Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.

We investigated the effects of targeting the mitotic regulators aurora kinase A and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Aurora protein expression levels in pediatric ALL and AML patient samples were determined by western blot and reverse phase protein array. Both kinases were overexpressed in ALL and AML patients ( P <0.0002), especially in E2A-PBX1-translocated ALL cases ( P <0.002), compared with normal bone-marrow mononuclear cells. Aurora kinase expression was silenced in leukemic cell lines using short hairpin RNAs and locked nucleic acid-based mRNA antagonists. Aurora B knockdown resulted in proliferation arrest and apoptosis, whereas aurora A knockdown caused no or only minor growth delay. Most tested cell lines were highly sensitive to the AURKB-selective inhibitor barasertib–hydroxyquinazoline–pyrazol–anilide (AZD1152-HQPA) in the nanomolar range, as tested with an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. But most importantly, primary ALL cells with a high aurora B protein expression, especially E2A-PBX1-positive cases, were sensitive as well. In adult AML early clinical trials, clear responses are observed with barasertib. Here we show that inhibition of aurora B, more than aurora A, has an antiproliferative and pro-apoptotic effect on acute leukemia cells, indicating that particularly targeting aurora B may offer a new strategy to treat pediatric ALL and AML.

Abstract Abstract 1465 Aurora kinases (AURK) A and B play distinct and important roles during mitosis, and many human cancer types are characterized by upregulated AURK expression. Several small-molecule inhibitors targeting AURKA, B or both, are in development and have shown promising anti-tumour activity in vitro and in vivo. However, most studies address the efficacy of these small-molecule inhibitors in adult cancer. Of all childhood cancers, acute leukemia is the most common type and there is a great medical need to improve outcome and side-effects of current treatment strategies. We therefore analyzed the effects of targeting AURKA and B in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Affymetrix microarray analysis of pediatric ALL (n=297) and AML cases (n=237), and normal bone marrow (nBM) samples (n=8), showed that AURKA and B gene expression was low in all patients. AURK protein expression was determined by western blot and reverse phase protein array. Protein levels of both kinases were significantly elevated in ALL and AML compared to nBM (p<0.0002), especially in patients with an E2A-PBX1 translocation (p<0.002). We used short hairpin RNAs and LNA-based mRNA antagonists to silence AURKA and B expression in three ALL and three AML cell lines of different genetic subtypes. Silencing of AURKA caused no or only minor growth delay, whereas AURKB knockdown resulted in proliferation arrest and apoptosis, as indicated by reduced cell counts and the presence of cleaved PARP. This suggests a pivotal role for AURKB in the survival and proliferation of leukemic cells. A panel of leukemic cell lines was exposed to several AURK-inhibitors, and cell viability was determined with an MTS assay. Comparable sensitivity in the low- to mid-nanomolar range was observed for the pan-Aurora inhibitor VX-680, AURKA-selective inhibitor MLN8237 and AURKB-selective inhibitor Barasertib-HQPA (AZD1152-HQPA), whereas cell lines were relatively resistant to the pan-Aurora inhibitor Danusertib. Since silencing experiments suggested that AURKB would be a suitable target, we tested the efficacy of Barasertib-HQPA in primary ALL cells. ALL patient cells with a high AURKB protein expression, especially E2A-PBX1-positive cases, were more sensitive to Barasertib-HQPA than those with a low expression (p<0.05), whereas nBM cells were resistant up to a concentration of 20μM. In conclusion, inhibition of AURKB, more than AURKA, has an anti-proliferative and pro-apoptotic effect on ALL and AML cells, suggesting that particularly targeting AURKB may be of benefit in the treatment of pediatric acute leukemia. Moreover, this study shows the potential of both LNA-containing mRNA antagonists and Barasertib, for which clear responses have been observed in adult AML early clinical trials, in the treatment of children with ALL and AML. Disclosures: Hedtjärn: Santaris Pharma: Employment. Hansen:Santaris Pharma: Employment. Koch:Santaris Pharma: Employment.

Abstract The inhibition of cellular processes associated with the malignant pheonotype is the goal many oncolytics. In the antisense oligonucleotide (ASO) approach, Watson and Crick base-pairing rules serve as the basis of rational drug design to create single-stranded oligonucleotides that are complementary and bind to RNAs critical for the malignant phenotype. The targets for antisense approaches can be mRNAs that encode for disease related proteins like those that control tumor growth, cell division, or survival. More recently, RNA targets in oncology have been expanded to include microRNAs. A successful oncolytic oligonucleotide must effectively bind and inhibit a target mRNA or miRNA that is critical for the malignant transformation or survival. Target identification presents the same challenge in antisense therapeutics as any other class of oncolytics, but drug design is based the known sequence of the target RNA. The first clinical trials to use antisense oligonucleotides in oncology produced only modest efficacy in clinical trials, but the therapeutic potential of these initial antisense drugs may have been hampered by their low stability and low binding affinity. Advancements in oligonucleotide chemistry have produced oligonucleotides with increased stability and increased affinities. This talk will focus on the use of oligonucleotides with the locked nucleic acid (LNA) modification. In these antisense constructs, some of the nucleotides in the sequence have a modification of the ribose sugar that includes a methylene bridge between the 2’ and 4’ positions. This bridge functions to lock the ribose into a (C3’-endo) configuration: a configuration that is optimized for binding to its cognate nucleotide. LNA-modified oligonucleotides bind to their target RNAs with higher affinities than most other oligonucleotide chemistries. One measure of affinity is the melting temperature (Tm) for binding to its complementary sequence. For each LNA-modified nucleotide added to an ASO sequence, Tm can increase Tm by over 5°. For example, an LNA-modified oligonucleotide currently in clinical development, miravirsen (SPC3649), with 9 LNA-modified nucleotides has a Tm of approximately 80° demonstrating that LNA-modified oligonucleotides have sufficient affinities to bind to and inhibit RNA function. Affinities like these have translated into increase potency for the inhibition of target RNAs in nonclinical models and should yield therapeutic benefit more robust than those seen with earlier generations of oligonucleotide therapeutics. Other drug-like properties of antisense therapeutics have also been improved by including LNA-modified nucleotides. For example, the pharmacokinetic properties of antisense oligonucleotides support their use in clinical medicine. Antisense oligonucleotides are single stranded and bind to proteins in circulation and on cell surfaces. After parenteral administration, antisense oligonucleotide are bound first to plasma proteins, limiting glomerular filtration, and then later the antisense oligonucleotides appear bound to cell surface (proteins), allowing them to be internalized in cells. Delivery into cells and tissues is achieved without the need for formulations more complex than just aqueous solutions. LNA modifications increase the metabolic stability in both circulation and inside of cells resulting in stable tissue concentrations and prolonged activities. This long tissue residence and stability, allows for constant exposure in tumor cells with dosing as infrequent as weekly or fortnightly. Again, this represents an improvement over antisense drugs used in previous oncology trials. Using in vitro and in vivo laboratory models it has been possible to demonstrate that treatment with LNA-modified oligonucleotides produces reductions in target gene expression and ultimately tumor growth that are sequence-, concentration-, and duration-of-therapy-dependent. Taken together the properties of LNA-modified oligonucleotides make them excellent candidates for inhibiting key RNA targets and at this time there are multiple LNA-modified oligonucleotides in clinical trials in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY31-02. doi:10.1158/1538-7445.AM2011-SY31-02

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.

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.

Inflammation induced by hypoxia–ischemia (HI) contributes to the development of injury in the newborn brain. In this study, we investigated the role of galectin-3, a novel inflammatory mediator, in the inflammatory response and development of brain injury in a mouse model for neonatal HI. Galectin-3 gene and protein expression was increased after injury and galectin-3 was located in activated microglia/macrophages. Galectin-3-deficient mice (gal3−/−) were protected from injury particularly in hippocampus and striatum. Microglia accumulation was increased in the gal3−/− mice but accompanied by decreased levels of total matrix metalloproteinase (MMP)-9 and nitrotyrosine. The protection and increase in microglial infiltration was more pronounced in male gal3−/− mice. Trophic factors and apoptotic markers did not significantly differ between groups. In conclusion, galectin-3 contributes to neonatal HI injury particularly in male mice. Our results indicate that galectin-3 exerts its effect by modulating the inflammatory response.

microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases.