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Principal Investigator: George, Jay
Institute Receiving Award Amelia Technologies, Llc
Location Washington, DC
Grant Number R44ES032522
Funding Organization National Institute of Environmental Health Sciences
Award Funding Period 12 Feb 2021 to 30 Jun 2025
DESCRIPTION (provided by applicant): Project Summary Current in vitro approaches for laboratory- and cell-based toxicology studies do not capture the interindividual variability in responses within the human population. Single nucleotide gene polymorphisms, gene heterozygosity, variations in gene expression and in some cases gene loss can yield highly variable responses to genotoxic compounds, ranging from hypersensitivity to complete resistance. Further, toxicological analysis based on model organisms such as bacteria, rats or mice do not adequately provide such response variability. A defined panel of human cells with appropriate genetic diversity, especially in genes and gene families that alter the response outcome to genotoxins, may begin to offer such toxicodynamic variability. Here, we propose to employ our Barcoded Exon Tagging And Gene (BETA-Gene) disruption platform to create barcoded control, heterozygous gene knockout (KO) and homozygous gene KO panels of diploid human cells for high-throughput, multiplexed genotoxin screens. The availability of panels of such cells would provide a level of genetic diversity currently unavailable for cyto-toxicological analysis. To address this significant need and in response to RFA-ES-20-008, we have outlined three specific aims. In Aim 1, we propose to develop a 99-cell panel of barcoded, human diploid RPE-1 cells engineered with a single or double allele gene disruption in genotoxin-response gene families: DNA damage response/repair, cell death and stress response. This approach, BETA-Gene disruption, utilizes the CRISPR/cas9 gene editing system for simultaneous exon deletion/disruption and gene-specific barcode tagging with preference for a single allele in diploid cells. This will then yield the development of a barcoded 48-cell line heterozygous gene KO panel, a barcoded 48-cell line homozygous gene KO panel and three barcoded, unmodified control cells amenable for multiplexed, cytotoxicity analysis. Goals of Aim 2 will include genetic validation and functional genotoxin-response testing of the RPE-1 BETA-Gene disrupted cell lines and in Aim 3, we will validate the barcoded, multiplex genotoxin screening platform to demonstrate the variability in response of the RPE-1 BETA-Gene disrupted heterozygous gene-KO and homozygous gene-KO cell line pools upon exposure to genotoxic and non-genotoxic compounds. This system will provide a rapid and high-throughput, barcode-based multiplex analysis of toxicodynamic variability coupled with mechanistic insight that contributes to the variability in genotoxin response.
Science Code(s)/Area of Science(s) Primary: 72 - Predictive Toxicology/Assay Development
Secondary: 03 - Carcinogenesis/Cell Transformation
Publications No publications associated with this grant
Program Officer Daniel Shaughnessy
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