microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease and therefore represent a potential new class of targets for therapeutic intervention. Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg(-1) LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg(-1) LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.

The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.

MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in many developmental and cellular processes. Moreover, there is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human diseases. Indeed, disease-associated miRNAs represent a new class of targets for the development of miRNA-based therapeutic modalities, which may yield patient benefits unobtainable by other therapeutic approaches. The recent explosion in miRNA research has accelerated the development of several computational and experimental approaches for probing miRNA functions in cell culture and in vivo. In this review, we focus on the use of antisense oligonucleotides (antimiRs) in miRNA inhibition for loss-of-function studies. We provide an overview of the currently employed antisense chemistries and their utility in designing antimiR oligonucleotides. Furthermore, we describe the most commonly used in vivo delivery strategies and discuss different approaches for assessment of miRNA inhibition and potential off-target effects. Finally, we summarize recent progress in antimiR mediated pharmacological inhibition of disease-associated miRNAs, which shows great promise in the development of novel miRNA-based therapeutics.

MicroRNAs are dysregulated in a setting of heart disease and have emerged as promising therapeutic targets. MicroRNA-34 family members (miR-34a, -34b, and -34c) are up-regulated in the heart in response to stress. In this study, we assessed whether inhibition of the miR-34 family using an s.c.-delivered seed-targeting 8-mer locked nucleic acid (LNA)-modified antimiR (LNA-antimiR-34) can provide therapeutic benefit in mice with preexisting pathological cardiac remodeling and dysfunction due to myocardial infarction (MI) or pressure overload via transverse aortic constriction (TAC). An additional cohort of mice subjected to MI was given LNA-antimiR34a (15-mer) to inhibit miR-34a alone as a comparison for LNAantimiR-34. LNA-antimiR-34 (8-mer) efficiently silenced all three miR-34 family members in both cardiac stress models and attenuated cardiac remodeling and atrial enlargement. In contrast, inhibition of miR-34a alone with LNA-antimiR-34a (15-mer) provided no benefit in the MI model. In mice subjected to pressure overload, LNAantimiR-34 improved systolic function and attenuated lung congestion, associated with reduced cardiac fibrosis, increased angiogenesis, increased Akt activity, decreased atrial natriuretic peptide gene expression, and maintenance of sarcoplasmic reticulum Ca 2+ ATPase gene expression. Improved outcome in LNA-antimiR-34-treated MI and TAC mice was accompanied by up-regulation of several direct miR-34 targets, including vascular endothelial growth factors, vinculin, protein O-fucosyltranferase 1, Notch1, and semaphorin 4B. Our results provide evidence that silencing of the entire miR-34 family can protect the heart against pathological cardiac remodeling and improve function. Furthermore, these data underscore the utility of seed-targeting 8-mer LNA-antimiRs in the development of new therapeutic approaches for pharmacologic inhibition of disease-implicated miRNA seed families.heart failure | PI3K | pathological hypertrophy | physiological hypertrophy | cardiomyocyte

MicroRNAs inhibit mRNA translation or promote mRNA degradation by binding complementary sequences in 3′ untranslated regions of target mRNAs. MicroRNA-21 (miR-21) is upregulated in response to cardiac stress, and its inhibition by a cholesterol-modified antagomir has been reported to prevent cardiac hypertrophy and fibrosis in rodents in response to pressure overload. In contrast, we have shown here that miR-21-null mice are normal and, in response to a variety of cardiac stresses, display cardiac hypertrophy, fibrosis, upregulation of stress-responsive cardiac genes, and loss of cardiac contractility comparable to wildtype littermates. Similarly, inhibition of miR-21 through intravenous delivery of a locked nucleic acid-modified (LNA-modified) antimiR oligonucleotide also failed to block the remodeling response of the heart to stress. We therefore conclude that miR-21 is not essential for pathological cardiac remodeling.

Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD.Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155-deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic antimiR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.

microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases.