This Small Business Innovation Research (SBIR) Phase II project proposes to develop a high-throughput assay incorporating 3-D skin models capable of accurately identifying and characterizing DNA damage. There is an urgent need for improved genotoxicity assays for safety screening in drug development. The process by which drug compounds are usually screened is expensive, time-consuming, and often does not provide an accurate depiction of in vivo behavior. Phase I of this project developed a full-thickness skin model that can be used in a range of toxicological assays. Phase II will address the limitations of current genotoxicity assays by incorporating fluorescent reporter constructs into the 96-well skin model to create an assay that is high-throughput and accurately distinguishes between classes of genotoxins.
The broader impacts of this research are to develop a genotoxicity screen that is more informative, accurate, and high-throughput than existing alternatives. Development of accurate in vitro assays not only reduces the need for animal testing, but can also reduce the risk to patients included in clinical trials by providing better predictions of the human response. Toxicity has become one of the leading reasons for product failure during drug development. The ability for this assay to identify and eliminate harmful compounds earlier in the development process could significantly reduce the costs and accelerate the timeline of drug development. In addition to these direct contributions for drug and chemical screening, the mechanistic data provided by this assay will provide a valuable tool for basic science research into DNA damage.
This Small Business Innovation Research Phase II project proposed to build upon the 96-well skin tissue platform technology developed in our Phase I project by incorporating fluorescent reporter constructs to allow for high-throughput screening of genotoxic potential. There is an urgent need for improved genotoxicity assays for safety screening in drug development. The process by which drug compounds are usually screened, characterized, and approved is expensive, time- consuming, and typically does not recapitulate the tissue architecture that regulates in vivo behavior. Phase I work developed a full-thickness skin model that can be used in a range of dermal irritancy and drug permeability assays. This Phase II project focused on development of a complete assay system that would: 1) be high-throughput, 2) distinguish between classes of genotoxins, 3) use a metabolically- competent, p53-wildtype human cell line, and 4) be made in a three-dimensional tissue format that more accurately models the in vivo response thus addressing the limitations of existing assays. During the course of this project, Stratatech made significant progress on the specific aims and milestones defined in the proposal. The project successfully generated multiple fluorescent keratinocyte clones driven by putative genotoxin-inducible regulatory elements. Each of the clones demonstrated the ability to generate 96-well skin models and express the selected fluorescent protein. Stratatech also developed an automated microscopy-based system to sensitively quantify fluorescent signal in full-thickness 96-well skin models. Development of that detection system was essential to making an efficient assay readout that would enable screening of large numbers of conditions. Development of specific assay parameters and conditions for screening of genotoxin-inducible chemicals is ongoing. When complete, this high-throughput assay will provide a greatly-needed tool for determining genotoxic potential. Importantly, this work also showed proof-of-concept for the development of a high-throughput, tissue-based, assay system. Three-dimensional tissue models have been shown to more accurately predict the in vivo response than traditional mammalian monolayer or bacterial cell-based assays. Although it was applied to detection of DNA damage in the current project, the various elements of the developed system could also be translated to other assays of interest. These results position Stratatech to develop future assays using this same platform technology.
