Evaluating miRNA Sequencing and Integrative Proteomics for Identifying Plasma miRNAs as Potential Pharmacodynamic...

We used miRNA sequencing to identify candidate plasma pharmacodynamic (PD) biomarkers of interferon beta-1a (IFNβ-1a) biologics and explored miRNA-mRNA targeted protein relationships and networks to support identified miRNA candidates. Plasma samples from 36 healthy subjects from a placebo-controlled randomized single-dose clinical study with IFNβ-1a and pegIFNβ-1a were used. Mature miRNAs were measured using an in-house miRNA-seq workflow at baseline, at 9 timepoints over 6 days in the IFNβ-1a group (n = 11 [30 μg]), and at 11 timepoints over 13 days in the pegIFNβ-1a group (n = 11 [125 μg]) and placebo-specific groups (n = 6 each). A miRNA was only considered expressed if it had a read count ≥ 10 in more than 50% of the samples across all treatment groups. Linear mixed-effects models (lmer) regressing miRNA changes with treatment, time, and their interaction were used to identify differentially expressed miRNAs, which were further prioritized based on the magnitude of response and biological relevance using in silico predicted miRNA-protein targets and regulatory network analyses. Ten and 13 miRNAs were impacted by IFNβ-1a and pegIFNβ-1a, respectively (lmer FDR-corrected p value < 0.1), compared to placebo, of which miR-223-3p and miR-21-5p were prioritized as candidate PD biomarkers for both products. Systems-level analysis of integrated proteomics data highlighted miRNAs' protein targets for both miR-223-3p and miR-21-5p that were linked to previously reported top proteomic response proteins and predicted regulatory networks including the IFN beta node. Using miRNA sequencing and linking candidates to predicted target proteins and regulatory networks, results suggest miR-223-3p and miR-21-5p as potential PD biomarkers of IFNβ-1a biologics for further investigation.