Genetically modified tissue engineered in vitro human models

描述(申请人提供)：当前提案的目标是应用最先进的基因修饰技术，包括慢病毒递送系统和RNA干扰(RNAi)技术，以生产具有先进功能特征的商业可用的体外组织工程人体模型。将开发的商业产品将包括有机类型的3-D皮肤和呼吸道上皮模型，这些模型将用于消费品/药物开发、免疫学和基础研究应用中的毒理学测试。将被纳入到体外转基因组织工程人体模型中的先进特征将包括：1)基因活性报告功能，用于监测具有毒理学、免疫学或发育意义的各种CEL信号通路的活性；以及2)体外转基因组织工程人体模型，具有特定遗传功能的敲除或过度表达。在一期工程期间，生产的可行性 这些模型是通过建立具有报告功能的器官型皮肤和呼吸道模型来建立的，以监测NF-B和Nrf2转录因子(TF)的活性。这些转录因子分别参与炎症反应和抗氧化反应的信号通路。用绿色荧光蛋白(GFP)荧光显微镜对报告基因的诱导活性进行定性评价，通过测定组织提取液中荧光素酶的活性进行定量评价。基因敲除模型的开发也取得了重大进展。对表皮细胞进行了基因改造，使其能够诱导白介素1、白介素1的基因敲除，白介素1是一种在上皮炎症、伤口愈合和环境因素诱导的疾病状态中发挥重要作用的细胞因子。IL-1基因和蛋白表达下调达72%。将这些细胞整合到器官模型中，进一步的功能测试正在进行中。在第二阶段项目期间，将生产一个由14个器官型皮肤和呼吸道上皮TF报告模型组成的小组，用于监测具有毒理学、免疫学或发育意义的细胞信号通路的活性。这些模型将被组装成商业产品，包括各种组合和配置，包括高吞吐量96孔格式，并用一套全面的参考化学品进行验证。这些模型将允许对工业化学品、消费品成分或环境制剂进行机械式毒理学评估。这些商业模型旨在供化学品生产商、消费品和化妆品制造商、制药公司以及工业、政府和学术环境毒理学家和制药研究人员使用。 与公众健康相关：该项目制作的器官型体外人体模型将允许对工业化学品、消费品成分或环境制剂进行机械性毒理学评估，以保护公众健康。这些商业模型旨在供化学品生产商、消费品和化妆品制造商、制药公司以及工业、政府和学术环境毒理学家和制药研究人员使用。

DESCRIPTION (provided by applicant): The goal of the current proposal is to apply state-of-the-art gene modification technology, including lentiviral delivery systems and RNA interference (RNAi) technology, to produce commercially available tissue engineered in vitro human models with advanced functional features. The commercial products to be developed will consist of organotypic 3-D skin and airway epithelial models that will be utilized for toxicology testing in consumer product/drug development, immunology and basic research applications. Advanced features that will be incorporated into the genetically modified tissue engineered in vitro human models will include: 1) gene activity reporter functions for monitoring the activity of various cel signaling pathways of toxicological, immunological or developmental significance; and 2) genetically modified tissue engineered in vitro human models with knockdown or over-expression of specific genetic functions. During the Phase I project, the feasibility of producing these models was established by development of organotypic skin and airway models with reporter functions for monitoring activity of NF B and Nrf2 transcription factors (TFs). These TFs are involved in inflammatory and antioxidant response signaling pathways, respectively. Induction of reporter activity was evaluated qualitatively by epifluorescence microscopy of green fluorescent protein (GFP), and quantitatively by measurement of luciferase activity in tissue extracts. Significant progress was also made in development of gene knockdown models. Epidermal cells were genetically modified to allow inducible knockdown of interleukin-1 IL-1 , a cytokine that plays an important role in epithelial inflammation, wound healing and disease states induced by environmental agents. Knockdown of IL-1 gene and protein expression of 72 % was achieved. Incorporation of these cells into organotypic models and further functional testing is in progress. A panel of 14 organotypic skin and airway epithelial TF reporter models for monitoring activity of cell signaling pathways of toxicological, immunological or developmental significance will be produced during the Phase II project. The models will be assembled into commercial products consisting of various combinations and configurations including high throughput 96-well formats, and validated with a comprehensive set of reference chemicals. These models will allow mechanistic toxicological evaluation of industrial chemicals, consumer product ingredients or environmental agents. The commercial models are intended for use by chemical producers, consumer product and cosmetic manufacturers, pharmaceutical companies, as well as industrial, governmental and academic environmental toxicologists and pharmaceutical researchers. PUBLIC HEALTH RELEVANCE: The organotypic in vitro human models produced by the project will allow mechanistic toxicological evaluation of industrial chemicals, consumer product ingredients or environmental agents for the protection of public health. These commercial models are intended for use by chemical producers, consumer product and cosmetic manufacturers, pharmaceutical companies, as well as industrial, governmental and academic environmental toxicologists and pharmaceutical researchers.