A lung targeted miR-29 mimic as a therapy for pulmonary fibrosis

Abstract

Background: MicroRNAs are non-coding RNAs that negatively regulate gene networks. Previously, we reported that systemically delivered miR-29 mimic MRG-201 reduced fibrosis in animal models, supporting the consideration of miR-29-based therapies for idiopathic pulmonary fibrosis (IPF).

Methods: We generated MRG-229, a next-generation miR-29 mimic based on MRG-201 with improved chemical stability due to additional sugar modifications and conjugation with the internalization moiety BiPPB (PDGFbetaR-specific bicyclic peptide)¹. We investigated the anti-fibrotic efficacy of MRG-229 on TGF-β1 treated human lung fibroblasts (NHLFs), human precision cut lung slices (hPCLS), and in vivo bleomycin studies; toxicology was assessed in two animal models, rats, and non-human primates. Finally, we examined miR-29b levels in a cohort of 46 and 213 patients with IPF diagnosis recruited from Yale and Nottingham Universities (Profile Cohort), respectively.

Findings: The peptide-conjugated MRG-229 mimic decreased expression of pro-fibrotic genes and reduced collagen production in each model. In bleomycin-treated mice, the peptide-conjugated MRG-229 mimic downregulated profibrotic gene programs at doses more than ten-fold lower than the original compound. In rats and non-human primates, the peptide-conjugated MRG-229 mimic was well tolerated at clinically relevant doses with no adverse findings observed. In human peripheral blood from IPF patients decreased miR-29 concentrations were associated with increased mortality in two cohorts potentially identified as a target population for treatment.

Interpretation: Collectively, our results provide support for the development of the peptide-conjugated MRG-229 mimic as a potential therapy in humans with IPF.

Funding: This work was supported by NIH NHLBI grants UH3HL123886, R01HL127349, R01HL141852, U01HL145567.

Keywords: Idiopathic pulmonary fibrosis; MicroRNA; RNA therapies; miR-29.

Figure 1

Overview of modifications differentiating second-geneneration miR-29 from first gen MRG-201/Remlarsen. Top, first-gen MRG-201, the parent compound, bottom, MRG-229 (Next generation miR-29 mimic), the second-gen compound. DNA bases: white circles = unmodified base, blue circles = 2’OMe, purple circles = 2’F, linkages: green circles = phosphorodiester linkage, orange circles = phosphorothioate (PS) linkage. NH2 terminus modification: MRG-201/Remlarsen = cholesterol, BiPPB = platelet-derived growth factor beta receptor (PDGFβR)-binding peptide (BiPPB).

Figure 2

miR-29 reverses fibrosis in vitro. (a) RT-PCR of the miR-29b target gene COL1A in NHLFs. (b) RT-PCR of the miR-29b target gene ACTA2 in NHLFs. (c) Cell viability in NHLFs treated with increasing concentrations of MRG-201 or MRG-229. (d) Reduction of procollagen I C-peptide secretion in NHLFs treated with increasing concentrations of MRG-201 or MRG-229. (e) Dose-dependent collagen secretion assessed by accumulation of hydroxyproline levels in LL29 cells at 72h treated with TGF-Beta to induce fibrosis and treated with increasing concentrations of MRG-229 as indicated. All experiments were performed on at least three biological replicates. Statistical analyses Ordinary one-way ANOVA (***P<0.001) were performed in GraphPad Prism.

Figure 3

MRG-229 reverses fibrosis in a human Precision-Cut Lung Slice (hPCLS) model (blinded experiment). (a) RT-PCR analysis of COL1A1 relative expression (b) RT-PCR analysis of COL3A1 relative expression. (c) RT-PCR analysis of miR-29 level in human PCLS after 120 hrs in media + FC. (d) Masson Trichrome on human PCLS from healthy donors, cultured with either a fibrosis-inducing cocktail or vehicle for 120 hrs with daily media changes, treated with a peptide or a cholesterol-conjugated MRG-229 mimic. (e) Graph depicts quantification of collagen levels after 120 hrs on histology. Statistical analyses Ordinary one-way ANOVA (***P<0.001) were performed in GraphPad Prism.

Figure 4

Day 14 analysis of prophylactic dosing of MRG-201 at 100 mpk and peptide-conjugated MRG-229 mimic at 10 mpk in the bleomycin-induced lung fibrosis model. (a) Schematic of prophylactic dosing paradigm. Bleomycin-treated mice were dosed through intravenous injection with saline, MRG-201 on days 3, 7, 10 and 13. On day 14, animals were sacrificed, and lung tissue analysed. (b) Mean % total lung collagen quantified by Orbit machine learning image analysis software in bleomycin-induced mice treated with MRG-201 or MRG-229. (c) qPCR analysis of downregulated gene expression levels of a panel of fibrosis-associated genes in lung harvested from bleomycin-induced mice treated with either MRG-201 or MRG-229. C. (d) qPCR analysis of downregulated gene expression levels of a panel of fibrosis-associated genes in lung harvested from bleomycin-induced mice treated with either saline or the MRG-229-unconjugated with all the stability modifications but lacking the BiPPB conjugate. Statistical analyses Ordinary one-way ANOVA (***P<0.001) were performed in GraphPad Prism. mpk - mg/Kg.

Figure 5

Day 21 analysis of therapeutic dosing of MRG-201 at 100 mpk and MRG-229 at 10 mpk in the bleomycin-induced lung fibrosis model (a) Schematic of therapeutic dosing paradigm. Bleomycin-treated mice were dosed through intravenous injection with saline, MRG-201 at 100 mpk, or MRG-229 at 10 mg/Kg, on days 10, 13, 17 and 20. On day 21, animals were sacrificed, and lung tissue analysed. (b) ELISA analysis of IGF-1 levels in bronchoalveolar lavage fluid (left) and TIMP-1 levels in serum (right) harvested from mice treated with either saline or MRG-229. (c) representative Masson Trichrome-stained histopathology images of saline-treated (top) and MRG-229-treated (bottom) lung sections. (d) mean % total lung collagen quantified by Orbit machine learning image analysis software in bleomycin-induced mice treated with MRG-201 or MRG-229. (e lung distribution of MRG-229 at different doses of either subcutaneous or intravenous injection. (f) lung distribution of MRG-229 at different doses of either subcutaneous or intravenous injection. Statistical analyses Ordinary one-way ANOVA (***P<0.001) were performed in GraphPad Prism. mpk - mg/Kg

Figure 6

Liver ALT and AST (a, b), and Kidney Urea and Creatinine c, d), assessments following miR-29 administration. Assessment of liver function enzymes and markers of kidney damage from the dose response and route of administration study showed no detrimental effect on liver or kidney function by MRG-229, even in the presence of bleomycin, up to 10 mg/kg biweekly administered. Statistical analyses Ordinary one-way ANOVA (***P<0.001) were performed in GraphPad Prism. mpk - mg/Kg

Figure 7

miR-29 survival in 2 IPF cohort: ROC identified similar miR-29b exosomal RNA thresholds for mortality prediction in the Yale (plasma level threshold of 4.84) and Profile (serum level threshold of 4.32) cohorts. After adjusting for the GAP severity index, exosomal miR-29b levels in plasma (≤ 4.86) and serum (≤ 4.32) were significantly associated with mortality in the Yale (HR:0.156, 95%CI: 0.0404-0.6066, Statistical analyses Ordinary one-way ANOVA P=0.0073) and Profile (HR:0.5066, 95%CI: 0.2984-0.8599, Statistical analyses Ordinary one-way ANOVA P=0.011) cohorts, respectively. Figures 7a and 7b.