Jens Bo Hansen

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.

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]

Protein Kinase C α is a marker for antiestrogen resistance and is involved in the growth of tamoxifen resistant human breast cancer cells

Development of resistance to antiestrogen treatment in breast cancer patients is a serious therapeutic problem. The molecular mechanisms contributing to resistance are currently unclear; however it is known that increased activation of growth signaling pathways is involved. Protein Kinase C α (PKCα) is associated with a diverse range of cancers and is previously shown to be overexpressed in three out of four antiestrogen resistant breast cancer cell lines. In this study we investigated whether PKCα contributes to antiestrogen resistant growth. A panel of nine resistant cell lines was investigated, all of which displayed elevated levels of PKCα expression relative to parental MCF-7 cells. Stable PKCα overexpression in MCF-7 cells significantly reduced sensitivity to the antiestrogens, tamoxifen and ICI 182,780. Two resistant cell lines were chosen for further studies: tamoxifen resistant MCF-7/TAMR-1 (TAMR-1) and ICI 182,780 resistant MCF-7/182R-6 (182R-6). Treatment with the PKCα inhibitor Ro-32–0432 resulted in a preferential growth inhibition of these two cell lines relative to MCF-7 cells. Moreover, transient down-regulation of PKCα resulted in a 30–40% growth inhibition of TAMR-1 and 182R-6, while MCF-7 remained unaffected. Stable PKCα knock-down in TAMR-1 using small hairpin RNA, resulted in a tamoxifen sensitive “MCF-7–like” growth phenotype, while the same approach in 182R-6 cells did not alter their sensitivity to ICI 182,780. These results demonstrate a functional contribution of PKCα to tamoxifen resistant growth. Furthermore, our data suggest the potential for PKCα as a marker for antiestrogen resistance and as a promising therapeutic target in the treatment of tamoxifen resistant breast cancer.

Survivin mRNA Antagonists Using Locked Nucleic Acid, Potential for Molecular Cancer Therapy

We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced synergistic effect when combining the mRNA antagonists against Survivin with the chemotherapeutic Taxol. This effect was demonstrated at concentrations of antagonists far lower than any previously demonstrated, indicating the high potential of locked nucleic acid for therapeutic use. Further characterisations in vivo are ongoing.

Antisense Knockdown of PKC-α Using LNA-Oligos

Abstract Full-length and 4 nucleotides truncated Locked Nucleic Acid (LNA) modifications of ISIS 3521 were compared for antisense properties in a cellular assay. ISIS 3521 is a 20-mer phosphorothioate designed to hybridise to human protein kinase C-α (PKC-α) mRNA and is currently submitted to clinical trials against cancer. We report that LNA can potentate this antisense oligo and retain the antisense potential with shorter oligos.

Synthesis and biological evaluation of LNA phosphoramidates.

The synthesis of LNA phosphoramidates is presented. The LNA phosphoramidates were evaluated for their ability to inhibit cell proliferation of the human prostate cancer cell line 15PC3. A number of the LNA phosphoramidates showed cell proliferation inhibition determined by the MTS assay.