Preclinical development of a miR-132 inhibitor for heart failure treatment

Abstract

Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound's therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme.

Fig. 1. Therapeutic potential of antimiR-132 treatment.

a Study outline of antimiR-132 application (20 mg/kg, i.p.) to miR-212/132 transgenic (TG) mice compared to placebo treatment (0.9% NaCl solution) and wildtype (WT) mice. b Functional miR-132 level. c MiR-132 target gene expression (Forkhead Box Protein O3, Foxo3). d Representative images of the parasternal long axis (PLAX) view of the left ventricle (LV) (scale bar = 2 mm) and heart weight (HW) vs. Tibia length ratio of mice. e Echocardiographic evaluation of ejection fraction and end-systolic volume. WT: n = 8, TG + NaCl: n = 7, TG + antimiR-132: n = 6. Data are mean ± s.e.m; **P < 0.01; unpaired two-sided Mann–Whitney U test.

Fig. 2. Functional properties of single cardiomyocytes.

a Representative action potential traces of adult ventricular cardiomyocytes derived from wildtype (WT), miR-212/132 transgenic (TG) mice treated with placebo (0.9% NaCl solution) or antimiR-132. b Resting membrane potential (RMP). c Action potential duration at 50% level of repolarization (APD50). d Action potential amplitude. e Upstroke velocity. (WT: n = 18 cells, TG + NaCl: n = 14 cells, TG + antimiR-132: n = 10 cells) f Representative normalized calcium transients. g, Time-to-peak (ttp) of calcium transients at 3 Hz stimulation frequency. (WT: n = 46 cells, TG + NaCl: n = 19 cells/, TG + antimiR-132: n = 26 cells) h Representative normalized sarcomere shortening. i Time-to-peak (ttp) of sarcomeric contraction at 3 Hz stimulation frequency. (WT: n = 52 cells, TG + NaCl: n = 57 cells, TG + antimiR-132: n = 41 cells). j MiR-132 target gene expression in heart tissue (Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ ATPase 2, Serca2a2). (WT: n = 8, TG + NaCl: n = 7, TG + antimiR-132: n = 5.) Data are mean ± s.e.m; *P < 0.05; **P < 0.01; unpaired two-sided Mann–Whitney U test.

Fig. 3. Pharmacokinetic properties of antimiR-132.

a Cardiac tissue levels of antimiR-132 in healthy pigs 48 h post treatment for different dose levels (Control: None; Low = 1 mg/kg, Medium = 5 mg/kg and High = 10 mg/kg antimiR-132, n = 3 respectively) by intravenous (IV) or intracoronary (IC) application. b Functional cardiac tissue level of miR-132 in healthy pigs 48 h post treatment. c Correlation between antimiR-132 and miR-132 cardiac tissue levels in healthy pigs 48 h post treatment. d Time course of cardiac tissue levels of antimiR-132 in healthy pigs at different timepoints post treatment (5 mg/kg antimiR-132, IV, n = 3 respectively). e Plasma levels of circulating antimiR-132 in healthy pigs at different timepoints post treatment (5 mg/kg antimiR-132, IV, n = 3 respectively). Data are mean ± s.e.m; *P < 0.05, **P < 0.01; Kruskal–Wallis test with Dunn’s multiple comparison (Control vs. treatment groups) and linear regression using non‐parametric Spearman correlation.

Fig. 4. Proof-of-concept study in a large animal model of post-MI HF.

a Study outline of treatment regimen (LAD = left anterior descending coronary artery). b Ejection fraction (EF) at baseline, day 3 and day 56 for different dosing groups of intracoronary/intravenous (ICIV) and intravenous/intravenous (IVIV) treated animals (Placebo: NaCl; Low = 1 mg/kg, Medium = 5 mg/kg and High = 10 mg/kg antimiR-132). c Functional improvement indicated by EF change from day 3 to day 56 (delta EF) for different dosing groups of ICIV and IVIV treated animals. d Responder analysis for different dosing groups of ICIV and IVIV treated animals. e N-terminal prohormone of brain natriuretic peptide (NT-proBNP) levels at baseline and day 56 for different dosing groups of ICIV and IVIV treated animals. f Quantification and representative micrographs of picrosirius red (PSR) staining of the left ventricular (LV) remote regions for different dosing groups of ICIV and IVIV treated animals (scale bar = 200 µm). g Quantification and representative micrographs of wheat germ agglutinin (WGA) staining for cardiac cell size measurement of the LV remote regions of IVIV treated placebo and high dose animals (scale bar = 50 µm). ICIV and IVIV: Placebo: n = 22, Low dose: n = 20, Medium dose: n = 20, High dose: n = 17. IVIV: Placebo n = 12, High dose: n = 7. Data are mean ± s.e.m; *P < 0.05, ***P < 0.001; unpaired two-sided Mann–Whitney U test (d3 vs. d56) or Kruskal–Wallis test with Dunn’s multiple comparison (Placebo vs. treatment groups).

Fig. 5. PK/PD relationship and target engagement panel.

a Tissue levels of antimiR-132 detected in the left ventricular (LV) remote region for different dosing groups of intracoronary/intravenous (ICIV) and intravenous/intravenous (IVIV) treated animals (Placebo: NaCl; Low = 1 mg/kg, Medium = 5 mg/kg and High = 10 mg/kg antimiR-132). b Correlation between antimiR-132 tissue levels and functional improvement (delta ejection fraction (EF) = EF_{day 56} – EF_{day 3}). c Functional tissue level of miR-132 detected in the LV remote region. d Correlation between antimiR-132 and miR-132 tissue levels. e Target de-repression in the LV remote region after antimiR-132 treatment (Forkhead Box Protein O3, FOXO3; Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ ATPase 2, SERCA2A; Endothelial Nitric Oxide Synthase 3, NOS3; SCL/TAL1 Interrupting Locus, STIL; TEK Receptor Tyrosine Kinase, TEK). Radar chart depicting the target engagement panel. ICIV and IVIV: Placebo: n = 22, Low dose: n = 20, Medium dose: n = 20, High dose: n = 17. Data are mean ± s.e.m; *P < 0.05, **P < 0.01, ***P < 0.001; Kruskal–Wallis test with Dunn’s multiple comparison and linear regression using non‐parametric Spearman correlation.