PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice.

Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3' untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.

A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo.

BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol. METHODOLOGY/PRINCIPAL FINDINGS: The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity. CONCLUSION/SIGNIFICANCE: LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF.

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.

MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction.

N-methyl-D-aspartate (NMDA) glutamate receptors are regulators of fast neurotransmission and synaptic plasticity in the brain. Disruption of NMDA-mediated glutamate signaling has been linked to behavioral deficits displayed in psychiatric disorders such as schizophrenia. Recently, noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators of neuronal functions. Here we show that pharmacological (dizocilpine) or genetic (NR1 hypomorphism) disruption of NMDA receptor signaling reduces levels of a brain-specific miRNA, miR-219, in the prefrontal cortex (PFC) of mice. Consistent with a role for miR-219 in NMDA receptor signaling, we identify calcium/calmodulin-dependent protein kinase II gamma subunit (CaMKIIgamma), a component of the NMDA receptor signaling cascade, as a target of miR-219. In vivo inhibition of miR-219 by specific antimiR in the murine brain significantly modulated behavioral responses associated with disrupted NMDA receptor transmission. Furthermore, pretreatment with the antipsychotic drugs haloperidol and clozapine prevented dizocilpine-induced effects on miR-219. Taken together, these data support an integral role for miR-219 in the expression of behavioral aberrations associated with NMDA receptor hypofunction.

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.

The utility of LNA in microRNA-based cancer diagnostics and therapeutics.

MicroRNAs (miRNAs) are a novel class of small endogenous non-coding RNAs that regulate gene expression post-transcriptionally by binding to their cognate target mRNAs. Emerging evidence implies that miRNAs play important roles in cancer and thus, miRNAs have rapidly emerged as valuable markers for cancer diagnostics and promising targets for therapeutics. Locked nucleic acid (LNA) is a conformational RNA analoque that binds complementary RNA with unprecedented affinity and specificity. These properties make LNA well suited for miRNA detection and analysis for cancer diagnostics. Furthermore, recent studies on LNA-mediated silencing of miRNA function in vitro and in vivo support the potential of LNA in therapeutic intervention of cancer-associated miRNAs.

Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver.

MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5' end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3' UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.

Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality.

Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform.

Locked nucleic acid containing antisense oligonucleotides enhance inhibition of HIV-1 genome dimerization and inhibit virus replication.

We have evaluated antisense design and efficacy of locked nucleic acid (LNA) and DNA oligonucleotide (ON) mix-mers targeting the conserved HIV-1 dimerization initiation site (DIS). LNA is a high affinity nucleotide analog, nuclease resistant and elicits minimal toxicity. We show that inclusion of LNA bases in antisense ONs augments the interference of HIV-1 genome dimerization. We also demonstrate the concomitant RNase H activation by six consecutive DNA bases in an LNA/DNA mix-mer. We show ON uptake via receptor-mediated transfection of a human T-cell line in which the mix-mers subsequently inhibit replication of a clinical HIV-1 isolate. Thus, the technique of LNA/DNA mix-mer antisense ONs targeting the conserved HIV-1 DIS region may provide a strategy to prevent HIV-1 assembly in the clinic.

Functional comparison of single- and double-stranded siRNAs in mammalian cells.

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs.

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.

A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors.

The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells. Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest. We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV). AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method. We show here that co-infection of mammalian producer cells with three viruses - a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene - produces infectious rAAV particles. This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production.