From Molecular Diagnostics to Therapeutics with Aptamers

从分子诊断到适体治疗

• 批准号: RGPIN-2015-05323
• 负责人: Berezovski, Maxim
• 金额: $ 2.55万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613485
• 关键词: Molecular; Diagnostics; Therapeutics; Aptamers

One of the most demanding and challenging tasks of bioanalytical chemistry and bioseparation is the replacement of animal- and hybridoma-made antibodies that have high production cost and short shelf life with artificial competitors that can be easily selected and chemically synthesized in high purity at low cost. Aptamers as “synthetic antibodies” have enormous potential. These small single-stranded DNA or RNA oligonucleotides can bind with high affinity and specificity to a variety of different targets ranging from small molecules to proteins. They are affinity ligands with significant potential to change the field of affinity probes and to replace antibodies as diagnostic and separation applications. I propose the development of three related research themes: 1) affinity purification of cells by switchable aptamers, 2) detection and imaging of rare cells by aptamers, and 3) discovery of rare cell biomarker by aptamers and mass spectrometry. Current antibody-based approaches for cell isolation and purification are not optimal because of the antibody's effect on cell viability and purity. In the first research theme, we will develop two types of DNA aptamers that bind specifically with high affinity to immune receptors and cancer stem cell receptors. The first type of aptamers will be EDTA-switchable DNA aptamers that bind to cells in the presence of Ca(II) or Mg(II) ions, and release the pure and intact cells upon addition of EDTA or EGTA in solution. The second type of aptamers will be “cold” switchable DNA aptamers that selectively bind to receptor positive cells at room temperature and release them at 0°C to yield aptamer-free cells. In the second theme, DNA aptamers selected for lung adenocarcinoma cells from postoperative tissues will be used for sensing and counting rare cancer cells (<100 cells/ml) in blood. The enumeration and molecular profiling of rare cells, such as cancer circulating tumour cells (CTCs) and cancer stem cell (CSCs) in biological fluids, is an emergent methodology for the detection and treatment of cancer. Such tumour specific aptamers can be produced for individual patients and synthesized many times during anticancer therapy, thereby opening up the possibility of personalized diagnostics. The third theme will use the findings of the first two technologies such as aptamers to rare cells, and a cell isolation procedure for identification of new protein biomarkers by mass spectrometry. The important feature of this theme is that it yields synthetic affinity probes (aptamers) simultaneously with biomarker discovery. The combined activities of this program over the next 5 years will make a significant contribution to the development of new in vitro evolution techniques for nucleic acid aptamers, and the design of new bioassays and biosensors.

生物分析化学和生物分离最苛刻和最具挑战性的任务之一是用易于选择并以低成本高纯度化学合成的人工竞争剂替代生产成本高和保质期短的动物和杂交瘤制造的抗体。适体作为“合成抗体”具有巨大的潜力。这些小的单链 DNA 或 RNA 寡核苷酸可以以高亲和力和特异性结合从小分子到蛋白质的各种不同靶标。它们是亲和配体，具有改变亲和探针领域并取代抗体作为诊断和分离应用的巨大潜力。我建议发展三个相关的研究主题：1）通过可切换适体对细胞进行亲和纯化，2）通过适体对稀有细胞进行检测和成像，3）通过适体和质谱法发现稀有细胞生物标志物。 由于抗体对细胞活力和纯度的影响，目前基于抗体的细胞分离和纯化方法并不是最佳的。在第一个研究主题中，我们将开发两种类型的DNA适体，它们能够以高亲和力与免疫受体和癌症干细胞受体特异性结合。第一种适体是 EDTA 可切换的 DNA 适体，在 Ca(II) 或 Mg(II) 离子存在的情况下与细胞结合，并在溶液中添加 EDTA 或 EGTA 后释放纯净且完整的细胞。第二种类型的适体是“冷”可切换DNA适体，它在室温下选择性地结合受体阳性细胞，并在0°C下释放它们以产生无适体的细胞。 在第二个主题中，为来自术后组织的肺腺癌细胞选择的DNA适体将用于感测和计数血液中的稀有癌细胞（<100个细胞/ml）。对生物体液中的癌症循环肿瘤细胞 (CTC) 和癌症干细胞 (CSC) 等稀有细胞进行计数和分子分析是一种新兴的癌症检测和治疗方法。这种肿瘤特异性适体可以为个体患者生产，并在抗癌治疗过程中多次合成，从而开启了个性化诊断的可能性。 第三个主题将利用前两项技术的发现，例如稀有细胞的适体，以及通过质谱法鉴定新蛋白质生物标志物的细胞分离程序。该主题的重要特征是它在发现生物标志物的同时产生合成亲和探针（适体）。 该计划未来5年的综合活动将为核酸适配体新的体外进化技术的开发以及新的生物测定和生物传感器的设计做出重大贡献。