Anita Seto

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.

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.

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

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.

Circulating microRNAs to identify human heart failure

MicroRNAs (miRNAs) are short, non-coding RNAs that act in a protein–nucleic acid complex, called the RNA-induced silencing complex (RISC), to repress gene expression—either through destabilization of an mRNA, or through inhibition of protein translation. The mechanism of miRNA action is mainly through complementary binding of the 5′ end of the miRNA to the 3′-untranslated region (UTR) of the targeted transcript. Although short in length, the effects of miRNA action are far reaching: some estimate that as much as 50% of the genome may be regulated by these petite RNAs. Further attesting to their important biological function, miRNAs are conserved from plants to human and are found in nearly all cell types (mirbase.org). In the last few years, the dysregulation of tissue expression levels of miRNAs has been causally linked to a wide variety of diseases, including cardiac disease. –5 Based on these findings, therapeutic applications for miRNA regulation are currently being explored. Recent rodent, non-human primate, and even human efficacy data (http://www.santaris.com/news/2011/ 10/03/santaris-pharmareport-new-clinical-data-miravirsen-phase-2a -study-treat-hepatitisc) indicate that antimiR compounds that inhibit specific miRNAs have the potential to become an entire new class of drugs. In addition to expression changes in tissues, more recent studies have indicated that miRNAs are detectable and highly stable in plasma, serum, and other biological fluids. Furthermore, the levels of circulating miRNAs have been shown to correlate with disease, thereby suggesting that miRNAs, in addition to being potential therapeutic targets, might also have utility as diagnostic biomarkers. For cardiovascular disease, circulating miRNAs so far have been shown to be potential biomarkers for acute myocardial infarction, heart failure, coronary artery disease, stroke, and type 2 diabetes. –14 Through expression profiling of human heart failure plasma samples compared with normal samples, two groups have identified microRNAs that appeared to correlate with N-terminal pro brain natriuretic peptide (NT-proBNP). Specifically, Corsten found miR-499 to be significantly increased in the plasma of acute heart failure patients, and Tijsen found miR-423-5p to be significantly increased with heart failure. Goren et al. have now reported a new constellation of miRNAs that have altered levels in serum of heart failure patients when compared with normal controls. This group utilized an approach similar to others described in the literature: microarray profiling of miRNAs from a small set of healthy and heart failure patients followed by quantitative PCR (qPCR) confirmation. A strength of this study compared with others is that 186 miRs were validated with qPCR whereas other reports have shown qPCR data from , 20 potentially interesting miRs. Unlike previous reports where individual miRNAs are shown to correlate with clinical parameters, Goren et al. show that the level of change of four serum-borne miRNAs combined (miR-423-5p, miR-92b, miR-320a, and miR-22), called the cumulative miRNAscore, can identify patients with heart failure compared with normals. These four microRNAs did not necessarily have the greatest fold change, but rather had differences with the highest statistical significance (P-value , 0.0005). The miRNA-score correlates with serum BNP levels and other clinical parameters, such as a widened QRS complex, an increase in end-diastolic diameter, and an increase in left atrial diameter. To date, these are the first miRNAs that show a correlation with cardiac function, and demonstrating the potential power of combining the changes for multiple circulating miRNAs. Not only is this the first report of such a method, but it also addresses the concern that the detection of small differences in individual miRNAs (often , 1.5-fold) may pose a challenge for accurate and reproducible measurement. The miRNA-score appears to improve the signal-to-noise ratio, thereby enabling a highly statistical probability of a difference between the heart failure group and the control group (P , 0.0005). As noted by the authors, the choice of these four particular miRNAs represents an initial proof-of-concept combination. The testing of the cumulative detection of other subsets of miRNAs remains to be done, and may reveal a more powerful diagnostic combination. The authors have focused on a set of 10 up-regulated miRNAs in the serum of heart failure patients, with a fold change . 1.2 and a P-value threshold of 7.20 × 10. Interestingly, the array data

The Efficacy of Cardiac Anti-miR-208a Therapy Is Stress Dependent

MicroRNAs (miRNAs) are important regulators of biology and disease. Recent animal efficacy studies validate the therapeutic benefit of miRNA modulation and underscore the therapeutic value of miRNA-targeting oligonucleotides. However, whether disease conditions (stress) influence the pharmacological effects of an anti-miR is currently unknown. To study the effect of disease on target regulation after anti-miR treatment, we injected animals with anti-miR-208a, a synthetic oligonucleotide that inhibits the cardiomyocyte-specific miR-208a. Our data indicate that the presence of stress increases the number of regulated miR-208a targets, and that higher stress levels correlate with stronger target derepression. Additionally, the type of stress also influences which targets are regulated upon miR-208a inhibition. Studies in a large animal model indicate a similar stress-dependent anti-miR effect. Subsequent in vitro studies suggest that the influence of stress on anti-miR efficacy depends at least in part on increased cellular anti-miR uptake. These data indicate that the pharmacological effect of anti-miRs is stronger under disease conditions, and that both the type and severity of disease determine the therapeutic outcome. These facts will be important for assessing the therapeutic dose and predicting the therapeutic outcome when applying anti-miRs in a clinical setting.

Cobomarsen, an oligonucleotide inhibitor of miR-155, co-ordinately regulates multiple survival pathways to reduce cellular proliferation and survival in cutaneous T-cell lymphoma

miR‐155, a microRNA associated with poor prognosis in lymphoma and leukaemia, has been implicated in the progression of mycosis fungoides (MF), the most common form of cutaneous T‐cell lymphoma (CTCL). In this study, we developed and tested cobomarsen (MRG‐106), a locked nucleic acid‐modified oligonucleotide inhibitor of miR‐155. In MF and human lymphotropic virus type 1 (HTLV‐1+) CTCL cell lines in vitro, inhibition of miR‐155 with cobomarsen de‐repressed direct miR‐155 targets, decreased expression of multiple gene pathways associated with cell survival, reduced survival signalling, decreased cell proliferation and activated apoptosis. We identified a set of genes that are significantly regulated by cobomarsen, including direct and downstream targets of miR‐155. Using clinical biopsies from MF patients, we demonstrated that expression of these pharmacodynamic biomarkers is dysregulated in MF and associated with miR‐155 expression level and MF lesion severity. Further, we demonstrated that miR‐155 simultaneously regulates multiple parallel survival pathways (including JAK/STAT, MAPK/ERK and PI3K/AKT) previously associated with the pathogenesis of MF, and that these survival pathways are inhibited by cobomarsen in vitro. A first‐in‐human phase 1 clinical trial of cobomarsen in patients with CTCL is currently underway, in which the panel of proposed biomarkers will be leveraged to assess pharmacodynamic response to cobomarsen therapy.