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Development and Evaluation of Novel Aptamer-based Therapeutics Targeting SARS-CoV-2 in a Physiologically-Relevant Model of Human Airway Epithelium

在人类气道上皮生理相关模型中针对 SARS-CoV-2 的新型适体疗法的开发和评估

基本信息

批准号：
10449392
负责人：
JEFFREY J DESTEFANO
金额：
$ 19.31万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-12 至 2024-06-30
项目状态：
已结题

项目摘要

The impact of SARS-CoV-2 on public health and the global economy cannot be overstated. As of September 28, 2020, 33,224,222 cases and 999,298 deaths worldwide have been linked to this emergent virus. This staggering number continues to grow, with the United States baring disproportionately high rates of morbidity and mortality. The virus targets the respiratory tract, leading to a wide range of clinical outcomes including mild upper respiratory tract illness and severe viral pneumonia with respiratory failure. To date, four SARS-CoV-2 vaccine candidates have entered phase 3 clinical trials and a massive parallel effort has been undertaken to repurpose already FDA-approved drugs for the treatment of COVID-19 or identify compounds with potential therapeutic activity. Despite this effort, remdesivir remains the only approved (with emergency use authorization) direct-acting antiviral for the treatment of COVID-19. Of critical importance: there is currently no vaccine or SARS-CoV-2-specific therapy approved for the prevention or treatment of disease. Furthermore, multiple antivirals may be required to avoid the rapid emergence of resistant SARS-CoV-2 strains. Thus, the development of novel therapeutics targeting SARS-CoV-2 are urgently needed. Infection requires interaction between the viral surface protein, spike (S), and a host protein, ACE2, that is expressed on type II alveolar cells and ciliated cells in the human airway epithelium (HAE), making these cells potentially vulnerable to infection. Thus, our goal is to develop a novel therapeutic that blocks this interaction between spike (on the virus) and ACE2 (on the host cell) to prevent infection and ameliorate disease. Aptamers are short nucleic acid-based sequences that bind with high affinity to their targets. Among other applications, aptamers have been shown to have potent antiviral activity and low toxicity in cell culture. While aptamers were originally made with RNA and DNA, Xeno-Nucleic Acids (XNA: nucleotide analogs with altered sugar, base, or phosphate backbones) have emerged as important new substrates and XNA aptamers often demonstrate enhanced target binding and greater stability compared to RNA and DNA aptamers. Thus, we hypothesize that aptamer technology, and specifically XNA aptamers, can be leveraged to inhibit spike-ACE2 interaction and propose to establish an innovative, in vitro screening platform that can serve to assess the efficacy of such aptamers, or other novel therapeutics, in blocking infection. This platform will utilize SARS-CoV-2 pseudoparticles (allowing work under Biosafety Level 2 containment) and a physiologically-relevant in vitro model of human airway epithelium that recapitulates the mucosal surface of the airway in vivo. Aptamers will also be tested using live virus infections of culture cells (Biosafety Level 3). This work is highly significant given the immediate need for novel therapeutics against SARS-CoV-2. Further, the development of a high-throughput, pseudoparticle-based assay to assess viral entry in a relevant culture system will have broad applications for additional drug screens and / or studies that aim to further understand SARSCoV- 2 virus-host interactions at the level of particle uptake.

SARS-CoV-2对公共卫生和全球经济的影响怎么强调都不为过。截至9月 2020年，全世界已有33,224,222例病例和999,298例死亡与这种紧急病毒有关。这令人震惊的数字继续增长，美国的发病率高得不成比例和死亡率。该病毒以呼吸道为目标，导致广泛的临床结果，包括轻微的上呼吸道疾病和严重病毒性肺炎合并呼吸衰竭。到目前为止，有四例SARS-CoV-2 候选疫苗已经进入第三阶段临床试验，并进行了大规模的平行努力，以调整已获美国食品和药物管理局批准的治疗新冠肺炎药物的用途或确定有潜力的化合物治疗活动。尽管做出了这样的努力，redesivir仍然是唯一获得批准(获得紧急使用授权)的药物。直接作用的抗病毒药物治疗新冠肺炎。至关重要的是：目前还没有疫苗或被批准用于预防或治疗疾病的SARS-CoV-2特异性疗法。此外，多个可能需要抗病毒药物以避免抗药性SARS-CoV-2毒株的迅速出现。因此，发展迫切需要针对SARS-CoV-2的新的治疗方法。感染需要病毒之间的相互作用表面蛋白，尖峰蛋白(S)和宿主蛋白，ACE2，表达在II型肺泡细胞和纤毛细胞上在人类呼吸道上皮(HAE)中，使这些细胞可能容易受到感染。因此，我们的目标是开发一种新的治疗方法，阻断(病毒上的)Spike和(宿主细胞上的)ACE2之间的这种相互作用预防感染，改善疾病。适配子是基于核酸的短序列，与对目标具有很高的亲和力。在其他应用中，适体已被证明具有强大的抗病毒活性在细胞培养中毒性低。虽然适配子最初是由RNA和DNA制成的，但异源核酸 (XNA：具有改变的糖、碱基或磷酸骨架的核苷酸类似物)已成为重要的新技术与RNA相比，底物和XNA适配子通常表现出更强的靶向结合和更高的稳定性和DNA适配子。因此，我们假设适配子技术，特别是XNA适配子，可以是利用来抑制Spike-ACE2的相互作用，并提议建立一个创新的体外筛选平台这可以用来评估这种适配子或其他新疗法在阻断感染方面的有效性。这平台将使用SARS-CoV-2伪劣物品(允许在生物安全二级控制下工作)和与生理相关的人呼吸道上皮细胞的体外模型，该模型重塑了呼吸道粘膜表面活体内的呼吸道。适体也将使用培养细胞的活病毒感染进行测试(生物安全3级)。这鉴于对抗SARS-CoV-2的新疗法的迫切需要，这项工作具有非常重要的意义。此外，建立一种高通量、基于伪粒子的检测方法以评估病毒在相关培养系统中的侵入将在更多的药物筛选和/或旨在进一步了解SARSCoV- 2病毒与宿主在颗粒摄取水平上的相互作用。