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， 国外该疾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、种族甚至是 存在于肠腔中的细菌与肠道炎症的起因有关。虽然一些治疗 尽管取得了进展，但大量患者对现有药物干预无反应 使许多人终身患病。这为改进现有疗法提供了强有力的理由， 发现替代和新的方法来处理肠道炎症状况。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， 生理相关性，不具有免疫细胞区室，这是一个缺失的关键组成部分 以获得人类炎症状况的精确模型。由于这些原因，有必要在 体外肠平台的治疗市场，该平台准确地再现了管腔上皮细胞， 肠道免疫细胞轴。为了满足这一需求，Altis Biosystems Inc.，一个早期的生物技术 该公司与学术实验室的科学家合作，开发了一种新颖的，基于初级干细胞的， 体外肠模型（称为肠）。我们已经完成了SBIR第一阶段计划，重点是转基因 以及对在去肠上培养的肠干细胞（ISCs）进行基因编辑。我们开发了一种基因免疫系统， 产生分化的上皮细胞，并实时感知和报告肠道炎症的关键特征，1）屏障 2）NF-κ B激活。在这里，我们扩大范围，以生成一个第一种共培养模型，称为， InflammaGutTM. InflammaGut是一个三方和灵活的系统，它使用了<$Gut的基本技术， 叠加炎症报告基因和人上皮细胞与同种异体肠相关 淋巴细胞（GAL）重建LEI轴。设想了四种产品：1）InflammaGut：ZO 1和 InflammaGut：NF-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）可商业化的人肠道相关淋巴细胞，以及4）新合同 研究服务（CRS）使用InflammaGut。有很强的工程和生物方法创新， 包括，1）开发Transwell插入物，使得能够可扩展地培养分化的人肠上皮细胞， 上皮覆盖水凝胶包封的淋巴细胞隔室，和2）分离、培养和冷冻库 人体器官捐献者的GAL InflammaGut将由学术合作者构建，验证和测试 （肠道技术的创始人），Altis Biosystems和两个行业合作者。 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