This Phase I project will demonstrate the feasibility for assessing the impact of genetic diversity onexposure and inferences to the corresponding risk of toxicity across the human population by generating doseresponse data for each test agent using the TempO-Seq gene expression assay for a panel of cells, each withdifferent pharmacogenomic gene (PGx) variant activities. As many as 10% to 20% of the population can expressa genetic variant, such as deficiency in CYP2D6, CYP2C19, or CYP2C9, but significant numbers of personshave functionally different activities resulting from genomic variants of many other pharmacogenomic genessuch as in ABC transporters, UGT transferases, and the ligand binding domains of PXR, AHR, and CAR. Thehuman-specific S1500v2 whole transcriptome surrogate TempO-Seq gene expression assay will be used toprofile HepaRG knockouts reflecting functional variation in activity of PGx gene products and 3D HEPATOPACco-cultures of primary human hepatocytes (PHH) from donors expressing functional variant activity. TheHepaRG 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 thestromal response. Dose response data for test agents (a set of reference compounds in Phase I), will beobtained after a 96-hr treatment, from which compound-specific signatures, mode of action and toxicitypathways will be identified using DESeq and pathway analysis. BMDExpress will be used to determine thebenchmark concentration (BMC) for each modulated pathway and gene. Differences between variants incompound signatures and gene and pathway BMCs will be identified and used to define the impact variantfunctional activity has on in vitro exposure and toxicity. These data will add to the field , since gene expressiondose response data for HepaRG knockouts, HEPATOPAC 3D cultured PHH, and functionally variant PHH havenot been published. Once feasibility is demonstrated in Phase I, the cell lines and assays will be marketed. Anexpanded 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 variantcells marketed so that the in vitro impact genetic variability across the population has on exposure and toxicityof agents being tested can be determined and used for in vitro-to-in vivo extrapolation (IVIVE) of exposure riskof 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 toxicityand BMCs: ii) Select the variant cells (e.g. high and low/no activity cells) for each identified pathway modulatedby test agent and determine variant toxicity profiles and BMCs to establish the in vitro range of exposure andtoxicity risk; iii) perform IVIVE risk assessment across the variability of exposure individuals may be subject todue to PGx variants. The variant cells can also be used to assess the impact of PGx variants on toxicokineticmetabolic clearance.
Public Health Relevance Statement: Narrative: This Phase I project will demonstrate the feasibility of using expression profiling for assessing the impact of genetic variation on response to compound exposure and inferences to the corresponding risk of toxicity across the human population. Using the TempO-Seq S1500 surrogate human whole transcriptome assay to profile all known biochemical pathways, together with HepaRG cells harboring knockouts of major xenobiotic clearance pathway genes, 3D HEPATOPAC co-cultures of primary human hepatocytes from donors with varying compound responses, and a set of reference compounds with known modes of action, compound- specific signatures and toxicity pathways will be identified for each genetic variant using DESeq2 and Pathway analysis, then quantified using BMDExpress to determine the benchmark concentration (BMC) for each modulated pathway and gene to define differences between variants in metabolism, exposure level, and toxicity pathways and dose. Once feasibility is demonstrated in Phase I, the use of the HEPATOPAC and HepaRG cell systems and the TempO-Seq assay will be marketed, an expanded panel of variant HepaRG variants and/or HEPATOPAC cultures will be established, and test process validated and commercialized in Phase II, so that the in vitro impact of genetic variability across the population on exposure and toxicity of test agents can be determined and used for in vitro-to-in vivo extrapolation of exposure risk of genetically diverse individuals.
Project Terms: CYP3A4 ; Cytochrome P450 3A4 ; Cytochrome P450, Subfamily IIIA, Polypeptide 4 ; Cytochrome P450PCN1 ; FAMILY III P450 ; Glucocorticoid-Inducible P450 ; Nifedipine Oxidase ; P450C3 ; P450PCN1 ; Steroid-Inducible P450- III ; CYP3A4 gene ; ABC15 ; ABCG2 ; ABCP ; ATP-Binding Cassette, Sub-Family G (WHITE), Member 2 Gene ; ATP-Binding Cassette, Sub-Family G, Member 2 ; ATP-Binding Cassette, Subfamily G, Member 2 ; BCRP ; BCRP1 ; Breast Cancer Resistance Protein ; EST157481 ; MRX ; MXR1 ; Mitoxantrone Resistance Protein ; Placenta-Specific ATP-Binding Cassette Transporter ; ABCG2 gene ; CYP2C ; CYP2C19 ; Cytochrome P450, Subfamily IIC, Polypeptide 19 ; Mephenytoin 4-Prime Hydroxylase ; P450C2C ; CYP2C19 gene ; CYP2C9 ; Cytochrome P450, Subfamily IIC, Polypeptide 9 ; CYP2C9 gene ; CPD6 ; CYP 2D6 ; CYP2D ; CYP2D6 ; CYP2DL1 ; CYPIID6 ; Cytochrome P-450 CYP2D6 ; Cytochrome P450 2D6 ; Cytochrome P450 Subfamily IID Polypeptide 6 ; Debrisoquine 4-Hydroxylase ; Debrisoquine 4-Monooxygenase ; Debrisoquine Hydroxylase ; Imipramine 2-Hydroxylase ; P450-2D6 ; P450-DB1 ; P450C2D ; P450DB1 ; Sparteine Monooxygenase ; Subfamily IID Cytochrome P450 ; Subfamily IID-Like 1 Cytochrome P450 ; CYP2D6 gene ; Dose ; Data ; Molecular Toxicology ; Proliferating ; in vitro Assay ; in vivo ; Cellular Assay ; cell assay ; Scientific Advances and Accomplishments ; scientific accomplishments ; scientific advances ; Molecular ; Knock-out ; Knockout ; Process ; Pathway interactions ; pathway ; genetic variant ; Gene variant ; allele variant ; allelic variant ; genomic variant ; Population ; innovation ; innovate ; innovative ; transcriptome ; global gene expression ; global transcription profile ; gene product ; three dimensional cell culture ; 3D cell culture ; 3D culture ; Expression Profiling ; in vivo evaluation ; in vivo testing ; phase 2 testing ; phase 2 evaluation ; phase II evaluation ; phase II testing ; Affect ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Budgets ; Cell Differentiation process ; Cell Differentiation ; Cell Line ; CellLine ; Strains Cell Lines ; cultured cell line ; Cells ; Cell Body ; Cultured Cells ; Gene Expression ; Genes ; Goals ; Human ; Modern Man ; In Vitro ; Literature ; Metabolism ; Intermediary Metabolism ; Metabolic Processes ; Mus ; Mice ; Mice Mammals ; Murine ; Persons ; Oligonucleotides ; Oligo ; oligos ; Publishing ; Risk ; Safety ; Specificity ; Testing ; Transferase ; Transferase Gene ; Genetic Variation ; Genetic Diversity ; Xenobiotics ; Measures ; 3T3 Cells ; Risk Assessment ; Stromal Cells ; Guidelines ; base ; detector ; Phase ; Variant ; Variation ; Hepatocyte ; Hepatic Cells ; Hepatic Parenchymal Cell ; Liver Cells ; Individual ; Policies ; ATP-Binding Cassette Transporters ; ABC Transport Protein ; ABC Transporter Protein ; ABC Transporters ; Toxicity Tests ; Toxicity Testing ; Biochemical Pathway ; Metabolic Networks ; Toxicokinetics ; Coculture Techniques ; Co-culture ; Cocultivation ; Coculture ; Metabolic ; Genetic ; gene function ; Exposure to ; tool ; programs ; System ; 3-D ; 3D ; three dimensional ; 3-Dimensional ; Best Practice Analysis ; Benchmarking ; success ; Toxicities ; Toxic effect ; expectation ; novel ; Binding Site Domain ; Ligand Binding Domain ; General Public ; General Population ; Gene Expression Monitoring ; Gene Expression Pattern Analysis ; Transcript Expression Analyses ; Transcript Expression Analysis ; gene expression analysis ; gene expression assay ; transcriptional profiling ; Gene Expression Profiling ; Network Analysis ; Pathway Analysis ; Modeling ; response ; cross reactivity ; Pharmacogenomics ; CP34 ; CYP3 ; CYP3A ;
