Abstract Drug-induced liver injury (DILI) has become a leading cause of acute liver failure, and it is the most common reason for regulatory actions after drug approval. Differences in the drug metabolism and drug disposition pathways used by humans and animal species have limited the predictive utility of animal toxicology studies. Drugs that produced minimal or no toxicity in animal studies have sometimes caused significant DILI in humans. Given the significant public health problem caused by DILI, and the difficulties associated with regulatory actions occurring after drug approval, there is a critical need for better methods to identify candidate medications that will cause DILI. This program will use 21st century genome engineering and a liver humanization model to improve drug safety. Since liver is the target organ for many drug-induced toxicities, toxicology studies using mice with `humanized' livers should improve the safety of drugs that will be tested in human subjects. Analyses of their response to fialuridine and bosentan treatment have indicated that TK-NOG mice with humanized livers could identify drugs that will cause human-specific liver toxicity. However, for this model to achieve its potential, its performance must be assessed with a larger number of drugs with different hepatotoxic potential in rodents and humans. Therefore, we will evaluate the response of control and humanized TK-NOG mice to 7 selected drugs, which include: (i) 3 nucleoside analogues (two were safe for humans and one caused human-specific liver toxicity); (ii) two drugs with the same molecular target to determine if chimeric mice can distinguish between drugs that will or will not cause human-specific liver toxicity; and (iii) two tyrosine kinase inhibitors that were hepatotoxic in rodents but not in humans to determine whether this platform can identify drugs that will be safe for humans, even though they caused rodent-specific toxicities. We have shown that bosentan-induced cholestatic liver toxicity (BICLT) develops in humanized (but not control) TK-NOG mice. However, we do not know why humanized mice have increased susceptibility to BICLT. Therefore, a series of mechanistic studies will be performed to determine why humanized mice are selectively susceptible to this toxicity. Lastly, residual murine hepatocytes in the chimeric liver produce mouse-specific drug metabolites, which can confound the results of toxicology studies that use humanized TK-NOG mice. To produce a liver humanization platform with better performance characteristics, mouse genome engineering will be used to inactivate key murine genes involved in phase I drug metabolism. A two-stage process will be used to ensure that murine phase I drug metabolism has been optimally reduced in these mice. Their response to 6 different drugs, which have different hepatotoxic potential in rodents and humans, will be assessed to determine if this next generation platform can better predict the human hepatotoxic potential of candidate medications.