Unexpected adverse drug responses (ADRs) including drug-induced liver injury (DILI) are the 4th leading cause of death in the U.S. In addition, DILI in individuals is one of the major reasons for drug withdrawal from the market and is difficult to predict using conventional in vitro hepatotoxicity tests and preclinical animal models. Due to the critical link between DILI and drug failure, there is an urgent need for improved human hepatotoxicity testing in the early stage of drug development by investigating the major pathogenic mechanisms of DILI, such as genetic variations in drug metabolizing enzymes (DMEs) and drug transporters. To address this need, we propose to develop genetically engineered human liver organoids (HLOs) on a pillar/perfusion plate using gene-edited, induced pluripotent stem cell (iPSC) lines carrying CRISPR/Cas9 synergistic activation mediator (CRISPR-SAM), inducible Csy4, and multiplexed guide RNA (gRNA), and recapitulate poor and ultrafast drug metabolizers in different ethnic groups. Using normal and engineered HLOs on the pillar/perfusion plate, together with high-throughput, high-content, HLO imaging assays, we propose to decipher the cellular and molecular mechanisms underlying the toxicity of drug candidates and chemicals and assess DILI potential. Our core hypotheses are: (i) overexpression of DMEs and drug transporters can recapitulate ultrafast drug metabolizers in different ethnic groups that may be critical in addressing ADRs; (ii) metabolism- induced hepatotoxicity can be established using normal and engineered HLOs with model compounds; and (iii) high-throughput, high-content analysis of HLOs on the pillar/perfusion plate can be used to identify DILI, which in turn can improve predictability of compound hepatotoxicity in vivo. The specific aims of the proposed work are to: (1) create genetically engineered HLOs containing doxycycline-inducible, CRISPR-SAM for overexpression of multiple hepatic genes to model ultrafast metabolizers; (2) validate normal and engineered HLOs with model compounds, which undergo metabolism and lead to toxic cellular responses in the human liver. Although several human hepatic cell/tissue models including primary hepatocytes (ATCC), engineered hepatoma cell lines (HepG2-CYP cell panel from Hera BioLabs), liver spheroids (3D InSight liver microtissues from InSphero), bioprinted liver tissues (ExVive human liver tissues from Organovo), and microfluidic liver chips (Liver-Chip from Emulate) are commercially available, these in vitro liver models have been used for assessing general hepatotoxicity of compounds for normal drug metabolizers and cannot easily simulate poor and ultrafast drug metabolizers in their assays who suffer the most from DILI. Thus, there is great potential to apply engineered HLOs on the pillar/perfusion plate as a safety assessment tool. Genetically engineered human organoids can be used to express any endogenous proteins of interest in the cells for disease modeling by using a combination of guide RNAs. There is a great potential for genetically engineered human organoids to be used to incorporate genetic diversity into toxicity testing and for disease modeling.
Public Health Relevance Statement: Project Narrative Bioprinting Laboratories Inc. proposes to leverage its unique âmicroarray three-dimensional (3D) bioprintingâ technology and associated pillar/perfusion plates and generate normal and genetically engineered human liver organoids (HLOs) on the pillar/perfusion plates for recapitulating poor and ultrafast metabolizers in different ethnic groups due to allelic variations in drug metabolizing enzymes (DMEs) and testing vulnerability of drug-induced liver injury (DILI). Genetically engineered HLOs containing doxycycline-inducible, CRISPR/Cas9 synergistic activation mediator (CRISPR-SAM) will be used to overexpress multiple DME genes in different ethnic groups to simulate ultrafast metabolizers. Since current in vitro liver models are used mainly for assessing general hepatotoxicity of compounds and cannot easily accommodate genetic diversity in their assays, there is high commercial potential for normal and engineered HLOs.
Project Terms: Allelomorphs; Alleles; Architecture; Engineering / Architecture; Biological Assay; Assay; Bioassay; Biologic Assays; Metabolic Biotransformation; Biotransformation; Cause of Death; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cells; Cell Body; Classification; Systematics; Cessation of life; Death; Diffusion; diffused; diffuses; diffusing; diffusions; Doxycycline; Vibramycin; alpha-6-Deoxyoxytetracycline; Drug toxicity; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Engineering; Enzymes; Enzyme Gene; Ethnic Population; Ethnic Group; Ethnic People; Ethnic individual; Ethnicity People; Ethnicity Population; ethnic subgroup; ethnicity group; Genes; Genetic Engineering; Genetic Engineering Biotechnology; Genetic Engineering Molecular Biology; Recombinant DNA Technology; genetically engineered; Goals; Hospitalization; Hospital Admission; Human; Modern Man; Human Engineering; In Vitro; Intestines; Intestinal; bowel; Laboratories; Liver; hepatic body system; hepatic organ system; Marketing; Metabolism; Intermediary Metabolism; Metabolic Processes; Morbidity - disease rate; Morbidity; mortality; Organoids; Oxygen; O element; O2 element; Patients; Perfusion; Genetic Polymorphism; polymorphism; Proteins; Stains; Staining method; Technology; Testing; Time; Tissues; Body Tissues; United States Food and Drug Administration; Food and Drug Administration; USFDA; Genetic Variation; Genetic Diversity; Caucasians; Caucasian; Caucasian Race; Caucasoid; Caucasoid Race; Occidental; white race; Work; matrigel; Measures; gRNA; Guide RNA; drug withdrawal; improved; Hepatic; Variation; Variant; Lentivirinae; Lentivirus; Link; Chemicals; Hepatic Cells; Hepatic Parenchymal Cell; Liver Cells; Hepatocyte; Failure; insight; Hepatotoxic effect; Liver Toxicity; Toxic effect on liver cells; hepatic toxicity; hepatoxicity; Hepatotoxicity; Individual; Toxicity Testing; Toxicity Tests; Nature; ABCB1 gene; ABC20; ABCB1; GP170; MDR-1; MDR1; MDR1 Protein; Multidrug Resistance 1; Multidrug Resistance Gene-1; Multidrug Resistance Gene-1s; Multidrug Resistance Proteins; Multidrug Resistant Proteins; P-GP; P-Glycoprotein; P-Glycoprotein 1 Gene; PGY-1 Protein; PGY1; Protocols documentation; Protocol; System; 3-Dimensional; 3-D; 3D; three dimensional; fetal; interest; drug efficacy; Animal Model; Animal Models and Related Studies; model of animal; Hydrogels; Toxic effect; Toxicities; hepatoma cell; Nutrient; drug metabolism; Disease model; disorder model; Modeling; response; drug development; high throughput screening; High Throughput Assay; Biomimetics; Biological Mimetics; Pathogenicity; CP34; CYP3; CYP3A; 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; µfluidic; Microfluidics; CYP2C19 gene; CYP2C; CYP2C19; Cytochrome P450, Subfamily IIC, Polypeptide 19; Mephenytoin 4-Prime Hydroxylase; P450C2C; CYP2D6 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; Mediator of activation protein; Mediator of Activation; CYP2B6 gene; CYP2B6; Cytochrome P450, Phenobarbital-Inducible; Cytochrome P450, Subfamily IIB, Polypeptide 6; Address; Affinity; Reproducibility; in vivo; Epigenetic Process; Epigenetic; Epigenetic Change; Epigenetic Mechanism; epigenetically; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Tissue Model; Molecular; Image; imaging; pre-clinical; preclinical; iPS; iPSC; iPSCs; induced pluripotent cell; inducible pluripotent stem cell; induced pluripotent stem cell; overexpress; overexpression; commercial scale manufacturing; manufacturing ramp-up; scale up batch; scale up production; upscale manufacturing; manufacturing scale-up; drug candidate; CRISPR; CRISPR/Cas system; Clustered Regularly Interspaced Short Palindromic Repeats; Hep G2; HepG2 cell line; HepG2; Assessment instrument; Assessment tool; CRISPR approach; CRISPR based approach; CRISPR method; CRISPR methodology; CRISPR technique; CRISPR technology; CRISPR tools; CRISPR-CAS-9; CRISPR-based method; CRISPR-based technique; CRISPR-based technology; CRISPR-based tool; CRISPR/CAS approach; CRISPR/Cas method; CRISPR/Cas9; CRISPR/Cas9 technology; Cas nuclease technology; Clustered Regularly Interspaced Short Palindromic Repeats approach; Clustered Regularly Interspaced Short Palindromic Repeats method; Clustered Regularly Interspaced Short Palindromic Repeats methodology; Clustered Regularly Interspaced Short Palindromic Repeats technique; Clustered Regularly Interspaced Short Palindromic Repeats technology; CRISPR/Cas technology; individual patient; bio-printing; bioprinting; individual heterogeneity; individual variability; individual variation; safety assessment; cost estimation; cost estimate; feasibility testing; drug induced liver injury; drug induced hepatotoxicity; drug induced liver disease
