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

战略优先领域：产业合作研究。关键词：药物发现，SARS-CoV2，呼吸道组织SARS-CoV2的大流行传播，新冠肺炎的主要病原体，已经确认迫切需要替代方法来快速筛选和鉴定限制人畜共患病病原体发病和传播的抗病毒化合物；尤其是在易患自身免疫和炎症性疾病的高危人群中。这一观察结果得到了疫苗生产和全球免疫的固有延迟的支持，这是在大流行爆发期间保护免疫幼稚人群所需的。识别小分子抑制物(SMI)的标准筛选方法严重依赖于基于二维(2D)细胞的感染分析。虽然这些2D系统适合高通量筛选，但它们不能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，需要广泛而耗时的二次验证分析。利用从健康供者获得的含有上皮细胞、成纤维细胞和杯状细胞的原代支气管细胞，我们已经展示了纤毛3D呼吸道上皮的分化，很容易支持SARS-CoV2感染。虽然适用于低通量的抑制剂研究和二次化验，但使用Transwell技术进行呼吸道细胞的分化是耗时的(至少4周)，劳动密集型，与高通量SMI筛查所需的自动化成像技术不兼容。因此，在新出现的病原体显示嗜好分化的呼吸道上皮的情况下，需要新的方法来促进直接在呼吸道组织中快速鉴定抗病毒化合物。3D生物打印的最新进展已经彻底改变了支持多种细胞类型分化的下一代生物分子支架的开发和大规模生产。该项目将建立在与Cellbricks的现有合作的基础上，一家中小型企业公司(https://cellbricks.com))，它利用专有的基于立体平版印刷的生物打印平台来生产支持细胞与3D生物聚合物基质的内部黏附的生物分子支架。与现有的Transwell或类有机物模型系统相比，这种支架的使用具有许多独特的优点：(1)可重复的细胞接种和下游感染动力学分析的内部尺寸受限；(2)用于促进细胞黏附和分化的内部3D微支架；(3)适用于高分辨率和高通量定量成像的光学清晰度；(4)由非感染的高风险供体组建立的直接在呼吸道组织中的药物分层。本项目的目的是建立这些预制的3D支架在高通量筛选、鉴定和分层中的使用，直接在呼吸道组织中对SARS-CoV2进行SMI化合物的筛选、鉴定和分层，从而避免在体内动物试验之前需要耗时的二次验证分析。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。该项目将开发适用于学术界和产业界的关键翻译研究技能，具有明确的研究目标和与全球健康、药物发现和精密医学相关的专业知识。该项目的成果将有助于开发与识别新出现的人畜共患病病原体相关的抗病毒化合物的新方法，这将支持其受训人员在学术界或产业界的职业发展。该项目的成果将有助于识别和分层治疗高危人群中感染SARS-CoV2的新冠肺炎患者的抗病毒抑制剂。

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.