Susanna Obad

LNA-mediated microRNA silencing in non-human primates

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.

Silencing of microRNA families by seed-targeting tiny LNAs

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.

Inhibition of microRNA function by antimiR oligonucleotides

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.

Therapeutic inhibition of the miR-34 family attenuates pathological cardiac remodeling and improves heart function

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 Ca2+ 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.

Silencing of microRNA-21 in vivo ameliorates autoimmune splenomegaly in lupus mice

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.

LNA-mediated anti–miR-155 silencing in low-grade B-cell lymphomas

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.

MicroRNA Profiling Identifies MicroRNA-155 as an Adverse Mediator of Cardiac Injury and Dysfunction During Acute Viral Myocarditis

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.

Regulation of acute graft-versus-host disease by microRNA-155

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.

The therapeutic potential of microRNAs in cancer.

AbstractMicroRNAs (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.

Pharmacological Inhibition of a MicroRNA Family in Nonhuman Primates by a Seed-Targeting 8-Mer AntimiR

Long-term treatment of obese, insulin-resistant nonhuman primates with a seed-targeting antimiR oligonucleotide against the microRNA-33 family derepresses hepatic expression of miR-33 targets, increases circulating HDL cholesterol, and has a clean safety profile. Little AntimiR Packs a Double Punch MicroRNAs (miRNAs) are a type of noncoding RNA that are about 22 nucleotides in length and affect a variety of cellular functions, including normal development and metabolism. These RNAs have also been implicated in many different diseases. Targeted inhibition of miRNAs can be achieved with antimiRs—RNA segments with complementary sequences to miRNAs of interest, which can bind and specifically inhibit their target miRNAs. In humans, miRNAs called miR-33a and miR-33b help control the homeostasis of cholesterol and other lipids, which are associated with cardiovascular disease. To inhibit both of these miRNAs at the same time, Rottiers and colleagues created an unusually short antimiR, only 8 nucleic acids in length, which targets the common portion of both miR-33a and miR-33b. They had tested it in mammalian cells and in mice, and now also confirmed that this short antimiR can be used in nonhuman primates. The authors demonstrated that their antimiR is safe in obese, insulin-resistant nonhuman primates, and that it increases high-density lipoprotein cholesterol. Additional studies will be necessary to learn more about the effects of this 8-mer antimiR on different parameters of metabolism, and to determine how it affects clinical outcomes, such as the risk of death from cardiovascular disease. Nevertheless, this work suggests that miRNA-based approaches could be specifically tailored and potentially safe for patient use, providing an alternative to standard pharmaceutical interventions. MicroRNAs (miRNAs) regulate many aspects of human biology. They target mRNAs for translational repression or degradation through base pairing with 3′ untranslated regions, primarily via seed sequences (nucleotides 2 to 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 of different miRNAs sharing the same seed sequence and the challenge of simultaneously targeting miRNAs that differ significantly in nonseed sequences complicate 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 nonhuman primates, have not been determined. 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 nonhuman primates results in derepression of miR-33 targets, such as ABCA1, increases circulating high-density lipoprotein cholesterol, and is well tolerated over 108 days of treatment. These findings demonstrate the efficacy and safety of an 8-mer LNA-antimiR against an miRNA family in a nonhuman 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.