Christoph Rosenbohm

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.

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 mgu2009kg−1 per day. These results indicate that α‐L‐LNA is a very promising member of the family of LNA analogues in antisense applications.

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]

Synthesis of 2′-amino-LNA: a new strategyElectronic supplementary information (ESI) available: further experimental details and the structure of compound S11 (.pdb file). See http://www.rsc.org/suppdata/ob/b2/b208864a/

In this paper we present revised and significantly improved synthetic routes to 2-amino-LNA (locked nucleic acid). The optimal route is convergent with the synthesis of LNA monomers (2-oxy-LNA) via a common intermediate obtained by a mild deacetylation for the liberation of the 2-hydroxy group to give compound 23 without the concomitant ring closure that affords the 2-oxy-LNA skeleton. After inversion of the stereochemistry at C2 and triflate formation at the 2-hydroxy group a new common intermediate 16 is obtained which gives easy access to a range of other analogues exemplified by the introduction of a sulfur nucleophile leading to the 2-thio-LNA structure. After substitution of the triflate with azide a basic reduction affords the desired 2-amino-LNA structure, i.e., compound 18. This new synthesis strategy towards 2-amino-LNA improves the overall yield significantly and converges the syntheses of 2-oxy-LNA and LNA analogues.

Introduction of a Universal Solid Support for Oligonucleotide Synthesis

Abstract Protection of 1,4-anhydro-D-ribitol with the bidentate reagent 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, followed by 4,4′-dimethoxytritylation, selective cleavage of the silyl group at the secondary oxygen, chloroacetylation, and desilylation at the 5-position, afforded 1,4-anhydro-3-O-chloroacetyl-2-O-(4, 4′-dimethoxytrityl)-D-ribitol. Derivatisation of the free hydroxyl group with LCAA-CPG gave a novel universal solid support for use in oligonucleotide synthesis and phosphoramidite-based combinatorial chemistry. This solid support was used for synthesis of a number of oligonucleotides, for which the purity and identity with oligonucleotides synthesised on commercial supports was demonstrated.

Parallel synthesis of a small library of novel aminoglycoside analogs based on 2-amino-2-deoxy-d-glucose and d-ribose scaffolds

Abstract The synthesis of a small library of aminoglycoside analogues based on 2-amino-2-deoxy- d -glucose and d -ribose was achieved using reductive amination reactions as key transformations.

Chapter 5:Locked Nucleic Acid: Properties and Therapeutic Aspects

In 1978 Zamecnik and Stephenson1 showed for the first time that messenger RNA (mRNA) repression could be achieved by single-stranded oligonucleotides (ONs). This mechanistic approach for gene inhibition was later called the antisense (AS) principle. The simplicity of this new principle captivated ma...

Synthesis of 2′-Amino-LNA Purine Nucleosides

The first reported synthesis of 2′-amino-LNA purine nucleosides via a transnucleosidation is accomplished enabling the preparation of oligonucleotides incorporating 2′-amino-LNA with all four natural bases.