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PDF file - 289K, Oligonucleotide sequences cloned into the pmirGLO vector (S1).

PDF file - 776K, Mouse SHH medulloblastomas do not show amplification of the miR-17~92 cluster (S1); 8-mer LNA-antimiRs inhibit miR-17 and miR-19a (S2); In vitro treatment with 8-mer LNA-antimiRs of MB cells from independently derived spontaneous MBs in Ptch1+/-;Cdkn2c-/- mice inhibits cell proliferation (S3); MiR-17~92 does not target Smad5 or Smad4 (S4); MiR levels of transplantable and non-transplantable Ptch1+/-;Cdkn2c-/- and Ptch1+/-;Trp53-/- flank tumors (S5); FAM-labeled tiny LNAs are present in tissues 14 days after MB cell pre-treatment and transplantation (S6).

Significance T cells control many immune functions, with Th17 cells critical in regulating inflammation. Following activation, T cells undergo metabolic reprogramming and utilize glycolysis to increase the ATP availability. Epigenetic mechanisms controlling metabolic functions in T cells are currently not well-defined. Here, we establish an epigenetic link between the histone H3K27me3 demethylases KDM6A/B and the coordination of a metabolic response. Inhibition of KDM6A/B leads to global increases in the repressive H3K27me3 histone mark, resulting in down-regulation of key transcription factors, followed by metabolic reprogramming and anergy. This work suggests a critical role of H3K27 demethylase enzymes in maintaining Th17 functions by controlling metabolic switches. Short-term treatment with KDM6 enzyme inhibitors may be useful in the therapy of chronic inflammatory diseases., T helper (Th) cells are CD4+ effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.

Key points MicroRNA (miRNA)-based therapies are in development for numerous diseases, including heart disease. Currently, very limited basic information is available on the regulation of specific miRNAs in male and female hearts in settings of disease. The identification of sex-specific miRNA signatures has implications for translation into the clinic and suggests the need for customised therapy. In the present study, we found that a miRNA-based treatment inhibiting miRNA-34a (miR-34a) was more effective in females in a setting of moderate dilated cardiomyopathy than in males. Furthermore, the treatment showed little benefit for either sex in a setting of more severe dilated cardiomyopathy associated with atrial fibrillation. The results highlight the importance of understanding the effect of miRNA-based therapies in cardiac disease settings in males and females. Abstract MicroRNA (miRNA)-34a (miR-34a) is elevated in the diseased heart in mice and humans. Previous studies have shown that inhibiting miR-34a in male mice in settings of pathological cardiac hypertrophy or ischaemia protects the heart against progression to heart failure. Whether inhibition of miR-34a protects the female heart is unknown. Furthermore, the therapeutic potential of silencing miR-34a in settings of dilated cardiomyopathy (DCM) and atrial fibrillation (AF) has not been assessed previously. In the present study, we examined the effect of silencing miR-34a in males and females in (1) a model of moderate DCM and (2) a model of severe DCM with AF. The cardiac disease models were administered with a locked nucleic acid-modified oligonucleotide (LNA-antimiR-34a) at 6-7 weeks of age when the models display cardiac dysfunction and conduction abnormalities. Cardiac function and morphology were measured 6 weeks after treatment. In the present study, we show that inhibition of miR-34a provides more protection in the DCM model in females than males. Disease prevention in LNA-antimiR-34a treated DCM female mice was characterized by attenuated heart enlargement and lung congestion, lower expression of cardiac stress genes (B-type natriuretic peptide, collagen gene expression), less cardiac fibrosis and better cardiac function. There was no evidence of significant protection in the severe DCM and AF model in either sex. Sex- and treatment-dependent regulation of miRNAs was also identified in the diseased heart, and may explain the differential response of males and females. These studies highlight the importance of examining the impact of miRNA-based drugs in both sexes and under different disease conditions.

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain–containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function.

Medulloblastoma, originating in the cerebellum, is the most common malignant brain tumor in children. Medulloblastoma consists of four major groups where constitutive activation of the Sonic Hedgehog (SHH) signaling pathway is a hallmark of one group. Mouse and human SHH medulloblastomas exhibit increased expression of microRNAs encoded by the miR-17∼92 and miR-106b∼25 clusters compared with granule progenitors and postmitotic granule neurons. Here, we assessed the therapeutic potential of 8-mer seed-targeting locked nucleic acid (LNA)-modified anti-miR oligonucleotides, termed tiny LNAs, that inhibit microRNA seed families expressed by miR-17∼92 and miR-106b∼25 in two mouse models of SHH medulloblastomas. We found that tumor cells (medulloblastoma cells) passively took up 8-mer LNA-anti-miRs and specifically inhibited targeted microRNA seed-sharing family members. Inhibition of miR-17 and miR-19a seed families by anti-miR-17 and anti-miR-19, respectively, resulted in diminished tumor cell proliferation in vitro. Treatment of mice with systemic delivery of anti-miR-17 and anti-miR-19 reduced tumor growth in flank and brain allografts in vivo and prolonged the survival of mice with intracranial transplants, suggesting that inhibition of the miR-17∼92 cluster family by 8-mer LNA-anti-miRs might be considered for the treatment of SHH medulloblastomas. Cancer Res; 73(23); 7068–78. ©2013 AACR.

MicroRNAs are important regulators of gene expression in normal development and disease. miR-9 is overexpressed in several cancer forms, including brain tumours, hepatocellular carcinomas, breast cancer and Hodgkin lymphoma (HL). Here we demonstrated a relevance for miR-9 in HL pathogenesis and identified two new targets Dicer1 and HuR. HL is characterized by a massive infiltration of immune cells and fibroblasts in the tumour, whereas malignant cells represent only 1% of the tumour mass. These infiltrates provide important survival and growth signals to the tumour cells, and several lines of evidence indicate that they are essential for the persistence of HL. We show that inhibition of miR-9 leads to derepression of DICER and HuR, which in turn results in a decrease in cytokine production by HL cells followed by an impaired ability to attract normal inflammatory cells. Finally, inhibition of miR-9 by a systemically delivered antimiR-9 in a xenograft model of HL increases the protein levels of HuR and DICER1 and results in decreased tumour outgrowth, confirming that miR-9 actively participates in HL pathogenesis and points to miR-9 as a potential therapeutic target.

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.

TRIM22 (Staf50) is an interferon (IFN)-inducible protein with unknown function. Recently, we identified TRIM22 as a novel p53 target gene and showed that overexpression of TRIM22 inhibits the clonogenic growth of monoblastic U937 cells. Moreover, expression of TRIM22 is high in lymphoid tissue, and levels decrease during T lymphocyte activation with CD3/CD2/CD28, suggesting that TRIM22 could exert antiproliferative effects. Here, a prominent increase in TRIM22 levels is observed during activation with interleukin-2 (IL-2) or IL-15 in contrast to the decrease observed during CD3/CD2/CD28-induced activation. However, stimulation of cells in these experiments was performed on crude T lymphocytes, allowing indirect regulation between different lymphocyte subtypes to take place. Therefore, to prevent interaction between different lymphocyte subtypes, expression of TRIM22 was examined during activation of sorted T lymphocyte subpopulations. In contrast to the marked changes of TRIM22 during activation of crude T lymphocytes, in isolated subpopulations, TRIM22 expression was not significantly affected in spite of IL-2-induced or CD3/CD2/CD28-induced activation. In addition, in contrast to the TRIM22 mouse ortholog Rpt-1, TRIM22 did not affect levels of CD25 (IL-2Ralpha) mRNA. Our data suggest a more complex role for TRIM22 during T lymphocyte activation than merely as an antiproliferative factor. TRIM22 probably has an activation stage-specific role connected to the paracrine crosstalk during T lymphocyte activation.

MicroRNAs are short non-coding RNAs that regulate gene expression. Previously, in a genome-wide screen, we found deregulation of microRNA expression in psoriasis skin. MicroRNA-21 (miR-21) is one of the microRNAs significantly up-regulated in psoriasis skin lesions. To identify the cell type responsible for the increased miR-21 level, we compared expression of miR-21 in epidermal cells and dermal T cells between psoriasis and healthy skin and found elevated levels of miR-21 in psoriasis in both cell types. In cultured T cells, expression of miR-21 increased markedly upon activation. To explore the function of miR-21 in primary human T helper cells, we inhibited miR-21 using a tiny seed-targeting LNA-anti-miR. Specific inhibition of miR-21 increased the apoptosis rate of activated T cells. Our results suggest that miR-21 suppresses apoptosis in activated T cells, and thus, overexpression of miR-21 may contribute to T cell-derived psoriatic skin inflammation.

Psoriasis is a common inflammatory skin disease with limited treatment options that is characterized by a complex interplay between keratinocytes, immune cells, and inflammatory mediators. MicroRNAs (miRNAs) are regulators of gene expression and play critical roles in many human diseases. A number of miRNAs have been described to be up-regulated in psoriasis, but their causal contribution to disease development has not been demonstrated. We confirm that miR-21 expression is increased in epidermal lesions of patients with psoriasis and that this leads to reduced epidermal TIMP-3 (tissue inhibitor of matrix metalloproteinase 3) expression and activation of TACE (tumor necrosis factor-α-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17). Using patient-derived skin samples and mouse models of psoriasis, we demonstrate that increased miR-21 may be a consequence of impaired transcriptional activity of Jun/activating protein 1 (AP-1), leading to activation of the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (Stat3) pathway. Inhibition of miR-21 by locked nucleic acid (LNA)-modified anti-miR-21 compounds ameliorated disease pathology in patient-derived psoriatic skin xenotransplants in mice and in a psoriasis-like mouse model. Targeting miR-21 may represent a potential therapeutic option for the treatment of psoriasis.

Therapeutic inhibition of the miR-34 family (miR-34a,-b,-c), or miR-34a alone, have emerged as promising strategies for the treatment of cardiac pathology. However, before advancing these approaches further for potential entry into the clinic, a more comprehensive assessment of the therapeutic potential of inhibiting miR-34a is required for two key reasons. First, miR-34a has ∼40% fewer predicted targets than the miR-34 family. Hence, in cardiac stress settings in which inhibition of miR-34a provides adequate protection, this approach is likely to result in less potential off-target effects. Secondly, silencing of miR-34a alone may be insufficient in settings of established cardiac pathology. We recently demonstrated that inhibition of the miR-34 family, but not miR-34a alone, provided benefit in a chronic model of myocardial infarction. Inhibition of miR-34 also attenuated cardiac remodeling and improved heart function following pressure overload, however, silencing of miR-34a alone was not examined. The aim of this study was to assess whether inhibition of miR-34a could attenuate cardiac remodeling in a mouse model with pre-existing pathological hypertrophy. Mice were subjected to pressure overload via constriction of the transverse aorta for four weeks and echocardiography was performed to confirm left ventricular hypertrophy and systolic dysfunction. After four weeks of pressure overload (before treatment), two distinct groups of animals became apparent: (1) mice with moderate pathology (fractional shortening decreased ∼20%) and (2) mice with severe pathology (fractional shortening decreased ∼37%). Mice were administered locked nucleic acid (LNA)-antimiR-34a or LNA-control with an eight week follow-up. Inhibition of miR-34a in mice with moderate cardiac pathology attenuated atrial enlargement and maintained cardiac function, but had no significant effect on fetal gene expression or cardiac fibrosis. Inhibition of miR-34a in mice with severe pathology provided no therapeutic benefit. Thus, therapies that inhibit miR-34a alone may have limited potential in settings of established cardiac pathology.

MicroRNAs (miRNAs) regulate many aspects of human biology. They target mRNAs for translational repression or degradation through base-pairing with 3’ UTRs, primarily via seed sequences (nucleotides 2-8 in the mature miRNA sequence). A number of individual miRNAs and miRNA families share seed sequences and targets, but differ in the sequences outside of the seed. miRNAs have been implicated in the etiology of a wide variety of human diseases and therefore represent promising therapeutic targets. However, potential redundancy and compensatory action of different miRNAs sharing the same seed sequence, and the challenge of simultaneously targeting miRNAs that differ significantly in non-seed sequences complicates therapeutic targeting approaches. We recently demonstrated effective inhibition of entire miRNA families using seed-targeting 8-mer locked nucleic acid (LNA)-modified antimiRs in short-term experiments in mammalian cells and in mice. However, the long-term efficacy and safety of this approach in higher organisms, such as humans and non-human primates, has not been determined. Here, we show that pharmacological inhibition of the miR-33 family, key regulators of cholesterol/lipid homeostasis, by a subcutaneously delivered 8-mer LNA-modified antimiR in obese and insulin-resistant non-human primates results in de-repression of miR-33 targets, such as ABCA1, increases circulating high-density lipoprotein-cholesterol (HDL-C), and is well tolerated over 108 days of treatment. These findings demonstrate the efficacy and safety of an 8-mer LNA-antimiR against a miRNA family in a non-human primate metabolic disease model, suggesting that this could be a feasible approach for therapeutic targeting of miRNA families sharing the same seed sequence in human diseases.

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-antimiR-34a (15-mer) to inhibit miR-34a alone as a comparison for LNA-antimiR-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, LNA-antimiR-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.

miR-155 acts as an oncogenic miR in B-cell lymphoproliferative disorders, including Waldenstrom macroglobulinemia (WM) and chronic lymphocytic leukemia, and is therefore a potential target for therapeutic intervention. However, efficient targeting of miRs in tumor cells in vivo remains a significant challenge for the development of miR-155–based therapeutics for the treatment of B-cell malignancies. In the present study, we show that an 8-mer locked nucleic acid anti–miR-155 oligonucleotide targeting the seed region of miR-155 inhibits WM and chronic lymphocytic leukemia cell proliferation in vitro. Moreover, anti–miR-155 delivered systemically showed uptake in the BM CD19+ cells of WM-engrafted mice, resulting in the up-regulation of several miR-155 target mRNAs in these cells, and decreased tumor growth significantly in vivo. We also found miR-155 levels to be elevated in stromal cells from WM patients compared with control samples. Interestingly, stromal cells from miR-155–knockout mice led to significant inhibition of WM tumor growth, indicating that miR-155 may also contribute to WM proliferation through BM microenvironmental cells. The results of the present study highlight the therapeutic potential of anti–miR-155–mediated inhibition of miR-155 in the treatment of WM.

Rationale: Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown. Objective: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. Methods and Results: Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. Conclusions: 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.

MicroRNAs (miRNAs) have been uncovered as important posttranscriptional regulators of nearly every biological process in the cell. Furthermore, mounting evidence implies that miRNAs play key roles in the pathogenesis of cancer and that many miRNAs can function either as oncogenes or tumor suppressors. Thus, miRNAs have rapidly emerged as promising targets for the development of novel anticancer therapeutics. The development of miRNA-based cancer therapeutics relies on restoring the activity of tumor suppressor miRNAs using double-stranded miRNA mimics or inhibition of oncogenic miRNAs using single-stranded antisense oligonucleotides, termed antimiRs. In the present review, we focus on recent advancements in the discovery and development of miRNA-based cancer therapeutics using these 2 approaches. In addition, we summarize selected studies, in which modulation of miRNA activity in preclinical cancer models in vivo has demonstrated promising therapeutic potential.

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 anti–miR-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.

MicroRNAs are short non-coding RNAs that regulate gene expression. Previously, in a genome-wide screen, we found deregulation of microRNA expression in psoriasis skin. MicroRNA-21 (miR-21) is one of the microRNAs significantly up-regulated in psoriasis skin lesions. To identify the cell type responsible for the increased miR-21 level, we compared expression of miR-21 in epidermal cells and dermal T cells between psoriasis and healthy skin and found elevated levels of miR-21 in psoriasis in both cell types. In cultured T cells, expression of miR-21 increased markedly upon activation. To explore the function of miR-21 in primary human T helper cells, we inhibited miR-21 using a tiny seed-targeting LNA-anti-miR. Specific inhibition of miR-21 increased the apoptosis rate of activated T cells. Our results suggest that miR-21 suppresses apoptosis in activated T cells, and thus, overexpression of miR-21 may contribute to T cell-derived psoriatic skin inflammation.

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 (miRNAs) have been implicated in B cell lineage commitment, regulation of T cell differentiation, TCR signalling, regulation of IFN signalling, and numerous other immunological processes. However, their function in autoimmunity, and specifically in systemic lupus erythematosus (SLE), remains poorly understood. B6.Sle123 is a spontaneous genetic mouse model of SLE characterized by autoantibody production, lymphosplenomegaly, and glomerulonephritis. We identified several differentially regulated miRNAs in B and T lymphocytes of B6.Sle123 mice. We found that miR-21 expression in lupus B and T cells is up-regulated and that in vivo silencing of miR-21 using a tiny seed-targeting LNA reversed splenomegaly, one of the cardinal manifestations of autoimmunity in B6.Sle123 mice, and de-repressed PDCD4 expression in vivo and in vitro. In addition, treatment with anti-miR-21 altered CD4/CD8 T cell ratios and reduced Fas receptor-expressing lymphocyte populations. Our study shows that tiny LNAs can be used to efficiently antagonize endogenous miRNAs in peripheral lymphocytes in vivo and in primary lymphocytes cultured ex vivo and can alter the course of a spontaneous genetic disease in mice.

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 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 wild. type 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. 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 wild-type 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.

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 interferon inducible protein TRIM22 has been identified as a p53 target gene, with possible involvement in proliferation and differentiation of leukaemia cells. Here, the expression levels of TRIM22 during haematopoietic differentiation are characterised. Expression of TRIM22 correlates inversely to differentiation, as TRIM22 is highly expressed in CD34(+) human bone marrow progenitor cells, but declines in mature populations. The erythroid lineage appears as a special case, as TRIM22 expression shows an extreme decrease during late erythroid maturation and is completely undetectable in nucleated erythroid populations in contrast to other lineages. In conclusion, our data could suggest lineage-specific roles for TRIM22 during haematopoietic differentiation.

The tumor suppressor gene p53 is a transcription factor that mediates both cell cycle arrest and apoptosis. Interestingly, p53 also induces differentiation of a number of tissues, including leukemic cells. However, although p53-mediated differentiation of leukemic U-937 cells depends on the transcriptional activity of p53, a p53 target gene mediating differentiation has hitherto not been identified. To screen for novel p53 target genes in leukemic cells, a cDNA microarray analysis was performed with U-937-4/ptsp53 cells, expressing a temperature-sensitive p53 mutant. We report that transcription of the Staf50 (stimulated transacting factor of 50 kDa) gene is upregulated in response to wild-type p53 in U-937-4, K562 and MCF-7 cells. Staf50 was directly activated by p53, as determined by the independence of de novo protein synthesis. Moreover, while the proximal promoter of Staf50 was found not to be p53 responsive, a functional enhancer-like p53-response element in intron 1 of the Staf50 gene was identified that was also transactivated by the p53-family member p73. Direct binding of p53 to the response element was shown by electrophoretic mobility shift analysis. Ectopic expression of Staf50 in U-937 cells resulted in reduced clonogenic growth. Moreover, levels of endogenous Staf50 mRNA correlated to all-trans retinoic acid-induced differentiation of promyelocytic NB-4 and HL60 cells, suggesting that Staf50 could be involved in proliferation and/or differentiation of leukemic cells.

Epigenetic dysregulation plays an important role in myeloma pathogenesis. Histone methyltransferases have been implicated in disease initiation and progression; for example MMSET, acting at H3K36 is the oncogenic driver in the 15% of patients with the initiating event t(4;14), whilst MLL, acting at H3K4, is recurrently mutated. It is logical to presume that enzymes with the opposite action at histone methyl marks, the demethylases, also play a role in myeloma and recurrent mutations and deletions are seen in KDM6A (UTX), a H3K27 demethylase. We further investigated the role of demethylases using linked gene expression and survival data from 3 large studies of newly diagnosed patients, a small molecule 2-oxoglutarate dependent histone demethylase inhibitor (GSK-J4, inhibitor of KDM5B+KDM6A/B) and knockdown with locked nucleic acids (LNAs), to define a potential new targeted therapeutic strategy for myeloma patients. Linked gene expression and survival data were available for the MRC Myeloma IX study (n=259), HOVON65/GMMG study (n=246) and Total Therapy 2+3 (TT2+3) studies (n=559). Patients with high expression of KDM5B, a H3K4 demethylase, had a significantly shorter overall survival (OS) than patients with lower expression. (Myeloma IX: high expression median OS 31.6 months vs 45.5 months, log rank p=0.020. HOVON65/GMMG-H4: high expression median OS 54.8 months vs median OS not reached, log rank p Inhibition of viability with GSK-J4 treatment occurred in a time and dose-dependent manner in a panel of 11 myeloma cell lines; GC50s ranging from 0.5 -3uM at 72 hours. Treatment induced apoptosis was demonstrated by increased annexin/PI staining, by flow cytometry, and increased caspase activity in the Caspase-Glo® 3/7 luminescent assay. Gene expression arrays (Illumina HumanHT12-v4), using RNA extracted from JJN-3 cells treated at 2uM for 6 and 24 hours, showed induction of expression of the metallothionein family of genes including MT1X, associated with intracellular metal-ion homeostasis, and genes associated with the cellular stress response including DDIT3 and INHBE, both ATF4 downstream target genes. These genes were validated by RT-PCR in 5 myeloma cell lines (e.g. RPMI-8226 24hrs 2.5uM GSK-J4 treatment mean RQ values: MT1X 176, DDIT 45, INHBE 70). Locked Nucleic Acid knockdown targeting KDM6A+6B reduced cell viability and induced the same transcriptional response seen with GSK-J4. Reduced expression of KDM5B was also detected following treatment with this LNA. Knockdown targeting KDM5B alone did not affect viability or induce the same transcriptional response, suggesting inhibition of all three demethylases is required for the response seen with GSK-J4. Chip-seq analysis of JJN-3 cells treated at 2uM GSK-J4 for 6 and 48 hours or LNA knockdown of KDM6A+B for 7 days, showed increased H3K4me3 marks globally, with specific peaks upstream of the metallothionein genes and ATF4 target genes, indicating that the effects seen with GSK-J4 treatment are mediated via an epigenetic mechanism. To evaluate the relevance of our findings to human disease, viability experiments using CD138+ plasma cells from myeloma patients were performed for cells alone and in co-culture with bone marrow stroma. GSK-J4 treatment reduced the viability of CD138 selected cells both alone and in co-culture compared to control. (Mean viability at 48hrs cells alone: 4uM GSK-J4 46%, 8uM 27%, 12uM 5%, co-cultured: 4uM 76%, 8uM 59%, 12uM 26%.) The transcriptional response seen in cell lines was conserved in primary patient cells following 24hr 2uM GSK-J4 treatment (Mean RQ values MT1X 105, INHBE 13, DDIT3 4). Treatment of bone marrow stromal cells and peripheral blood mononuclear cells with GSK-J4 demonstrated a therapeutic index between normal and malignant tissue. This work demonstrates a pro-apoptotic effect of demethylase inhibition in myeloma, a class of drug targets not previously investigated for this disease. A complex interplay between the demethylases affected by the GSK-J4 chemical probe is demonstrated and suggests that the modulation of KDM5B, KDM6A and KDM6B simultaneously delivers the apoptotic signal. The enrichment of H3K4me3 marks at genes involved in the transcriptional response along with survival data suggests KDM5B might be key to the response. Disclosures Lindow: Santaris: Employment. Obad:Santaris: Employment. Oerum:Santaris: Employment.

e13567 Background: MicroRNAs (miRs) have emerged as powerful regulators of cellular processes and are frequently deregulated in cancer. Our previous work showed that miR-31 was overexpressed in human and murine lung cancers and repression of miR-31 inhibits lung cancer proliferation and tumorigenesis. Novel miR-31-targeting compounds were developed using a locked nucleic acid (LNA) drug platform. Compounds were tested in cell-based assays to assess levels of miR-31, miR-31-target genes and cell proliferation. A lead compound was chosen and we are currently examining its in vivo activity in human lung cancer cells and in murine transgenic lung tumor models. Methods: We previously developed a murine model of lung cancer by specifically overexpressing human cyclin E in the lung (CEO mice). The ED1 cell line was derived from a CEO mouse lung tumor. ED1 cells were transfected with the miR-31-LNA-targeting compounds and miR-31 levels and target genes quantified versus controls using RT-PCR assays. Cell proliferation was assessed using the CellTiter-Glo assay. Microarray analysis was performed to further investigate novel miR-31 target genes that were validated using independent RT-PCR assays. A pharmacodynamic study (5 daily tail vein injections) was performed in CEO mice that develop lung tumors spontaneously. Lung tissues were harvested 24 hours after the final dose and analyzed for immunohistochemical biomarkers of drug response (Ki67 and cyclin E). Results: Novel LNA anti-miR-31 compounds decreased miR-31 levels and proliferation rates in murine and human lung cancer cells. The compound, as expected, augmented expression of a known miR-31 target, LATS2. The microarray analysis identified over 100 genes with 2-fold elevated expression in anti-miR-31 treated cells. Validated novel miR-31 targets include GADD45a, Trp53inp1, Txnip and Slc40a1. Preclinical studies in mouse models are ongoing. Conclusions: Targeting miR-31 using LNA–based therapy inhibits cell growth in lung cancer cells and is a promising therapeutic approach for the treatment of lung cancer. Successful preclinical activity of these compounds will warrant translation into the clinic in proof of principle trials to assess pharmacodynamic and pharmacokinetic outcomes.

Abstract 2728 Background. We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods. WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results. The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn't exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary. A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures: Roccaro: Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.