PROJECT SUMMARY Transplantation of solid organs (liver, pancreas, spleen, adrenal glands, heart, lungs) can save the lives of many patients with irreversible organ disease or injury. In the U.S., more than 40,000 transplants were performed in 2021 alone. After a solid organ transplant, lifelong immunosuppressant therapy must be used to prevent and treat organ rejection. The calcineurin inhibitor tacrolimus is a major therapeutic in such cases. Due to the risk of organ rejection, infection, and drug toxicity, therapeutic drug monitoring is advised to ensure pharmacokinetic (PK) targets. Typically, the need for precise dosing has been linked to genetic differences, particularly those affecting drug PK. Despite the important role of pharmacogenomics in precision dosing, large expression variability occurs within each genotype, which can be captured by expression data. Moreover, no known genetic signatures define the wide expression variations for certain key enzymes, such as CYP3A4. Hence, assessing variability in expression of gut and hepatic CYP3A4 is a promising alternative to inform drug development and precision medicine. This promising approach may capture transcriptional regulation effects, such as the downregulation of CYP3A4 by proinflammatory cytokines. Recent exploratory studies showed the application of plasma-derived extracellular vesicles (EV) and omics technologies deployed as a liquid biopsy to assess the expression of drug-metabolizing enzymes and transporters. Hence, EV are gaining traction as new phenotyping biomarkers. Various EVs are shed into the blood and contain cargo representing their tissue of origin. Building upon earlier findings, we will answer the following research question: Can we use liver-derived EV as in vivo CYP3A4/5 expression biomarkers to inform tacrolimus dosing in liver transplant patients? A prospective clinical study will be conducted in patients undergoing liver transplants to explore whether liver and gut-derived EV expression data can be used to predict the variability in tacrolimus pharmacokinetics and ultimately to propose an optimal dosing regimen. In summary, we will integrate PK, transcriptomic data, and modeling and simulation to establish an innovative strategy to individualize tacrolimus dosing. The use of expression data to support precision dosing can potentially overcome the exclusion of under-represented population subgroups (e.g. rare genetic variants of CYP3A4/5 and P- glycoprotein (P-gp). Thus, this proposal will promote diversity in immunosuppression therapy by enhancing the number of subjects responding to tacrolimus efficacy and reducing the risk of adverse reactions. Our research will develop an integrated package of highly predictive mathematical models and dosing optimization strategies for tacrolimus. By tracking different sources of variability in tacrolimus pharmacokinetics, we will inform precision public health interventions in solid organ transplant patients. Importantly, our liquid biopsy approach can be further evaluated and applied in other solid organ transplant patients such as heart, lung, and kidneys.

PROJECT NARRATIVE We will investigate whether expression data from serum-derived extracellular vesicles can be used to predict the in vivo pharmacokinetics of the CYP3A4/5 substrate tacrolimus. Using a multi- pronged strategy, we will integrate pharmacokinetics, liquid biopsy technology, genotyping, and activity data to propose individualized doses, based on the individual expression of hepatic and gut enzymes. Data will be integrated into model-informed Bayesian estimation tool to guide individual tacrolimus dosing in post-liver transplant patients.