A liquid biopsy-RNAseq method for monitoring the expression of genes involved in drug disposition: Proof-of-concept application to cholestatic liver disease.

Between-patient variability in metabolism and disposition of therapeutic drugs is associated with variable expression and activity of enzymes and transporters. Direct measurement of the expression of such proteins in relevant organs is limited by access to tissue biopsies. Plasma-derived extracellular vesicles (EVs), sampled as liquid biopsy, offer a minimally invasive and widely accessible alternative to tissue biopsy. This report provides a technical account for the extraction, characterization and RNAseq of EVs from plasma from healthy donors and donors with cholestatic liver disease (n = 3 in each group). Quality control (QC) steps implemented at different stages of the workflow ensured quality of the EVs and extracted RNA for downstream analysis. These QCs included visualization, size distribution, protein content and RNA quality and yield. Extracted EVs were a mixture of small and large vesicles (35-350 nm). Replicate RNAseq data reflected strong correlation (Pearson's r = 1, p < 0.001), low bias (average fold error = 1) and limited scatter (absolute average fold error = 1.34) across replicates. The method enabled monitoring RNA expression of 656 pharmacologically relevant genes, including 199 xenobiotic-metabolizing enzymes, 444 transporters, and 11 transcription factors. Disease perturbation in expression from healthy baseline was estimated for 93 % of monitored pharmacokinetic pathways. The data were used, in combination with demographic and serum albumin changes, to generate a cholestatic liver disease pharmacokinetic model. The model reflected exposure to budesonide and cyclosporine consistent with early-stage disease. The model also reflected increased total serum bilirubin, likely due to dysregulation of hepatobiliary transport.