Niels Fisker

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 1–2u2009mg/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.

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. To obtain this, it is necessary to identify and inhibit molecular targets on which the cancer cells are strongly dependent. Survivin represents such a target, and it has been published previously that peptide vaccines, the small-molecule YM155, and the antisense molecule LY2181308/ISIS23722, via different mechanisms, have been used as survivin inhibitors. In this article, a new potent antisense inhibitor of survivin, SPC3042, is presented, and the properties of SPC3042 are compared with the previously published antisense drug, LY2181308/ISIS23722. SPC3042 is a 16-mer locked nucleic acid (LNA) oligonucleotide and designed as a fully phosphorothiolated gapmer containing 7 LNA nucleotides in the flanks. The LNA nucleotides in SPC3042 provide nuclease stability and higher potency for survivin mRNA inhibition compared with earlier generations of antisense reagents. It is shown that the down-regulation of survivin with SPC3042 leads to cell cycle arrest, pronounced cellular apoptosis, and down-regulation of Bcl-2. It is also shown that SPC3042 is a sensitizer of prostate cancer cells to Taxol treatment in vitro and in vivo. [Mol Cancer Ther 2008;7(9):2736–45]

Risk factors for respiratory syncytial virus hospitalisation in children with heart disease

Objective: To assess the risk and risk factors for respiratory syncytial virus (RSV) hospitalisation and determinants of the severity of RSV disease in children with heart disease. Methods: By using a database on RSV tests in Denmark all children with RSV diagnosed with heart disease in Denmark from January 1996 to April 2003 were identified. For each case child one control child matched for age and centre was drawn from the population of children with heart disease. Clinical information was obtained through a review of all records. Results: Data were obtained on 313 pairs. Median age at admission was 280 days (range 15–2379). In the multivariate analysis predictors of RSV hospitalisation were Down syndrome (odds ratio (OR) 3.24, 95% CI 1.80 to 5.80), cardiomyopathy (OR 5.84, 95% CI 1.26 to 27.16) and haemodynamically significant heart disease (OR 1.53, 95% CI 1.04 to 2.26). During RSV hospitalisation predictors of the need for respiratory support (supplemental oxygen, nasal continuous positive airway pressure or mechanical ventilation) were young age (relative risk (RR) 0.47, 95% CI 0.32 to 0.67 per additional year of age) and cardiac decompensation (RR 1.81, 95% CI 1.02 to 3.23). The incidence rate of RSV hospitalisation among children with any heart disease aged 0–23 months was 5.65 per 100 child-years. Conclusion: In children with heart disease risk factors for RSV admission are Down syndrome, cardiomyopathy and haemodynamically significant heart disease. Young age and cardiac decompensation are associated with a more severe course of RSV disease.

Blood borne viral infections among Danish health care workers frequent blood exposure but low prevalence of infection

Denmark is a country with low prevalence and incidence of blood borne viral infections. Among health care workers (HCWs) vaccination for hepatitis B is only offered to high-risk groups. The aims of this cross sectional survey were to determine the prevalence of hepatitis B, -C, and human immunodeficiency virus (HIV) among the staff at a Danish University hospital and to correlate this with risk factors for transmission. Additionally, we wanted to examine the current frequency of blood exposure, reporting habits and hepatitis B vaccination status in the staff. Of 1439 eligible hospital staffs included, 960 (67%) were HCWs. The overall human immunodeficiency virus (HIV)-, hepatitis C Virus (HCV)- and hepatitis B Virus (HBV)-prevalence was 0% (0/1439), 0.14% (2/1439) and 1.6% (23/1439), respectively. Twenty-three percent of HCWs were vaccinated against HBV. Age, blood transfusion and stay in endemic areas were associated independently to HBV infection as opposed to job-category, duration of employment, HBV vaccination status and blood exposure. Based on a 4-week recall period, the incidence of percutaneous blood exposure was 1.5/person-year. In conclusion the HIV and hepatitis prevalence was low despite frequent blood exposure and the principal risk factors were unrelated to work. Danish HCWs do not seem to be at increased risk of hepatitis B even though universal HBV vaccination has not been implemented.

A novel mechanism for NETosis provides antimicrobial defense at the oral mucosa.

Neutrophils are essential for host defense at the oral mucosa and neutropenia or functional neutrophil defects lead to disordered oral homeostasis. We found that neutrophils from the oral mucosa harvested from morning saliva had released neutrophil extracellular traps (undergone NETosis) in vivo. The NETosis was mediated through intracellular signals elicited by binding of sialyl Lewis(X) present on salival mucins to l-selectin on neutrophils. This led to rapid loss of nuclear membrane and intracellular release of granule proteins with subsequent neutrophil extracellular trap (NET) release independent of elastase and reduced NAD phosphate-oxidase activation. The saliva-induced NETs were more DNase-resistant and had higher capacity to bind and kill bacteria than NETs induced by bacteria or by phorbol-myristate acetate. Furthermore, saliva/sialyl Lewis(X) mediated signaling enhanced intracellular killing of bacteria by neutrophils. Saliva from patients with aphthous ulcers and Behçet disease prone to oral ulcers failed to induce NETosis, but for different reasons it demonstrated that disordered homeostasis in the oral cavity may result in deficient saliva-mediated NETosis.

Clinical Review of Nontyphoid Salmonella Infections from 1991 to 1999 in a Danish Country

To determine outcomes of nontyphoid Salmonella infections, we reviewed all 3328 cases of infection registered in a large Danish county from 1991 to 1999. The hospitalization rate was 27% among patients <5 years old, 22% among patients 5-59 years old, and 54% among patients > or =60 years old; complications were present in 7% and extraintestinal disease in 4% of patients. We conclude that nontyphoid Salmonella infections may frequently cause severe disease even among patients without predisposing conditions.

Immunodeficiency Associated with a Nonsense Mutation of IKBKB

We report an infant of consanguineous parents of Turkish decent with a novel immunodeficiency associated withxa0homozygosity for a nonsense mutation of the gene encoding Inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKβ). At five months, she presented with respiratory insufficiency and Pneumocystis jirovecii pneumonia which was successfully treated. At nine months, iatrogenic systemic infection with Mycobacterium bovis was found and eventually led to her death at age 14xa0months. Laboratory findings were reminiscent of hyper-IgM syndrome, but genetic testing gave no explanation before whole exome sequencing revealed a novel mutation abrogating signaling through the canonical NF-κB pathway.

Identifying a hepatitis B outbreak by molecular surveillance: a case study

Infection outbreaks are usually recognised when an unexpectedly high number of cases of a clinical illness are observed. With hepatitis B infection, the incubation period is long and a substantial proportion of infected people—especially children and the immunocompromised host—may remain asymptomatic.1–3 These factors increase the risk of prolonged and unrecognised outbreaks. Denmark has not implemented universal childhood hepatitis B immunisation, and vaccination is not required for health workers performing invasive procedures.nnPhylogenetic analysis of hepatitis B virus have been used to investigate recognised outbreaks,4 5 to reject or confirm suspected chains of transmission,6 and to prove unusual reservoirs or routes of infection.7 8 We present the unravelling of an outbreak of asymptomatic hepatitis B on a paediatric ward after the incidental identification of a single case of hepatitis B. The recognition and subsequent halting of the spread of this outbreak were possible owing to the availability of a DNA sequences library compiled from patients with hepatitis B in the locality.9nnDuring preparation for bone marrow transplantation, a child was diagnosed with an acute asymptomatic hepatitis B infection at the paediatric haematology and oncology department at Odense University Hospital. Screening for hepatitis B had been negative two years previously, and serology testing and polymerase chain reaction in repository blood donation samples and follow-up samples from all involved blood donors were negative for the virus. The patient had had no contact with known hepatitis B cases during the suspected incubation period.nnAs part of a research programme, strains of the hepatitis B virus consecutively detected in the area had been partially sequenced. The patients sequence (the hepatitis B epi strain) was identical to that of a known hepatitis B carrier who had been …