Research Project
10112083
Summary: This Phase I project will demonstrate the feasibility for assessing the impact of genetic diversity on exposure and inferences to the corresponding risk of toxicity across the human population by generating dose response data for each test agent using the TempO-Seq gene expression assay for a panel of cells, each with different pharmacogenomic gene (PGx) variant activities. As many as 10% to 20% of the population can express a genetic variant, such as deficiency in CYP2D6, CYP2C19, or CYP2C9, but significant numbers of persons have functionally different activities resulting from genomic variants of many other pharmacogenomic genes such as in ABC transporters, UGT transferases, and the ligand binding domains of PXR, AHR, and CAR. The human-specific S1500v2 whole transcriptome surrogate TempO-Seq gene expression assay will be used to profile HepaRG knockouts reflecting functional variation in activity of PGx gene products and 3D HEPATOPAC co-cultures of primary human hepatocytes (PHH) from donors expressing functional variant activity. The HepaRG cells will be grown and profiled in the proliferative and differentiated states. The HEPATOPAC PHH (co-cultures with mouse 3T3 ?stromal? cells) will also be profiled using the mouse S1500 assay to measure the stromal response. Dose response data for test agents (a set of reference compounds in Phase I), will be obtained after a 96-hr treatment, from which compound-specific signatures, mode of action and toxicity pathways will be identified using DESeq and pathway analysis. BMDExpress will be used to determine the benchmark concentration (BMC) for each modulated pathway and gene. Differences between variants in compound signatures and gene and pathway BMCs will be identified and used to define the impact variant functional activity has on in vitro exposure and toxicity. These data will add to the field , since gene expression dose response data for HepaRG knockouts, HEPATOPAC 3D cultured PHH, and functionally variant PHH have not been published. Once feasibility is demonstrated in Phase I, the cell lines and assays will be marketed. An expanded panel of variant HepaRG and/or (depending on the utility of each determined in Phase I) HEPATOPAC co-cultures will be established in Phase II, the test process validated, and the additional variant cells marketed so that the in vitro impact genetic variability across the population has on exposure and toxicity of agents being tested can be determined and used for in vitro-to-in vivo extrapolation (IVIVE) of exposure risk of individuals with such genetic variants. The test process envisioned will be to i) profile the test agent in (e.g.) 3D PHH culture with ?normal? average PGx function to identify the pathways of metabolism, MoA, and toxicity and BMCs: ii) Select the variant cells (e.g. high and low/no activity cells) for each identified pathway modulated by test agent and determine variant toxicity profiles and BMCs to establish the in vitro range of exposure and toxicity risk; iii) perform IVIVE risk assessment across the variability of exposure individuals may be subject to due to PGx variants. The variant cells can also be used to assess the impact of PGx variants on toxicokinetic metabolic clearance.
