A Multifunctional LNA Oligonucleotide-Based Strategy Blocks AR Expression and Transactivation Activity in PCa Cells.

The androgen receptor (AR) plays a critical role in the development of prostate cancer (PCa) through the activation of androgen-induced cellular proliferation genes. Thus, blocking AR-mediated transcriptional activation is expected to inhibit the growth and spread of PCa. Using tailor-made splice-switching locked nucleic acid (LNA) oligonucleotides (SSOs), we successfully redirected splicing of the AR precursor (pre-)mRNA and destabilized the transcripts via the introduction of premature stop codons. Furthermore, the SSOs simultaneously favored production of the AR45 mRNA in lieu of the full-length AR. AR45 is an AR isoform that can attenuate the activity of both full-length and oncogenic forms of AR by binding to their common N-terminal domain (NTD), thereby blocking their transactivation potential. A large screen was subsequently used to identify individual SSOs that could best perform this dual function. The selected SSOs powerfully silence AR expression and modulate the expression of AR-responsive cellular genes. This bi-functional strategy that uses a single therapeutic molecule can be the basis for novel PCa treatments. It might also be customized to other types of therapies that require the silencing of one gene and the simultaneous expression of a different isoform.

Refining LNA safety profile by controlling phosphorothioate stereochemistry.

Drug discovery with phosphorothioate oligonucleotides is an area of intensive research. In this study we have controlled the stereochemistry of the phosphorothioate backbone of LNA oligonucleotides to investigate the differences in safety profile, target mRNA knock down, and cellular uptake in vitro. The study reveals that controlling only four stereocenters in an isomeric phosphorothioate mixture can improve the therapeutic index significantly by improving safety without compromising activity.

In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3' and 5' thiophosphate linkages.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

Protein Kinase C-α is a Critical Protein for Antisense Oligonucleotide-mediated Silencing in Mammalian Cells.

We have identified the existence of a productive, PKC-α-dependent endocytotic silencing pathway that leads gymnotically-delivered locked nucleic acid (LNA)-gapmer phosphorothioate antisense oligonucleotides (ASOs) into late endosomes. By blocking the maturation of early endosomes to late endosomes, silencing the expression of PKC-α results in the potent reduction of ASO silencing ability in the cell. We have also demonstrated that silencing of gene expression in the cytoplasm is vitiated when PKC-α expression is reduced. Restoring PKC-α expression via a reconstitution experiment reinstates the ability of ASOs to silence. These results advance our understanding of intracellular ASO trafficking and activity following gymnotic delivery, and further demonstrate the existence of two distinct silencing pathways in mammalian cells, one in the cytoplasmic and the other in the nuclear compartment.

The synergism of MCL1 and glycolysis on pediatric acute lymphoblastic leukemia cell survival and prednisolone resistance.

In vitro and in vivo resistance to prednisolone are predictive for an adverse prognosis in pediatric precursor B-acute lymphoblastic leukemia. Causes of resistance are still poorly understood. In this study, we observed that prednisolone exposure of prednisolone-sensitive patients' leukemic cells decreased anti-apoptotic MCL1 protein levels by 2.9-fold, while MCL1 protein expression in prednisolone-resistant leukemic patients' cells was unaffected (P<0.01). Locked nucleic acid oligonucleotides directed against MCL1 reduced MCL1 protein levels by 82±16% (P<0.05) in leukemic cells, decreased proliferation by 9-fold and sensitized to prednisolone up to 80.8-fold, compared to a non-silencing-control locked nucleic acid (P<0.05). Remarkably, we discovered that MCL1-silencing up-regulated the glucose consumption of leukemic cells by 2.5-fold (P<0.05), suggesting a potential rescue mechanism mediated by glycolysis. Targeting glycolysis by 2-deoxyglucose synergistically inhibited leukemic survival by 23.2-fold in MCL1-silenced cells (P<0.05). Moreover, 2-deoxyglucose and MCL1 locked nucleic acid concomitantly sensitized leukemic cells to prednisolone compared to MCL1 locked nucleic acid or 2-deoxyglucose alone (P<0.05). In conclusion, these results indicate the need to target both MCL1 and glycolysis simultaneously to inhibit leukemic survival and sensitize acute leukemia patients towards prednisolone.

Silencing of gene expression by gymnotic delivery of antisense oligonucleotides.

Antisense oligodeoxyribonucleotides have been used for decades to achieve sequence-specific silencing of gene expression. However, all early generation oligonucleotides (e.g., those with no other modifications than the phosphorothioate backbone) are inactive in vitro unless administered using a delivery vehicle. These delivery vehicles are usually lipidic but can also be polyamines or some other particulate reagent. We have found that by employing locked nucleic acid (LNA) phosphorothioate gap-mer nucleic acids of 16 mer or less in length, and by carefully controlling the plating conditions of the target cells and duration of the experiment, sequence-specific gene silencing can be achieved at low micromolar concentrations in vitro in the absence of any delivery vehicle. This process of naked oligonucleotide delivery to achieve gene silencing in vivo, which we have termed gymnosis, has been observed in many both adherent and nonadherent cell lines against several different targets genes.

Knockdown of survivin (BIRC5) causes apoptosis in neuroblastoma via mitotic catastrophe.

BIRC5 (survivin) is one of the genes located on chromosome arm 17q in the region that is often gained in neuroblastoma. BIRC5 is a protein in the intrinsic apoptotic pathway that interacts with XIAP and DIABLO leading to caspase-3 and caspase-9 inactivation. BIRC5 is also involved in stabilizing the microtubule-kinetochore dynamics. Based on the Affymetrix mRNA expression data, we here show that BIRC5 expression is strongly upregulated in neuroblastoma compared with normal tissues, adult malignancies, and non-malignant fetal adrenal neuroblasts. The over-expression of BIRC5 correlates with an unfavorable prognosis independent of the presence of 17q gain. Silencing of BIRC5 in neuroblastoma cell lines by various antisense molecules resulted in massive apoptosis as measured by PARP cleavage and FACS analysis. As both the intrinsic apoptotic pathway and the chromosomal passenger complex can be therapeutically targeted, we investigated in which of them BIRC5 exerted its essential anti-apoptotic role. Immunofluorescence analysis of neuroblastoma cells after BIRC5 silencing showed formation of multinucleated cells indicating mitotic catastrophe, which leads to apoptosis via P53 and CASP2. We show that BIRC5 silencing indeed resulted in activation of P53 and we could rescue apoptosis by CASP2 inhibition. We conclude that BIRC5 stabilizes the microtubules in the chromosomal passenger complex in neuroblastoma and that the apoptotic response results from mitotic catastrophe, which makes BIRC5 an interesting target for therapy.

Silencing of microRNA families by seed-targeting tiny LNAs.

The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.

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.

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.

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.

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.

Design, synthesis, and biological evaluation of LNA nucleosides as adenosine A3 receptor ligands.

We have prepared a series of adenosine analogs based on the bicyclo[2.2.1]heptane scaffold of locked nucleic acid (LNA) and tested them for both agonist and antagonist activity at the adenosine A(3) receptor. The design of these derivatives was based on the known A(3) agonist IB-MECA and related compounds. Modifications thus include the 5'-uronamides and N(6)-(3-iodobenzyl) derivatives. In this way we have prepared analogs of known A(3) agonists with the sugar ring restricted in an N-conformation. For comparison we have also prepared 2'-O-methyl derivatives of IB-MECA. The LNA nucleosides showed no agonist activity but some of them are potent antagonists. The 2'-O-methyl derivative of IB-MECA is an agonist with similar potency as the parent compound.

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.

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). 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 alpha-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 alpha-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 alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.

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. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform.

Evaluation of LNA-modified DNAzymes targeting a single nucleotide polymorphism in the large subunit of RNA polymerase II.

Allele-specific inhibition (ASI) is a new strategy to treat cancer through a vulnerability created by the loss of large segments of chromosomal material by loss of heterozygosity (LOH). Using antisense approaches, it is possible to target single nucleotide polymorphisms (SNP) in the remaining allele of an essential gene in the tumor, thus killing the tumor while the heterozygous patient survives at the expense of the other nontargeted allele lost by the tumor. In this study, the feasibility of using locked nucleic acid (LNA)-modified DNAzymes (LNAzymes) of the 10-23 motif as allele-specific drugs was investigated. We demonstrate that incorporation of LNA into 10-23 motif DNAzymes increases their efficacy in mRNA degradation and that, in a cell-free system, the 10-23 motif LNAzyme can adequately discriminate and recognize an SNP in the large subunit of RNA polymerase II (POLR2A), an essential gene frequently involved in LOH in cancer cells. However, the LNAzymes, optimized under in vitro conditions, are not always efficient in cleaving their RNA target in cell culture, and the efficiency of RNA cleavage in cell culture is cell type dependent. The cleavage rate of the LNAzyme is also much slower than RNase H-recruiting DNA phosphorothioate antisense oligonucleotides. Moreover, compared with DNA phosphorothioates, the ability of the LNAzymes to differentially knock down two POLR2A alleles in cultured cancer cells is limited.