Together, these data indicate that unformulated miR‐29b mimic can increase the miRNA level with tissue‐dependent clearance and delivery efficiency, without any clear effect on gene expression under baseline conditions.

To gain more insights into the in vivo stability of miRNA mimics, we injected 125 mpk of miR‐29b mimic and sacrificed the mice 1, 2, 4, or 7 days later. Robust presence of miR‐29b mimic could be detected by both Northern blot and real‐time PCR analysis 1 day after injection in all tissues examined; however, tissue clearance greatly differed thereafter (Fig 1 E and F). Liver and kidney rapidly cleared miR‐29b mimic with minimal detection after day 1. Lung and spleen demonstrated the most pronounced detection of miR‐29b mimic over time, which sustained at least 2–4 days post‐treatment (Fig 1 E and F). The increase was specific for miR‐29b without any effect on miR‐29a and miR‐29c levels as measured by real‐time PCR ( Supplementary Fig S3 ). Also, here real‐time PCR analysis of miR‐29 targets showed no downregulation at the mRNA level in non‐stressed animals ( Supplementary Fig S4 ).

To start exploring the in vivo applicability and distribution of miR‐29 mimic, we injected mice intravenously with 10, 50, 100, or 125 mg per kg (mpk) and sacrificed them 4 days later. Northern blot analysis on multiple tissues indicated little to no increase in miR‐29b in kidney or liver samples compared to saline control. Cardiac distribution was detected; however, this appeared to be quite variable and spleen delivery could be observed at the highest dose only. In contrast, delivery to the lungs could be observed at all 3 of the highest doses 4 days after injection (Fig 1 C). No effects on liver function (transaminase, ALT) were observed in the plasma, indicating that these miRNA mimics are well tolerated at these doses ( Supplementary Fig S1 ). Real‐time PCR demonstrated similar results with robust dose‐dependent distribution of the miR‐29b mimic to the lung compared to saline‐injected animals (Fig 1 D). Additionally, real‐time PCR analysis of miR‐29 targets showed no regulation at the mRNA level in the treated animals except for Col3a1 at the highest dose in the spleen ( Supplementary Fig S2 ). This suggests that the target genes are either at steady state in non‐stressed animals and that mimics lower target genes when they are elevated, or that functional delivery was inadequate or insufficient.

To test for functional efficacy, we transfected miR‐29b mimic into a mouse fibroblast cell line (NIH 3T3) and measured the effect on Collagen1a1 ( Col1a1 ) expression, a known direct target gene of miR‐29 (van Rooij et al , 2008 ). Increasing amount of miR‐29b mimic showed a dose‐dependent decrease in Col1a1 , compared to either untreated or control oligo treated cells, indicating the miR‐29b mimic to be functional. An siRNA directly targeting Col1a1 was taken along as a positive control (Fig 1 B).

Synthetic RNA duplexes can be used to therapeutically mimic or increase the level of a miRNA to enhance the endogenous activity of the miRNA of interest. These miRNA mimics harbor chemical modifications for stability and cellular uptake. We designed double‐stranded miR‐29 mimics utilizing lessons learned from antisense and siRNA technologies. The “guide strand” or “antisense strand” is identical to the miR‐29b, with a UU overhang on the 3′ end, modified to increase stability, and chemically phosphorylated on the 5′ end. Since the guide strand has to function as a miRNA and the RISC machinery in the cell needs to recognize it as such, the allowed chemical modifications are limited. The 2′‐F modification helps to protect against exonucleases, hence making the guide strand more stable, while it does not interfere with RISC loading. The “passenger strand” or the “sense strand” contains 2′‐O‐Me modifications to prevent loading into RNA‐induced silencing complex (RISC) as well as increase stability and is linked to cholesterol for enhanced cellular uptake. Several mismatches are introduced to prevent this strand from functioning as an antimiR and to lessen hybridization affinity for the guide strand (Fig 1 A).

miR‐29b mimic blunts bleomycin‐induced pulmonary fibrosis

Current treatments of tissue fibrosis mostly rely on targeting the inflammatory response; however, these are ultimately ineffective in preventing progression of the disease, underscoring the need for new mechanistic insights and therapeutic approaches (Friedman et al, 2013). Recent studies indicate the involvement of miRNAs in pulmonary fibrosis (Pandit et al, 2011).

Due to the preferential lung distribution of our mimic, we set out to explore whether stress and subsequent induction of target gene expression would allow for detectable changes in mRNA target genes and downstream therapeutic effects in response to miR‐29b mimic. To this end, we used the bleomycin‐induced model of pulmonary fibrosis as described (Pandit et al, 2010) and injected the mice with 100 mpk miR‐29b mimic, control or a comparable volume of saline at two time‐points: 3 and 10 days after bleomycin treatment. As expected, 14 days after bleomycin treatment, miR‐29 levels were reduced, while miR‐29b mimic treatment resulted in the increased detection of miR‐29b levels compared to either control or saline‐injected animals as measured by real‐time PCR, albeit with a high level of variation (Fig 2A). It is currently unclear why the increase in miR‐29b levels is less than we detected in baseline mice (Fig 1). A comparable decline in miR‐29 levels was observed in pulmonary biopsies of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Fig 2B). Histological examination by trichrome staining showed a clear and robust fibrotic and inflammatory reaction in response to bleomycin, which was blunted by miR‐29b mimic treatment (Fig 2C). Additionally, hydroxyproline measurements to assay for total collagen content indicated a significant increase following bleomycin treatment in both saline and control‐treated groups, while there was no statistical difference in the miR‐29 mimic‐treated group between saline and bleomycin‐treated mice, indicating that miR‐29b mimic treatment blunts bleomycin‐induced pulmonary fibrosis (Fig 2D).

Figure 2.miR‐29b mimic blunts pathological signs of bleomycin‐induced pulmonary fibrosis A. Real‐time PCR analysis indicates a reduction in all miR‐29 family members in response to bleomycin, while miR‐29 mimic treatment resulted in the increased detection of miR‐29b levels compared to either control‐ or saline‐injected animals. * P < 0.05 versus Saline/Saline.

B. Real‐time PCR analysis indicated a comparable decline in miR‐29 levels in pulmonary biopsies of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls. * P < 0.05 versus Normal.

C. Histological examination by trichrome staining showing pronounced fibrotic and inflammatory response in response to bleomycin, which was blunted by miR‐29b mimic treatment. Scale bar indicates 100 μm.

D. Hydroxyproline measurements to assay for total collagen content showed a significant increase following bleomycin treatment in both saline‐ and control‐treated groups, while there was no statistical difference in the miR‐29 mimic‐treated group between saline‐ and bleomycin‐treated mice.

E–G. Cytokine measurements on bronchoalveolar lavage (BAL) fluids indicated a significantly higher concentrations of IL‐12 (E), IL‐4 (F), and G‐CSF (G) were detectable in BAL fluids from lungs from bleomycin‐treated mice, which was reduced with miR‐29b mimic ( n = 4). * P < 0.05.

H. Bleomycin treatment increases the detection of immune cells in BAL fluids which was significantly reduced in the presence of miR‐29b mimic, while the control mimic had no effect (n = 4)., *P < 0.05 versus Saline/Bleo, ^P < 0.05 versus Control/Bleo.

Innate immune effector signaling pathways act as important drivers of myofibroblast transdifferentiation by provoking fibrosis. To further characterize the therapeutic effects of miR‐29b mimic in the setting of bleomycin‐induced pulmonary fibrosis, we performed bronchoalveolar lavage (BAL) on these mice and assessed cytokine levels. Significantly higher concentrations of IL‐12, IL‐4, and G‐CSF were detectable in BAL fluids from lungs from bleomycin‐treated mice, which were reduced with miR‐29b mimic (Fig 2E–G). Additionally, the bleomycin‐induced elevation of detectable immune cells in BAL fluids was significantly reduced in the presence of miR‐29b mimic (Fig 2H), indicating an inhibitory effect on the immune response by miR‐29b, which is likely secondary to the antifibrotic‐effect. To determine if miR‐29 mimicry has a direct effect on macrophages, we transfected miR‐29b mimic and control into macrophage cells, RAW 264.7, and harvested the supernatant at 24 and 48 h after transfection. IFN‐r, IL‐1B, IL‐2, IL‐4, IL‐5, IL‐6, KC, IL‐10, IL‐12P70, and TNF‐α were assessed, with no significant differences observed between miR‐29b mimic and control (P. Latimer and R. Montgomery, unpublished data). By real‐time PCR analysis, there were no significant differences in Tgfb1, Ctgf, FGF1, or PDGF expression; however, we did observe a significant difference in Csf3, Igf1, and Kc expression (Supplementary Fig S5 and P. Latimer and R. Montgomery, unpublished data).

Since it has been well validated that miR‐29 functions through the regulation of many different extracellular matrix related genes (van Rooij & Olson, 2012), we confirmed the regulation of a subset of these target genes. While a significant increase in Col1a1 and a trend increase in Col3a1 expression were observed with bleomycin treatment in both saline and control‐treated groups, the detection of Col1a1 and Col3a1 was significantly blunted in the presence of miR‐29b mimic in the bleomycin‐treated mice (Fig 3A and B). Interestingly, the increase in Igf1 levels in BAL fluids following bleomycin treatment was significantly blunted in the presence of miR‐29 mimic compared to both saline and control‐treated mice (Fig 3C). Furthermore, immunohistochemistry for Igf1 demonstrated robust reductions in Igf1 after bleomycin in miR‐29b mimic‐treated groups compared to saline or controls (Fig 3D).

Figure 3.In vivo mimicry of miR‐29b represses the induction of miR‐29 target genes during pulmonary fibrosis A, B. Bleomycin treatment increases the expression of Col1a1 (A) and Col3a1 (B), and the presence of miR‐29b mimic inhibits Col1a1 and Col3a1 as measured by real‐time PCR. MiR‐29b mimicry has no effect on target repression under baseline conditions. ( n = 6–8), * P < 0.05.

C. IGF1 levels in BAL fluids increase following bleomycin treatment which were significantly blunted in the presence of miR‐29 mimic compared to both saline‐ and control mimic‐treated mice ( n = 4). * P < 0.05.

D. Immunohistochemistry demonstrated robust detection of IGF1 after bleomycin treatment, which was reduced in the miR‐29b mimic‐treated group compared to saline‐ or control mimic‐treated mice. Scale bar indicates 50 μm.

After establishing that early (days 3 and 10) miR‐29 mimicry was sufficient to prevent bleomycin‐induced fibrosis, we sought to determine if miR‐29 mimicry affects established fibrosis. For that purpose, we started the miR‐29b mimic administration at day 10 post‐bleomycin and repeated the doses at days 14 and 17 after which we harvested the lungs at day 21. Hydroxyproline assessment of the right lung showed a significant increase with bleomycin in both saline and control‐treated lungs; however miR‐29b mimic treatment blunted this effect (Fig 4A). Furthermore, bleomycin treatment resulted in significant increases in Col1a1 and Col3a1 expression, which was also normalized with miR‐29b mimic treatment (Fig 4B and C). Histological assessment by trichrome staining corroborated this effect, whereby bleomycin induced significant fibrosis with saline or control treatment which was blunted with miR‐29b mimicry (Fig 4D).

Figure 4.Therapeutic mimicry of miR‐29 attenuates bleomycin‐induced fibrosis A. Hydroxyproline assessment showed a significant increase following bleomycin treatment in both saline‐ and control‐treated groups; however, there was no statistical difference in the miR‐29 mimic‐treated group between saline‐ and bleomycin‐treated mice. * P < 0.05 ( n = 8).

B, C. Real‐time PCR analysis showed a significant increase of Col1a1 (B) and Col3a1 (C) after bleomycin treatment. miR‐29b mimic treatment normalized both Col1a1 and Col3a1 to vehicle‐treated expression levels. * P < 0.05 ( n = 8).

D. Histological examination by trichrome staining showing robust fibrosis in response to bleomycin, which was blunted by miR‐29b mimic treatment. Scale bar indicates 50 μm.

E, F. Primary pulmonary fibroblasts from patients with IPF were treated with vehicle or TGF‐β and transfected with control mimic or miR‐29b mimic. Real‐time PCR was performed for Col1a1 (E) and Col3a1 (F). miR‐29b mimic treatment showed a dose‐dependent reduction in both collagens.

G, H. A549 cells were treated with vehicle or TGF‐β and transfected with control mimic or miR‐29b mimic. Real‐time PCR was performed for Col1a1 (G) and Col3a1 (H). miR‐29b mimic treatment showed a dose‐dependent reduction in expression of both Col1a1 and Col3a1.

While we believe these effects are mediated through regulation of collagen production from lung fibroblasts, we are not able to rule out effects from other collagen producing cells. To address this, we assessed miR‐29b mimic effects in vitro from different lung cells, including primary fibroblasts from IPF patients and A549 cells, a lung epithelial cell line. As expected, primary pulmonary fibroblasts from IPF patients show an increase in Col1a1 and Col3a1 in response to TGF‐β (Fig 4E and F). This effect was dose‐dependently blunted with miR‐29b mimic treatment at both 24 and 48 h (Fig 4E and F and P. Latimer and R. Montgomery, unpublished data). Similarly, A549 cells respond to TGF‐β with robust increases in Col1a1and Col3a1 expression (Fig 4G and H). Again, miR‐29b mimic treatment is able to block collagen induction, in both TGF‐β treated as well as baseline conditions (Fig 4G and H). The effects on collagen induction are much more robust in the A549 cells compared to primary IPF cells; however, this is likely due to the already high expression in primary fibroblasts from IPF patients. While A549 cells are epithelial cells and the contribution of these cells to pulmonary fibrosis is still debated, these data do show that miR‐29 mimicry can also block collagen induction in this cell type. Additionally, we looked at miR‐29 effects in the macrophage line, Thp‐1, but could not observe collagen expression in the cells, regardless of stimulation (P. Latimer and R. Montgomery, unpublished data). These data suggest miR‐29b mimicry is able to blunt collagen‐induced expression in fibroblasts and epithelial cells. These data are all in line with a recent paper by Xiao et al (2012), in which they showed that gene transfer of miR‐29 using a Sleeping Beauty‐transposon system was capable of preventing and treating bleomycin‐induced pulmonary fibrosis, further underscoring the therapeutic potential for increasing miR‐29.

Our data suggests the feasibility of using microRNA mimics to restore the function of lost or downregulated miRNAs. However, it is important to note that because RISC incorporation is required for appropriate miRNA function, the allowed chemical modifications are limited; thus, miRNA mimics are far less stable than antimiR chemistries, and dosage and administration regiments need to be worked out in detail as the doses used in most animal studies to date are probably significantly higher than what would be therapeutically acceptable. Another potential issue with miRNA replacement therapies is the challenge of restoring the level of a downregulated miRNA while preventing the introduction of supraphysiological levels of the miRNA. Additionally, although a miRNA mimic can have therapeutic use, potential off target effects of miRNA mimicry can occur as a result of delivery to tissues or cells that do not normally contain the miRNA of interest. Thus, targeting those patients with exceedingly low levels of the miRNA and delivery to the appropriate cell type or tissue are important aspects of effective miRNA mimicry. In the case of pulmonary fibrosis, this may suggest that direct delivery through the inhaled route may be an appealing alternative to traditional routes of administration. Lastly, it should also be noted that double‐stranded miRNA mimics can potentially induce a non‐specific interferon response through toll‐like receptors (Peacock et al, 2011), and thus careful assessment of dosing and off target effects will be required.

Despite the fact that the currently available mimic chemistries require further optimization to increase stability and efficacy, our data clearly support the notion that miRNA mimics can be used to therapeutically increase miRNA levels and that miR‐29 is a potent therapeutic miRNA for treating pulmonary fibrosis. Validating these data in additional models of pulmonary fibrosis will be important before we can translate these data into a clinical setting. The lack of observable effects on gene expression under baseline conditions might relieve some of the concerns regarding the systemic gene regulatory effects of miRNA mimics. Our data, combined with the fact that the first synthetic formulated miRNA mimic for miR‐34 is currently entering a Phase 1 trial in patients with primary liver cancer (Bouchie, 2013), provides great promise for mimics as novel miRNA therapeutics.