Accelerating SARS-CoV2 antiviral drug discovery using next generation 3D bioprinted scaffolds

战略重点领域：产业协同研究。SARS-CoV2是COVID-19的主要病原，其大流行的传播已经确定了迫切需要替代方法来快速筛选和鉴定抗病毒化合物，以限制人畜共患病原体的发病和传播；特别是在易患自身免疫性和炎症性疾病的高危人群中。在大流行爆发期间保护immuno-naïve人口所需的疫苗生产和全球免疫的固有延迟支持了这一观察结果。鉴别小分子抑制剂（SMIs）的标准筛选方法严重依赖于二维（2D）细胞感染试验。虽然适用于高通量筛选，但这些2D系统难以反映呼吸道的三维生理、混合细胞类型群体或药代动力学特性。因此，在SMI临床前动物试验之前，需要进行广泛且耗时的二次验证分析。利用从健康供体获得的原代支气管细胞，其中包含上皮细胞、成纤维细胞和杯状细胞的混合物，我们已经证明了纤毛三维呼吸道上皮的分化，可以很容易地支持SARS-CoV2感染。虽然适用于低通量抑制剂研究和二次分析测试，但使用Transwell技术进行呼吸细胞分化耗时（至少4周），劳动密集型，并且与高通量SMI筛选所需的自动化成像技术不兼容。因此，在新出现的病原体表现出分化呼吸道上皮的倾向的情况下，需要新的方法来促进直接在呼吸组织内快速鉴定抗病毒化合物。生物3D打印的最新进展彻底改变了“下一代”生物分子支架的开发和大规模生产，这些支架可以支持多种细胞类型的分化。该项目将建立在与Cellbricks的现有合作基础上，Cellbricks是一家中小企业公司（https://cellbricks.com），该公司利用专有的基于立体光刻的生物打印平台来生产生物分子支架，支持细胞在3D生物聚合物基质上的内部粘附。与现有的Transwell或类器官模型系统相比，使用这种支架具有许多明显的优势：(1)可重复的细胞播种和下游感染动力学分析的内部尺寸受限；(2)内部三维微凹槽，促进细胞粘附和分化；(3)光学清晰度适合高分辨率、高通量定量成像；(4)直接在非感染高危供体人群呼吸道组织中进行药物分层。该项目的目标是建立这些预制3D支架在呼吸组织内直接对SARS-CoV2的SMI化合物进行高通量筛选、鉴定和分层的使用；从而避免了在动物体内试验之前进行耗时的二次验证分析的需要。学员和项目成果：该项目将提供传染病研究（MRC-UoG CVR）和生物分子工程（Cellbricks）方面的广泛培训。该项目将培养适用于学术界和工业界的关键转化研究技能，具有明确的研究目标和与全球健康，药物发现和精准医学相关的专业知识。该项目的产出将有助于开发与鉴定新出现的人畜共患病原体的抗病毒化合物有关的新方法，这将支持其受训人员在学术界或工业界的职业发展。本项目的成果将有助于识别和分层抗病毒抑制剂，用于治疗高危人群感染SARS-CoV2的COVID-19患者。

Strategic priority area:Industrial collaborative research. Keywords:Drug discovery, SARS-CoV2, respiratory tissueThe pandemic spread of SARS-CoV2, the principle etiological agent of COVID-19, has identified an urgent need for alternative methodologies for the rapid screening & identification of antiviral compounds that limit the pathogenesis & transmission of zoonotic pathogens;specifically in high-risk category groups susceptible to auto-immune and inflammatory disease.This observation is supported by the inherent delays in vaccine production & global immunization required to protect an immuno-naïve population during a pandemic outbreak.Standard screening approaches for the identification of small molecule inhibitors (SMIs) have relied heavily on two-dimensional (2D) cell-based infection assays.While amenable to high-throughput screening, these 2D systems poorly reflect the three-dimensional physiology,mixed cell-type population,or pharmacokinetic properties of the respiratory airway.Consequently, extensive & time-consuming secondary validation assays are required prior to SMI pre-clinical animal testing.Utilizing primary bronchial cells obtained from healthy donors,which contain a mixture of epithelial, fibroblast & goblet cells,we have shown the differentiation of ciliated 3D respiratory epithelium to readily support SARS-CoV2 infection.While amenable to low-throughput inhibitor studies & secondary assay testing,the use of Transwell technology for the differentiation of respiratory cells is time consuming (minimum 4 weeks),labour intensive, & incompatible with automated imaging techniques required for high-throughput SMI screening.Thus, in the case of emerging pathogens that show tropism for differentiated respiratory epithelia new methodologies are required to facilitate the rapid identification of antiviral compounds directly within respiratory tissue.Recent advances in 3D bioprinting have revolutionized the development & mass production of 'next generation' biomolecular scaffolds that can support the differentiation of multiple cell-types.This project will build on an existing collaboration with Cellbricks, a SME company (https://cellbricks.com) which utilizes a proprietary stereolithography-based bioprinting platform to produce biomolecular scaffolds that support the internal adhesion of cells onto a 3D biopolymer matrix.The use of such scaffolds has many distinct advantages over that of existing Transwell or organoid model systems: (1) Constrained internal dimensions for reproducible cell seeding and downstream infection kinetic assays; (2) Internal 3D micro-bays to promote cell adhesion and differentiation; (3) Optical clarity suitable to high-resolution and high-throughput quantitative imaging; (4) stratification of drugs directly in respiratory tissue established from non-infected high-risk donor groups.The objective of this project is to establish the use of these prefabricated 3D scaffolds in the high-throughput screening,identification & stratification of SMI compounds to SARS-CoV2 directly within respiratory tissue;thereby circumventing the need for time consuming secondary validation assays prior to animal testing in vivo. Trainee and project outcomes:This project will provide extensive training in infectious disease research (MRC-UoG CVR) & biomolecular engineering (Cellbricks).This project will develop key translational research skills applicable to both academia & industry,with clearly defined research aims & expertise pertinent to global health,drug discovery, and precision medicine.Outputs from this project will aid the development of novel methodologies relevant to the identification of antiviral compounds to emerging zoonotic pathogens that will support the career progression of its trainee in academia or industry. Outputs from this project will facilitate the identification and stratification of antiviral inhibitors in the treatment of SARS-CoV2 infected COVID-19 patients from high risk groups.