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

• 批准号: 10287842
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 23.18万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-12 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10287842
• 关键词: 2019-nCoV; ACE2; Affinity; Alveolar; Antibodies; Antiviral Agents; Aptamer Technology; Base Sequence; Binding; Biochemical; Biological Assay; Biosensor; COVID-19; COVID-19 therapeutics; COVID-19 treatment; COVID-19 vaccine; Cell Culture Techniques; Cells; Cessation of life; Chemical Structure; Clinical; Collaborations; Containment; Coronavirus; DNA; Development; Diagnostic; Disease; Drug Screening; Enzyme-Linked Immunosorbent Assay; Evaluation; Evolution; FDA Emergency Use Authorization; FDA approved; Fatality rate; Feedback; Future; Generations; Goals; Human; In Vitro; Infection; Infection prevention; Letters; Ligands; Link; Membrane Proteins; Middle East Respiratory Syndrome; Modeling; Modernization; Modification; Molecular Biology; Morbidity - disease rate; Mucous Membrane; Nucleic Acids; Nucleotides; Outcome; Pharmacotherapy; Phase III Clinical Trials; Physiological; Prevention; Property; Proteins; Public Health; RNA; Race; Resistance; Respiratory Failure; Respiratory System; Ribonucleases; SARS coronavirus; Societies; Surface; System; Testing; Therapeutic; Toxic effect; United States; Upper respiratory tract; Vaccines; Vertebral column; Viral; Viral Pneumonia; Virus; Virus Diseases; Virus Inhibitors; Work; airway epithelium; alveolar type II cell; aptamer; base; human model; human pathogen; immunogenicity; improved; in vitro Model; in vivo; innovation; inorganic phosphate; mortality; new therapeutic target; novel; novel therapeutics; nucleotide analog; pandemic disease; particle; rapid test; receptor binding; remdesivir; respiratory virus; screening; small molecule; sugar; technology development; therapeutic target; tool; uptake; vaccine candidate; virus host interaction

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过分。截至9月 2020年，全球有33，224，222例病例和999，298例死亡与这种新出现的病毒有关。这 惊人的数字继续增长，美国的发病率不成比例地高 and mortality.该病毒以呼吸道为目标，导致广泛的临床结果，包括轻度 上呼吸道疾病和严重病毒性肺炎伴呼吸衰竭。迄今为止， 候选疫苗已进入3期临床试验，同时也在进行大规模的努力， 重新利用已经FDA批准的药物治疗COVID-19或识别具有潜在治疗作用的化合物 治疗活性。尽管做出了这些努力，但Remdesivir仍然是唯一获得批准的（紧急使用授权） 用于治疗COVID-19的直接抗病毒药物。重要的是：目前没有疫苗或 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耐药SARS-CoV-2菌株的迅速出现。因此，发展 因此，迫切需要针对SARS-CoV-2的新疗法。感染需要病毒和病毒之间的相互作用 表面蛋白，刺突（S）和宿主蛋白，ACE 2，在II型肺泡细胞和纤毛细胞上表达 在人气道上皮细胞（HAE）中，使这些细胞潜在地易受感染。因此，我们的目标是 开发一种新的治疗方法，阻断刺突（病毒上）和ACE 2（宿主细胞上）之间的相互作用 以预防感染和改善疾病。适体是短的基于核酸的序列，其与 对它们的目标有很高的亲和力。在其他应用中，适体已被证明具有有效的抗病毒活性 并且在细胞培养中毒性低。虽然适体最初是由RNA和DNA制成的，但异种核酸 (XNA：具有改变的糖、碱基或磷酸骨架的核苷酸类似物）已经成为重要的新的 与RNA底物和XNA适体相比， 和DNA适体。因此，我们假设，适体技术，特别是XNA适体，可以被应用于临床。 利用抑制加标物-ACE 2相互作用，并建议建立一个创新的体外筛选平台 其可用于评估此类适体或其它新型治疗剂在阻断感染中的功效。这 该平台将利用SARS-CoV-2伪粒子（允许在生物安全2级控制下工作）和 人气道上皮的生理学相关体外模型，其重现了气道上皮的粘膜表面， 体内气道。还将使用培养细胞的活病毒感染检测适体（生物安全等级3）。这 考虑到对新的抗SARS-CoV-2疗法的迫切需要，这项工作是非常重要的。此夕h 开发一种高通量、基于假粒子的检测方法，以评估相关培养系统中的病毒进入 将广泛应用于其他药物筛选和/或旨在进一步了解SARSCoV的研究- 2病毒-宿主相互作用在颗粒摄取水平。