Genetically modified tissue engineered in vitro human models

描述（由申请人提供）：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，以生产具有高级功能特征的市售组织工程体外人体模型。将开发的商业产品将包括器官型3-D皮肤和气道上皮模型，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。将被纳入遗传修饰的组织工程化体外人模型的先进特征将包括：1）用于监测毒理学、免疫学或发育意义的各种细胞信号传导途径的活性的基因活性报告功能;和2）具有特定遗传功能的敲低或过表达的遗传修饰的组织工程化体外人模型。 在一期工程中， 这些模型是通过开发具有用于监测NF B和Nrf 2转录因子（TF）活性的报告功能的器官型皮肤和气道模型来建立的。这些TF分别参与炎症和抗氧化反应信号通路。通过绿色荧光蛋白（GFP）的落射荧光显微镜定性评价报告活性的诱导，并通过测量组织提取物中的荧光素酶活性定量评价报告活性的诱导。在基因敲除模型的开发方面也取得了重大进展。对表皮细胞进行遗传修饰以允许诱导性敲低白细胞介素-1 IL-1，这是一种在环境因子诱导的上皮炎症、伤口愈合和疾病状态中起重要作用的细胞因子。IL-1基因的敲除和蛋白表达达到72%。将这些细胞纳入器官型模型和进一步的功能测试正在进行中。 在第二阶段项目期间，将产生一组14个器官型皮肤和气道上皮TF报告模型，用于监测毒理学，免疫学或发育意义的细胞信号传导途径的活性。这些模型将被组装成商业产品，由各种组合和配置组成，包括高通量96孔格式，并使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、消费品成分或环境因子进行机械毒理学评估。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.