SBIR-STTR Award

?The objective of this application is to develop an accurate high-throughput genotoxicity screening system with high specificity and accuracy using our established and well-validated toxicogenomic biomarker in cultured human cells. Genotoxicity represented by chromosome damage and mutations in DNA is considered to be the hallmark of carcinogenic risk. The standard genotoxicity assays, especially in the case of in vitro chromosome aberration assays, have a high false positive rate which results in costly and time consuming follow up assays that increase the cost of drug development and chemical safety assessment. Hence, gaining insight into genotoxic mechanisms and distinguishing those "false" positive genotoxicity findings caused by nongenotoxic mechanisms is of great value, so a simple, reliable technology proposed here would be sought after by pharmaceutical and chemical companies. Our biomarker, TGx-28.65, is capable of recognizing incorrectly identified compounds. The specificity of genotoxicity prediction by TGx-28.65, the intra- and inter- laboratory. In the Phase I feasibility project reproducibility, and the reproducibility on differen technical platforms have been carefully validated by us and by a second laboratory in follow-up studies. Our TGx-28.65 biomarker recently was incorporated into the "1500 Genes" panel for the Tox21 Phase III high throughput transcriptomics project we propose to develop a commercially viable and efficient high-throughput genotoxicity screening system using TGx-28.65, which has shown remarkable specificity and robustness for genotoxicity prediction. Our technical approach will employ direct digital counting technology to achieve high levels of precision, linearity, and reproducibility in measuring the expression levels of 65 genes in TGx-28.65 simultaneously. The proposed approach will provide significant benefits in comparison to the current genotoxicity battery and is poised to be commercially successful.

Public Health Relevance Statement:


Public Health Relevance:
Genotoxicity testing is an essential component of the safety assessment paradigm required by regulatory agencies world-wide for drug candidates, industrial chemicals and environmental pollutants. However, the current genotoxicity testing battery features high incidence of false positive finding for in vitro chromosome damage assays, that provides a challenge to both industry and regulatory agencies. This proposal addresses the high Incidence of false positive findings by applying the genomic biomarker TGx-28.65, which was identified by the Fornace laboratory, and that is capable of identifying relevant genotoxic responses. The Phase I proposal will primarily be a feasibility and proof-of-principle project for developing a high-throughput TGx-28.65-based screening service and later a genotoxicity kit. The successful completion of this project will enable a broad application of the first toxicogenomics assay for genotoxicity, i.e. DNA damage, assessment in the pharmaceutical and chemical industry. Furthermore, the automated screening system is expected to improve drug discovery and risk assessment of industrial chemicals.

Project Terms:
abstracting; Address; adverse outcome; Ames Assay; Animal Testing; base; Biological Assay; Biological Markers; Cancer Etiology; cell bank; Cell Culture Techniques; Cells; Chemical Exposure; Chemical Industry; Chemicals; Chromosome abnormality; Chromosomes; cost; cost effective; Coupling; design; Detection; Development; digital; DNA; DNA Damage; drug candidate; drug development; drug discovery; Drug Industry; Economics; Environment; Environmental Pollutants; Event; Exposure to; Finding by Cause; follow-up; Future; Gene Expression; Genes; genomic biomarker; Genomics; genotoxicity; Health; Human; improved; In Vitro; in vitro Assay; in vivo; Incidence; Industry; insight; knowledge base; Laboratories; Lead; Mammalian Chromosomes; Measures; Metabolic Activation; Methods; Micronucleus Tests; Molecular Biology; Molecular Toxicology; Mutagenicity Tests; Mutation; Names; nano-string; novel; Pathway interactions; Performance; Pharmacologic Substance; Phase; Procedures; public health relevance; Rattus; Registries; Reproducibility; Research; response; Risk; Risk Assessment; Risk Management; RNA; Running; Safety; Salmonella; screening; Sensitivity and Specificity; Services; Specificity; System; Technology; Testing; Time; Toxic effect; Toxicogenetics; Toxicogenomics; Toxicology; Training; transcriptomics; Validation; Weight

Regulatory agencies including EPA, FDA, ECHA, and EMA require thorough assessment of the health effects of chemicals present in the environment and marketplace. Interpretation of positive genotoxicity findings using the current standard in vitro testing battery is a major challenge to both industry and regulatory agencies. These tests have high sensitivity, but suffer from low specificity, leading to high rates of irrelevant positive findings (i.e., positive results in vitro that are not relevant or reproduced in vivo); this leads to unnecessary and costly follow- up as well as exclusion of potentially beneficial agents from further development. We have developed an in vitro transcriptomic biomarker-based approach that provides pathway-based mechanistic context to positive genotoxicity assay data, particularly for in vitro chromosome damage assays that suffer from a high frequency of irrelevant (false positive) results. Our transcriptomic biomarker, TGx-28.65, readily distinguishes DNA damage-inducing agents from other agents with much higher accuracy. In Phase I we have successfully integrated TGx-28.65 with nCounter® technology, which is based on direct multiplexed measurement of gene expression. nCounter offers high levels of precision, linearity, reproducibility, and sensitivity (<1 copy per cell). These unique characteristics of nCounter make it an ideal technology platform for our biomarker-based genotoxicity screening assay. We now show that that our nCounter approach can be used directly with cell lysates and adapted to high-throughput screening (HTS); this addresses another critical need since the standard in vitro genotoxicity panel is typically not amenable to HTS. The objective of this application is to develop and commercialize this high-throughput genotoxicity screening system using our well-validated TGx- 28.65 toxicogenomic biomarker. We will further assess our approach with priority environmental agents using 200 chemicals from the Toxcast collection. While many aneugens are not DNA-damaging, they can also trigger chromosome aberrations and micronucleus formation, so there is a need to also assess for this property. We have already observed transcriptomic responses for a limited number of aneugens and will now assess with a larger panel of aneugens with known modes of action. The proposed approach will employ our standard human TK6 cell approach with or without metabolic activation, and will be extended to HepaRG 3D spheroid culture to more closely model in vivo exposure; primary cells, such as human hepatocytes, will also be employed using the conditionally-reprogramed-cell approach developed at Georgetown. Our proposed approach can be integrated into genetic safety hazard assessment as a follow-up to positive chromosome damage findings, as well as a stand-alone for in vitro genotoxicity assessment. Considering the high cost of animal testing and now an E.U. regulatory ban for some product applications, development of accurate and cost-effective in vitro approaches is critical. This proposal should significantly benefit safety assessment by providing highly specific genotoxicity HTS, and is poised to become a commercially successful screening service.

Thesaurus Terms:
Address; Aneugens; Animal Testing; Base; Biological; Biological Assay; Biological Markers; Biomarker Panel; Businesses; Cell Culture Techniques; Cells; Characteristics; Chemical Agents; Chemicals; Chromosome Abnormality; Chromosomes; Collection; Commerce; Complex; Computer Software; Cost; Cost Effective; Data; Data Analyses; Development; Digital; Dna Damage; Drug Candidate; Environment; Environmental Agent; Environmental Pollutants; Equipment And Supply Inventories; Evaluation; Exclusion; Exposed Human Population; Exposure To; Follow-Up; Frequencies; Gene Expression; Genetic; Genetic Transcription; Genomics; Genotoxicity; Hazard Assessment; Health; Health Assessment; Hepatocyte; High Throughput Screening; Human; In Vitro; In Vitro Assay; In Vitro Testing; In Vivo; In Vivo Model; Incidence; Industrialization; Industry; International; Laboratories; Manufacturer Name; Measurement; Measures; Metabolic Activation; Methods; Micronucleus; Modeling; Mutagenicity Tests; Mutagens; Nature; Notification; Novel; Pathway Interactions; Phase; Poisons; Preparation; Programs; Property; Prototype; Public Health; Reporting; Reproducibility; Research; Response; Risk; Risk Assessment; Rna; Safety; Screening; Screening Program; Services; Small Business Innovation Research Grant; Small Molecule Libraries; Specificity; System; Technology; Testing; Testing Services; Tissues; Toxic Effect; Toxicant; Toxicogenomics; Toxicology; Training; Transcript; Transcriptomics; Work;