MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear. Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK-MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK-MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK-MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction. These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.

All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA 'seed' region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.

MicroRNAs (miRNAs) are increasingly implicated in regulating metastasis. Despite progress in silencing miRNAs in normal tissues of rodents and non-human primates, the development of effective approaches for sequence-specific inhibition of miRNAs in fast-growing tumors remains a significant scientific and clinical challenge. Here we show that systemic treatment of tumor-bearing mice with miR-10b antagomirs – a class of chemically modified anti-miRNA oligonucleotides – suppresses breast cancer metastasis. Silencing of miR-10b both in vitro and in vivo with antagomirs significantly decreases miR-10b levels and increases levels of a functionally important miR-10b target, Hoxd10. Administration of miR-10b antagomirs to mice bearing highly metastatic cells does not reduce primary mammary tumor growth but instead markedly suppresses formation of lung metastases. This metastasis-suppressing effect is sequence-specific. The miR-10b antagomir, which is well tolerated by normal animals, appears to be a promising candidate and a starting point for the development of new anti-metastasis agents.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50 -70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia. plasma PCSK9 ͉ tissue LDLR levels P roprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the mammalian serine proprotein convertase family that typically functions in the proteolytic processing and maturation of secretory proteins (1, 2). PCSK9 was the first family member to be implicated in a dominantly inherited form of hypercholesterolemia (3). Mechanistic studies addressing the function of PCSK9 in mice and humans have demonstrated that overexpression or gain-of-function mutations in PCSK9 reduced low density lipoprotein receptor (LDLR) protein levels in liver, which significantly increased circulating plasma cholesterol both in mice and humans (4). Additional studies showed that the deletion of Pcsk9 in mice resulted in increased LDLR levels, accelerated the clearance of low density lipoprotein cholesterol (LDLc), and reduced circulating cholesterol levels (5). Recently, studies in mice have also shown that lowering PCSK9 transcript levels by antisense oligonucleotides resulted in reduced total cholesterol, LDLc, and HDL cholesterol (HDLc) in blood and increased LDLR levels in liver after 6 weeks of treatment (6). This effect was very similar to that observed in the Pcsk9 Ϫ/Ϫ mice (5). Collectively, these studies have clearly established a role for PCSK9 in cholesterol homeostasis.Validation of PCSK9 as an attractive therapeutic target for the treatment of hypercholesterolemia has come from genetic studies in humans. Cohen et al. (7) first identified loss-of-function mutations in PCSK9...

SUMMARY A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. Here we demonstrate that a small regulatory microRNA, miR-296 has a major role in this process. Glioma cells and angiogenic growth factors elevate the level of miR-296 in primary human brain microvascular endothelial cells in culture. The miR-296 level is also elevated in primary tumor endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFR-β. Furthermore, inhibition of miR-296 with antagomirs reduces angiogenesis in tumor xenografts in vivo.