InflammaGut: a drug-screenable co-culture system using gut-associated lymphocytes and autologous primary human gut epithelium that reports inflammation

项目摘要 广泛的炎症性胃肠道疾病影响着美国数百万人和 在国外。该病的病因是多因素和异质性的，但共同的潜在特征是 过度活跃的免疫系统。遗传，环境因素，年龄，性别，种族，甚至 肠腔中的细菌与肠道炎症的原因有关。虽然有些治疗方法 已经取得了进展，大量患者对现有的药物干预没有反应 使许多人患上终生疾病。这为改进现有疗法提供了强有力的理由和 发现替代的和新的方法来处理肠道的炎症状况。临床前模型 用于药物研究和发现的药物在历史上一直很差。它们是由癌细胞组成的，这些癌细胞具有低 与生理相关，并且不具有免疫细胞隔间，而免疫细胞隔间是缺失的关键组成部分 以实现对人体炎症状况的准确模型。出于这些原因，有必要在 治疗市场的体外肠道平台，准确地重述腔上皮- 肠道的免疫细胞轴。为了满足这一需求，早期生物技术公司Altis BiosSystems Inc. 公司与学术实验室的科学家合作，开发了一种基于原代干细胞的新型 体外肠道模型(RepliGut)。我们已经完成了以转基因为重点的SBIR第一阶段计划 以及在RepliGut上培养的肠道干细胞(ISCs)的基因编辑。我们开发了报告基因ISCs 产生分化上皮并实时检测和报告肠道炎症的关键特征，1)屏障 功能；2)核因子-kB活化。在这里，我们扩展范围以产生一个首创的共同文化模型，称为， InflammaGutTM.InflammaGut是一个三方灵活的系统，它使用了RepliGut和 炎症报告基因叠加及人上皮与异体肠相关细胞的共培养 淋巴细胞(GAL)重建LEI轴。设想了四种产品：1)InflammaGut：ZO1和 InflammaGut：核因子-kB报告基因上皮，2)InflammaGut：共培养：上皮/肠道相关淋巴细胞 (GAL)共培养系统，3)可商业化的人类肠道相关淋巴细胞，以及4)新合同 使用InflammaGut的研究服务(CRS)。有强大的工程和生物方法创新， 包括，1)开发Transwell插入物，使RepliGut分化的人能够进行可扩展的培养 水凝胶包裹的淋巴细胞室上皮，以及2)分离、培养和冷冻库 来自人体器官捐赠者的半乳糖。InflammaGut将由学术合作者构建、验证和测试 (RepliGut技术的创始人)、阿尔蒂斯生物系统公司和两家行业合作者。 1

Project Summary A broad range of inflammatory gastrointestinal conditions impact millions of people in the United States and abroad. The etiology of the disease is multifactorial and heterogeneous, but the common underlying feature is an overactive immune system. Genetics, environmental factors, age, sex, race and even the composition of bacteria that reside in the gut lumen are associated with causation of gut inflammation. While some therapeutic advances have been made, a large number of patients do not respond to existing pharmaceutical interventions leaving many to have life-long morbidity. This provides strong rationale to improve existing therapies and discover alternative and novel approaches to deal with inflammatory conditions of the gut. Preclinical models used for drug research and discovery have been historically poor. They are comprised cancer cells that have low physiological relevance and do not possess the immune cell compartment, which is a missing pivotal component to achieve an accurate model of human inflammatory conditions. For these reasons, there is a need in the therapeutics marketplace for an in vitro intestinal platform that accurately recapitulates luminal-epithelial- immune cell axis of the intestines. To meet this need, Altis Biosystems Inc., an early-stage biotechnology company, has collaborated with scientists at academic laboratories to develop a novel, primary-stem cells-based, in vitro intestinal model (termed RepliGut). We have finished an SBIR Phase I program focused on transgenesis and gene editing of intestinal stem cells (ISCs) cultured on RepliGut. We developed reporter-gene ISCs that generate differentiated epithelium and sense and report in real-time key features of gut inflammation, 1) barrier function, and 2) NF-kB activation. Here we expand the scope to generate a first-in-kind co-culture model called, InflammaGutTM. InflammaGut is a tripartite and flexible system that uses fundamental technology of RepliGut and superimposes inflammation reporter genes and co-culture of human epithelium with allogeneic gut-associated lymphocytes (GAL) to recreate the LEI-axis. Four products are envisioned: 1) InflammaGut:ZO1 and InflammaGut:NF-kB reporter gene epithelium, 2) InflammaGut:Co-Culture: an epithelial/gut-associate lymphocyte (GAL) co-culture system, 3) commercializable human gut-associated lymphocytes, and 4) a new contract research service (CRS) using InflammaGut. There is strong engineering and biological method innovation, including, 1) development of Transwell insert enabling scalable culture of RepliGut differentiated human epithelium over a hydrogel encapsulated lymphoid-cell compartment, and 2) isolation, culture, and cryobanking of GAL from human organ donors. InflammaGut will be built, validated, and tested by academic collaborators (founders of the RepliGut technology), Altis Biosystems, and two industry collaborators. 1