; Serum; Small Business Innovation Research Grant; Specimen; Surface; Technology; Testing; base; cancer biomarkers; circulating microRNA; clinical application; colorectal cancer screening; commercialization; cost; cost effective; disease diagnosis; improved; microRNA biomarkers; new technology; novel; success

Abstract This direct SBIR Phase II project aims to develop a novel technology and associated assays for efficiently and robustly extracting circulating miRNAs from blood (i.e. plasma or serum), which could transform the utility of miRNA testing in the diagnosis and monitoring of major diseases such as cancer and cardiovascular disease. Our competitive advantage lies in the ability to retrieve circulating miRNAs more efficiently and robustly than current extraction methods, which has been a critical barrier in implementing miRNA testing clinically. This advantage is achieved by implementing sequence specific capture in conjunction with a propriety method of making novel capture beads. Circulating miRNAs are potential disease biomarkers. However, to date, there are no FDA approved miRNA tests available. A bottleneck problem is unreliable circulating miRNA extraction. Current extraction methods are based on adsorption of polar molecules on polar surfaces, which were originally developed to extract large DNA and RNA molecules. However, because miRNAs are so small (~22nt), their interaction with polar surfaces is much weaker, therefore their adsorption on polar surfaces can be easily interrupted by other molecules present in sample. Since the weak adsorption of miRNAs on polar surfaces is an inherent problem that cannot be fully solved even if all other conditions were optimized, new methods for retrieving circulating miRNAs based on a different mechanism are clearly needed. Sequence-specific capture (SSC) is another method to extract nucleic acids, but historically it performs poorly when used to extract circulating nucleic acids from clinical samples due to the lack of effective capture beads. In addition, the cost of making capture beads by current methods is very high, making SSC too expensive for clinical use. Recently, we made a major breakthrough by developing a proprietary method of making capture beads that can transform SSC from a concept to a practical method for extracting circulating miRNAs in an efficient, robust, and cost-effective manner. Therefore, we employed our capture beads to develop our own SSC assays, and conducted a systematic study to examine the feasibility of using our SSC assay in extracting circulating miRNAs. Our SSC assay was indeed found to efficiently and robustly extract circulating miRNAs. In the study, we also found that extraction efficiency of current kits varied by as much as 60-fold by plasma sample, further confirming that current methods are not robust. Considering the problem of current methods, unparalleled features of our SSC technology, and success of our early study, we propose this Phase II SBIR project to further develop and validate our SSC technology for extracting circulating miRNAs from plasma/serum, providing a foundation for its commercialization.

摘要 这个直接SBIR第二阶段项目旨在开发一种新的技术和相关的测定方法， 从血液（即血浆或血清）中稳健地提取循环miRNA，这可以改变 miRNA检测在癌症和心血管疾病等重大疾病的诊断和监测中的应用。 我们的竞争优势在于能够更有效和稳健地回收循环miRNA， 目前的提取方法，这已经成为临床上实施miRNA检测的关键障碍。这 通过结合适当的方法实施序列特异性捕获， 制造新型捕获珠。 循环miRNA是潜在的疾病生物标志物。然而，迄今为止，没有FDA批准的miRNA 测试可用。一个瓶颈问题是不可靠的循环miRNA提取。目前的提取方法 基于极性分子在极性表面上的吸附，其最初被开发用于提取大的 DNA和RNA分子。然而，由于miRNAs是如此之小（~22 nt），它们与极性分子的相互作用， 表面是弱得多，因此它们在极性表面上的吸附可以很容易地被其他 样品中存在的分子。由于miRNAs在极性表面上的弱吸附是一个固有的问题， 即使优化所有其他条件也不能完全解决的问题， 显然需要基于不同机制的miRNA。序列特异性捕获（SSC）是另一种 方法提取核酸，但在历史上，当用于提取循环核酸时，其表现不佳 由于缺乏有效的捕获珠粒，此外，捕获的成本 通过目前的方法，微珠的SSC非常高，使得SSC对于临床使用来说太昂贵。 最近，我们通过开发一种制造捕获珠的专有方法取得了重大突破， 可以将SSC从概念转变为实用的方法，用于以高效，稳健， 成本效益的方式。因此，我们采用捕获珠来开发我们自己的SSC测定， 进行了系统的研究，以检查使用我们的SSC测定在提取循环的可行性 miRNAs。我们的SSC测定确实被发现有效且稳健地提取循环miRNA。在这项研究中， 我们还发现，当前试剂盒的提取效率因血浆样品而变化多达60倍， 这证实了目前的方法并不可靠。 考虑到当前方法的问题，我们SSC技术无与伦比的特点，以及我们的成功， 在早期研究中，我们提出了第二阶段SBIR项目，以进一步开发和验证我们的SSC技术， 从血浆/血清中提取循环miRNA，为其商业化提供基础。