Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules.

Quantum mechanical studies of DNA and LNA.

Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials.

Hepatotoxic potential of therapeutic oligonucleotides can be predicted from their sequence and modification pattern.

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood.

A locked nucleic acid oligonucleotide targeting microRNA 122 is well-tolerated in cynomolgus monkeys.

MicroRNA 122 (miR-122) is liver specific, fine-tunes lipid metabolism, and is required for hepatitis C virus (HCV) abundance. Miravirsen, an oligonucleotide with locked nucleic acid, binds to miR-122, potently inhibiting its activity. We aimed at determining the safety of the miR-122 antagonism in vivo in 6 to 10 cynomolgus monkeys/group intravenously treated with a range of dose levels twice weekly for 4 weeks.

MicroRNAs in liver disease.

MicroRNAs are small noncoding RNA molecules that regulate gene expression posttranscriptionally through complementary base pairing with thousands of messenger RNAs. They regulate diverse physiological, developmental, and pathophysiological processes. Recent studies have uncovered the contribution of microRNAs to the pathogenesis of many human diseases, including liver diseases.

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).

Silencing the expression of Ras family GTPase homologues decreases inflammation and joint destruction in experimental arthritis.

Changes in the expression and activation status of Ras proteins are thought to contribute to the pathological phenotype of stromal fibroblast-like synoviocytes (FLS) in rheumatoid arthritis, a prototypical immune-mediated inflammatory disease. Broad inhibition of Ras and related proteins has shown protective effects in animal models of arthritis, but each of the Ras family homologues (ie, H-, K-, and N-Ras) makes distinct contributions to cellular activation. We examined the expression of each Ras protein in synovial tissue and FLS obtained from patients with rheumatoid arthritis and other forms of inflammatory arthritis.

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates.

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides.

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.

Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.

The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology.

Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection).

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.

A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth factor pathways, is associated with poor prognosis in many types of cancer. Therefore, down-regulation of HIF-1alpha protein by RNA antagonists may control cancer growth.

SPC3042: a proapoptotic survivin inhibitor.

The ability to regulate the cellular homeostasis of a higher organism through tight control of apoptosis and cell division is crucial for life. Dysregulation of these mechanisms is often associated with cancerous phenotypes in cells. Optimal cancer therapy is a fine balance between effective cancer cell killing and at the same time minimizing, or avoiding, damage to the surrounding healthy tissue.

An Endogenous TNF-α Antagonist Induced by Splice-switching Oligonucleotides Reduces Inflammation in Hepatitis and Arthritis Mouse Models.

Tumor necrosis factor-α (TNF-α) is a key mediator of inflammatory diseases, including rheumatoid arthritis (RA), and anti-TNF-α drugs such as etanercept are effective treatments. Splice-switching oligonucleotides (SSOs) are a new class of drugs designed to induce therapeutically favorable splice variants of targeted genes. In this work, we used locked nucleic acid (LNA)-based SSOs to modulate splicing of TNF receptor 2 (TNFR2) pre-mRNA.

Locked nucleic acid holds promise in the treatment of cancer.

Providing novel treatments to help cancer patients live longer and have better lives remains one of the biggest challenges of the pharmaceutical industry. Today much is known about the molecular and genetic causes of cancers thus facilitating the development of novel targeted cancer drugs with improved risk-benefit ratios compared to contemporary broadly-acting, cytotoxic cancer drugs. Antisense therapy, e.

An endogenous TNF-alpha antagonist induced by splice-switching oligonucleotides reduces inflammation in hepatitis and arthritis mouse models.

Tumor necrosis factor-alpha (TNF-alpha) is a key mediator of inflammatory diseases, including rheumatoid arthritis (RA), and anti-TNF-alpha drugs such as etanercept are effective treatments. Splice-switching oligonucleotides (SSOs) are a new class of drugs designed to induce therapeutically favorable splice variants of targeted genes. In this work, we used locked nucleic acid (LNA)-based SSOs to modulate splicing of TNF receptor 2 (TNFR2) pre-mRNA.

The application of locked nucleic acids in the treatment of cancer.

Locked nucleic acid (LNA) is a novel high-affinity and biologically stable RNA analog in which the normally flexible ribose sugar ring is fixed in a rigid conformation through a methylene 2'-O, 4'-C linkage. This fixed conformation brings substantial advantages to the design of effective RNA binding drugs, and enables single-stranded LNA oligonucleotides, termed 'RNA antagonists', to have superior efficacies in vivo in downregulating target mRNA when compared to oligonucleotides based on other chemistries or to published short interfering RNA. The features that allow LNA to be a valuable drug platform include unprecedented RNA binding affinity, excellent specificity, resistance to enzymatic degradation, safety, and ease of manufacture.

Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides.

Aptamers interacting with RNA hairpins through loop-loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer.

Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice.

Locked nucleic acid (LNA) oligomers were found to be very effective in their ability to modulate alternative splicing in vivo in transgenic mice that ubiquitously express a modified EGFP pre-mRNA containing an aberrantly spliced beta-globin intron (IVS2-654). Following intraperitoneal injections, the splice-switching oligonucleotide LNA SSO-654 targeted to the aberrant 5' splice site in EGFP-654 pre-mRNA corrected aberrant splicing and increased production of repaired EGFP mRNA mainly in the liver, colon, and small intestine. Little or no effect was detected in heart, lung, or kidney, the organ where most of the oligonucleotide was distributed after four consecutive daily injections.

On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide.

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design).

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.

LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA.