Henrik Ørum

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

Anti-MicroRNA Antiviral MicroRNAs (miRNAs) are small noncoding RNAs found in eukaryotes and viruses. They are critical regulators of a wide range of cellular processes. The highly conserved miRNA miR-122 is required for infection by hepatitis C virus (HCV), a leading cause of liver disease in humans. Present HCV treatment regimes can have serious side effects and are effective in only 50% of cases. In order to try to tackle HCV infection, Lanford et al. (p. 198, published online 3 December) targeted miR-122 using a complementary locked nucleic acid (LNA) oligonucleotide. Treatment of chimpanzees infected by HCV with the LNA antagonist resulted in a long-term reduction of disease symptoms without the concomitant appearance of resistant strains of the virus. Targeting a microRNA required for hepatitis C virus infection reduces disease symptoms in chimpanzees. 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. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.

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 γ subunit (CaMKIIγ), 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.

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). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

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. Moreover, microRNAs have been identified as biomarkers that can often be detected in the systemic circulation. We review the role of microRNAs in liver physiology and pathophysiology, focusing on viral hepatitis, liver fibrosis, and cancer. We also discuss microRNAs as diagnostic and prognostic markers and microRNA-based therapeutic approaches for liver disease.

A RNA antagonist of hypoxia-inducible factor-1α, 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 α-subunit of HIF-1 (HIF-1α), 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-1α protein by RNA antagonists may control cancer growth. EZN-2968 is a RNA antagonist composed of third-generation oligonucleotide, locked nucleic acid, technology that specifically binds and inhibits the expression of HIF-1α mRNA. In vitro, in human prostate (15PC3, PC3, and DU145) and glioblastoma (U373) cells, EZN-2968 induced a potent, selective, and durable antagonism of HIF-1 mRNA and protein expression (IC50, 1-5 nmol/L) under normoxic and hypoxic conditions associated with inhibition of tumor cell growth. Additionally, down-regulation of HIF-1α protein by EZN-2968 led to reduction of its transcriptional targets and of human umbilical vein endothelial cell tube formation. In vivo, administration of EZN-2968 to normal mice led to specific, dose-dependent, and highly potent down-regulation of endogenous HIF-1α and vascular endothelial growth factor in the liver. The effect can last for days after administration of single dose of EZN-2968 and is associated with long residence time of locked nucleic acid in certain tissues. In efficacy studies, tumor reduction was found in nude mice implanted with DU145 cells treated with EZN-2968. Ongoing phase I studies of EZN-2968 in patients with advanced malignancies will determine optimal dose and schedule for the phase II program. [Mol Cancer Ther 2008;7(11):3598–608]

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. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.

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 β-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 ∼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 ∼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.

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.

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. Survival, body weights, clinical signs, and cardiovascular and ophthalmologic parameters were unaffected. Anticipated hypolipidemia due to the inhibition of miR-122 was observed in all treated animals. Only the highest dose level produced distinct transient prolongations of clotting times, slight alternative complement pathway activation, and a reversible increase of hepatic transaminases. Distribution half-life was 10-20 minutes, and accumulation was mainly in the kidney and liver with slow elimination. Microscopic examinations revealed granulated Kupffer cells and lymph node macrophages, cytoplasmic vacuolation in proximal renal tubules, and hepatocytes. The granules were most likely phagolysosomes containing miravirsen. A slightly increased incidence of hepatocyte apoptosis was observed in some monkeys given the highest dose; otherwise, there was no evidence of treatment-related degenerative changes in any organ. In conclusion, the maximal inhibition of miR-122 was associated with limited phenotypic changes, indicating that the clinical assessment of miravirsen as host factor antagonist for treatment of HCV infections is warranted.

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 (β‐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, β‐D‐amino LNA (amino‐LNA), β‐D‐thio LNA (thio‐LNA), β‐D‐LNA (LNA), and its stereoisomer α‐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). The resulting gapmers were tested in cancer‐cell cultures and in a nude‐mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H‐Ras ODNs were very efficient in H‐Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti‐H‐Ras ODN containing α‐L‐LNA had clearly the highest efficacy in H‐Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino‐LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both α‐L‐LNA and LNA gapmer ODNs had a high efficacy of tumor‐growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor‐growth inhibition could be observed at dosages as low as 0.5 mg kg−1 per day. These results indicate that α‐L‐LNA is a very promising member of the family of LNA analogues in antisense applications.