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.