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

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-ß1 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.

A synthetic microRNA-92a inhibitor (MRG-110) accelerates angiogenesis and wound healing in diabetic and nondiabetic wounds.

There is a strong unmet need for new therapeutics to accelerate wound healing across both chronic and acute indications. It is well established that local tissue hypoxia, vascular insufficiency, and/or insufficient angiogenesis contribute to inadequate wound repair in the context of diabetic foot ulcers as well as to other chronic wounds such as venous stasis and pressure ulcers. microRNA-92a-3p (miR-92a) is a potent antiangiogenic miRNA whose inhibition has led to increases in angiogenesis in multiple organ systems, resulting in an improvement in function following myocardial infarction, limb ischemia, vascular injury, and bone fracture. Due to their pro-angiogenic effects, miR-92a inhibitors offer potential therapeutics to accelerate the healing process in cutaneous wounds as well. This study investigated the effect of a development stage locked nucleic acid-modified miR-92a inhibitor, MRG-110, in excisional wounds in db/db mice and in normal pigs. In both acute and chronic wounds, MRG-110 increased granulation tissue formation as assessed by histology, angiogenesis as assessed by immunohistochemistry and tissue perfusion, and wound healing as measured by time to closure and percent closure over time. The effects of MRG-110 were greater than those that were observed with the positive controls rhVEGF-165 and rhPDGF-BB, and MRG-110 was at least additive with rhPDGF-BB when co-administered in db/db mouse wounds. MRG-110 was found to up-regulate expression of the pro-angiogenic miR-92a target gene integrin alpha 5 in vitro in both human vascular endothelial cells and primary human skin fibroblasts and in vivo in mouse skin, demonstrating its on-target effects in vitro and in vivo. Additional safety endpoints were assessed in both the mouse and pig studies with no safety concerns noted. These studies suggest that MRG-110 has the potential to accelerate both chronic and acute wound healing and these data provide support for future clinical trials of MRG-110.

MicroRNA-214 antagonism protects against renal fibrosis.

Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti-miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-ß signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-ß blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-ß signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney.

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure.

AIMS: Recent studies have shown that microRNAs (miRNAs), besides being potent regulators of gene expression, can additionally serve as circulating biomarkers of disease. The aim of this study is to determine if plasma miRNAs can be used as indicators of disease progression or therapeutic efficacy in hypertension-induced heart disease. METHODS AND RESULTS: In order to define circulating miRNAs that change during hypertension-induced heart failure and that respond to therapeutic treatment, we performed miRNA arrays on plasma RNA from hypertensive rats that show signs of heart failure. Array analysis indicated that approximately one-third of the miRNAs on the array are detectable in plasma. Quantitative real-time polymerase chain reaction (PCR) analysis for a selected panel of miRNAs indicated that circulating levels of miR-16, miR-20b, miR-93, miR-106b, miR-223, and miR-423-5p were significantly increased in response to hypertension-induced heart failure, while this effect was blunted in response to treatment with antimiR-208a as well as an ACE inhibitor. Moreover, treatment with antimiR-208a resulted in a dramatic increase in one miRNA, miR-19b. A time course study indicated that several of these miRNA changes track with disease progression. CONCLUSIONS: Circulating levels of miRNAs are responsive to therapeutic interventions and change during the progression of hypertension-induced heart disease.

Inhibition of miR-15 protects against cardiac ischemic injury.

RATIONALE: Myocardial infarction (MI) is a leading cause of death worldwide. Because endogenous cardiac repair mechanisms are not sufficient for meaningful tissue regeneration, MI results in loss of cardiac tissue and detrimental remodeling events. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in a sequence dependent manner. Our previous data indicate that miRNAs are dysregulated in response to ischemic injury of the heart and actively contribute to cardiac remodeling after MI. OBJECTIVE: This study was designed to determine whether miRNAs are dysregulated on ischemic damage in porcine cardiac tissues and whether locked nucleic acid (LNA)-modified anti-miR chemistries can target cardiac expressed miRNAs to therapeutically inhibit miR-15 on ischemic injury. METHODS AND RESULTS: Our data indicate that the miR-15 family, which includes 6 closely related miRNAs, is regulated in the infarcted region of the heart in response to ischemia-reperfusion injury in mice and pigs. LNA-modified chemistries can effectively silence miR-15 family members in vitro and render cardiomyocytes resistant to hypoxia-induced cardiomyocyte cell death. Correspondingly, systemic delivery of miR-15 anti-miRs dose-dependently represses miR-15 in cardiac tissue of both mice and pigs, whereas therapeutic targeting of miR-15 in mice reduces infarct size and cardiac remodeling and enhances cardiac function in response to MI. CONCLUSIONS: Oligonucleotide-based therapies using LNA-modified chemistries for modulating cardiac miRNAs in the setting of heart disease are efficacious and validate miR-15 as a potential therapeutic target for the manipulation of cardiac remodeling and function in the setting of ischemic injury.

Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure.

BACKGROUND: Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown. METHODS AND RESULTS: Here, we show that systemic delivery of an antisense oligonucleotide induces potent and sustained silencing of miR-208a in the heart. Therapeutic inhibition of miR-208a by subcutaneous delivery of antimiR-208a during hypertension-induced heart failure in Dahl hypertensive rats dose-dependently prevents pathological myosin switching and cardiac remodeling while improving cardiac function, overall health, and survival. Transcriptional profiling indicates that antimiR-208a evokes prominent effects on cardiac gene expression; plasma analysis indicates significant changes in circulating levels of miRNAs on antimiR-208a treatment. CONCLUSIONS: These studies indicate the potential of oligonucleotide-based therapies for modulating cardiac miRNAs and validate miR-208 as a potent therapeutic target for the modulation of cardiac function and remodeling during heart disease progression.

Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication.

To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNA-treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation.

Selection, design, and characterization of a new potentially therapeutic ribozyme.

An in vitro selection was designed to identify RNA-cleaving ribozymes predisposed for function as a drug. The selection scheme required the catalyst to be trans-acting with phosphodiesterase activity targeting a fragment of the Kras mRNA under simulated physiological conditions. To increase stabilization against nucleases and to offer the potential for improved functionality, modified sequence space was sampled by transcribing with the following NTPs: 2'-F-ATP, 2'-F-UTP, or 2'-F-5-[(N-imidazole-4-acetyl) propylamine]-UTP, 2'-NH2-CTP, and GTP. Active motifs were identified and assessed for their modified NMP and divalent metal dependence. The minimization of the ribozyme's size and the ability to substitute 2'-OMe for 2'-F and 2'-NH2 moieties yielded the motif from these selections most suited for both nuclease stability and therapeutic development. This motif requires only two 2'-NH2-Cs and functions as a 36-mer. Its substrate sequence requirements were determined to be 5'-Y-G-H-3'. Its half-life in human serum is >100 h. In physiologically relevant magnesium concentrations [approximately 1 mM] its kcat = 0.07 min(-1), Km = 70 nM. This report presents a novel nuclease stable ribozyme, designated Zinzyme, possessing optimal activity in simulated physiological conditions and ready for testing in a therapeutic setting.