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

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

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

A Locked Nucleic Acid Antisense Oligonucleotide (LNA) Silences PCSK9 and Enhances LDLR Expression In Vitro and In Vivo

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.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.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

Nucleic acid based therapeutic approaches

Nucleic acid therapy comprises several approaches based on nucleic acid as the active therapeutic component to treat human disease. Antisense and short interfering RNA (siRNA) are two gene-silencing techniques in this category. Based on nucleic acid sequence recognition both techniques inhibit gene expression. This is highly attractive whenever there is deleterious expression of genetic material, as for example in viral diseases or cancer. in many of these diseases effective treatments are limited and/or toxic; therefore there is a need for new alternative therapeutics. We aimed to improve these genesilencing techniques to facilitate future therapeutic application. We have made efforts in facilitating the use of viral vectors for nucleic acid delivery, but in particular, we have used the synthetic nucleotide analogue locked nucleic acid (LNA) in both antisense oligonucleotides and siRNA. As therapeutic targets we inhibited the human pathogens human immunodeficiency virus type 1 (H IV-1) and severe acute respiratory syndromeassociated corona virus (SARS-CoV) in cell culture. The results demonstrate that LNA is advantageous in both antisense and siRNA techniques. Starting with delivery, as one major obstacle for nucleic acid based therapeutics, we aimed to facilitate the use of recombinant adeno-accosiated virus (AAV). In certain applications viruses can serve as suitable delivery vehicles for gene-silencing techniques. AAV has favourable properties for use as delivery vehicle, however there has been limited use of AAV due to production difficulties. We developed an alternative method for production that utilized baculoviruses for transfer of needed helper genes. We showed expression of the helper genes from the baculovirus and production of infectious recombinant AAV. The protocol may lead to improved AAV production and facilitate increased use of recombinant AAV. Continuing with the development of synthetic antisense oligonucleotides, we targeted the conserved HIV-1 dimerization initiation site with LNA modified antisense oligonucleotides, LNA/DNA mix-mers. We showed improved inhibition of dimerization by using LNA/DNA mix-mers and activation of RNase H by LNA/DNA mix-mers containing at least six consecutive DNA bases. We subsequently demonstrated inhibition of HIV-1 replication. LNA improves the antisense oligonuleotides and can function on a therapeutic target. Using the more recently discovered genesilencing technique siRNA, we targeted the SARS-CoV RNA dependent RNA polymerase. The siRNA inhibited SARSCoV replication both when delivered pre and post infection. This study shows an example of how genetic information on an emerging pathogen can rapidly be converted to an antiviral tool by the means of siRNA. Finally, we combined LIMA with siRNA to a compound we term siLNA. We showed siLNA compatibility with the siRNA machinery in association with higher nuclease resistance and enhanced strand bias. siLNA provides a possibility to reduce undesired, off-target effects of siRNA. We also applied siLNA to inhibit SARS-CoV and showed improved efficiency over unmodified siRNA on certain target sites. LNA brings many favourable features to siRNA beneficial to future therapeutic use. Taken together, these studies improve certain technical aspects of gene-silencing techniques and show potential applications of nucleic acid based therapeutics

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

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

Sense and antisense strand activity of siRNA and 5′ sense end modified siLNA

<p><b>Copyright information:</b></p><p>Taken from "Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality"</p><p>Nucleic Acids Research 2005;33(1):439-447.</p><p>Published online 14 Jan 2005</p><p>PMCID:PMC546170.</p><p>© 2005, the authors © </p> () Schematic representation of the SARS 3 target cloned in the sense (pS3Xs) or the antisense (pS3Xas) direction behind firefly luciferase. Also shown is the parental luciferase plasmid without the SARS 3 target (pGL3). () Activity of siRNA and siLNA against sense (pS3Xs), antisense (pS3Xas) or control target (pGL3-Control). Mean and SD values are from two experiments performed in duplicate