Solid-Phase Separation of Toxic Phosphorothioate Antisense Oligonucleotide-Protein Nucleolar Aggregates Is Cytoprotective.

Phosphorothioate antisense oligonucleotides (PS-ASOs) interact with proteins and can localize to or induce the formation of a variety of subcellular PS-ASO-protein or PS-ASO-ribonucleoprotein aggregates. In this study, we show that these different aggregates that form with varying compositions at various concentrations in the cytosol, nucleus, and nucleolus may undergo phase separations in cells. Some aggregates can form with both nontoxic and toxic PS-ASOs, such as PS bodies, paraspeckles, and nuclear filaments. However, toxic PS-ASOs have been shown to form unique nucleolar aggregates that result in nucleolar dysfunction and apoptosis. These include liquid-like aggregates that we labeled "cloudy nucleoli" and solid-like perinucleolar filaments. Toxic nucleolar aggregates may undergo solid-phase separation and in the solid phase, protein mobility in and out of the aggregates is limited. Other aggregates appear to undergo liquid-phase separation, including paraspeckles and perinucleolar caps, in which protein mobility is negatively correlated with the binding affinity of the proteins to PS-ASOs. However, PS bodies and nuclear filaments are solid-like aggregates. Importantly, in cells that survived treatment with toxic PS-ASOs, solid-like PS-ASO aggregates accumulated, especially Hsc70-containing nucleolus-like structures, in which modest pre-rRNA transcriptional activity was retained and appeared to mitigate the nucleolar toxicity. This is the first demonstration that exogenous drugs, PS-ASOs, can form aggregates that undergo phase separations and that solid-phase separation of toxic PS-ASO-induced nucleolar aggregates is cytoprotective.

Application of hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the retention and sensitivity studies of antisense oligonucleotides.

The aim of the present investigation was application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the study of antisense oligonucleotides. The influence of several mobile phases, differing with the salt type, their concentration and pH value on the retention and the separation of antisense oligonucleotides has been examined for this purpose. Four different stationary phases were also applied including unmodified silica, silica modified with the use of sulfobetaine groups, polyhydroxy and aminopropyl groups. Such wide range of tested conditions has been useful in better understanding of the retention mechanism of tested compounds. The results obtained during this investigation indicated that greater retention, greater peaks symmetry, as well as more effective separation of oligonucleotides, were obtained for the zwitterionic stationary phase. Moreover, the optimization of tandem mass spectrometry parameters with the use of Central Composite Design was performed and different mobile phases were tested to choose that one, which provided the greatest antisense oligonucleotides peak areas in Multiple Reaction Monitoring mode and consequently, the greatest possible sensitivity. Hydrophilic interaction liquid chromatography was compared with the ion pair chromatography, commonly used in the analysis of oligonucleotides. Both techniques were compared in terms of selectivity of separation as well as the sensitivity of their determination. Obtained results proved that ion pair chromatography provided better results in terms of separation efficiency and peak areas in Multiple Reaction Monitoring for tested conditions. However, these results do not preclude application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the oligonucleotides analysis especially when a mobile phase without ion pair reagents is required.

Hydrophilic interaction in solid-phase extraction of antisense oligonucleotides.

The presented studies aimed to develop a new and simple extraction method based on hydrophilic interaction for antisense oligonucleotides with different modifications. For this purpose, solid-phase extraction cartridges with unmodified silica were used. All extraction steps were performed by utilizing water, acetonitrile, acetone or their mixtures. The results obtained show that a high content (95%) of organic solvent, used during sample loading, is critical to achieve a successful extraction, while elution with pure water allows effective oligonucleotides desorption. The recovery values were greater than 90% in the case of unmodified DNA, phosphorothioate, 2'-O-(2-methoxyethyl) and 2'-O-methyl oligonucleotides. For the mixture of phosphorothioate oligonucleotide and its two synthetic metabolites, the recovery values for the standard solutions were in the range of 70-75%, while for spiked human plasma, 45-50%. The developed method is simple, may be performed in a short time and requires simple solvents like water or acetonitrile/acetone, thus showing promise as an alternative to chaotropic salt-based or ion pair-based SPE methods.

Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides.

Methods for the quantification of antisense oligonucleotides (AONs) provide insightful information on biodistribution and intracellular trafficking. However, the established methods have not provided information on the absolute number of molecules in subcellular compartments or about how many AONs are needed for target gene reduction for unconjugated AONs. We have developed a new method for nuclear AON quantification that enables us to determine the absolute number of AONs per nucleus without relying on AON conjugates such as fluorophores that may alter AON distribution. This study describes an alternative and label-free method using subcellular fractionation, nucleus counting, and locked nucleic acid (LNA) sandwich enzyme-linked immunosorbent assay to quantify absolute numbers of oligonucleotides in nuclei. Our findings show compound variability (diversity) by which 247,000-693,000 LNAs/nuclei results in similar target reduction for different compounds. This method can be applied to any antisense drug discovery platform providing information on specific and clinically relevant AONs. Finally, this method can directly compare nuclear entry of AON with target gene knockdown for any compound design and nucleobase sequence, gene target, and phosphorothioate stereochemistry.

Review on sample preparation methods for oligonucleotides analysis by liquid chromatography.

Antisense oligonucleotides have been successfully investigated for the treatment of different types of diseases. Detection and determination of antisense oligonucleotides and their metabolites are necessary for drug development and evaluation. This review focuses mainly on the first step of the analysis of oligonucleotides i.e. the sample preparation stage, and in particular on the techniques used for liquid chromatography and liquid chromatography coupled with mass spectrometry. Exceptional sample preparation techniques are required as antisense oligonucleotides need to be determined in complex biological matrices. The text discusses general issues in oligonucleotide sample preparation and approaches to their solution. The most popular techniques i.e. protein precipitation, protein enzyme digestion and liquid-liquid extraction are reviewed. Solid phase extraction methods are discussed and the issues connected with the application of each method are highlighted. Other newly reported promising techniques are also described. Finally, there is a summary of actually used techniques and the indication of the direction of future research.

Identification of GalNAc-Conjugated Antisense Oligonucleotide Metabolites Using an Untargeted and Generic Approach Based on High Resolution Mass Spectrometry.

Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceutical companies. Antisense oligonucleotides may be coupled to high-affinity ligands (triantennary N-acetyl galactosamine = GalNAc) for hepatocyte-specific asialoglycoprotein receptors (ASGPR) to enhance uptake to hepatocytes and to increase potency. Since disposition and biotransformation of GalNAc-conjugated oligonucleotides is different from unconjugated oligonucleotides, appropriate analytical methods are required to identify main cleavage sites and degradation products of GalNAc conjugated and unconjugated oligonucleotides in target cells. A highly sensitive method was developed to identify metabolites of oligonucleotides using capillary flow liquid chromatography with column switching coupled to a high resolution Orbitrap Fusion mass spectrometer. Detection of GalNAc-conjugated oligonucleotides and their metabolites was achieved by combining full scan MS with two parallel MS2 experiments, one data-dependent scan and an untargeted MS2 experiment (all ion fragmentation) applying high collision energy. In the all ion fragmentation scan, a diagnostic fragment originating from the phosphorothioate backbone (O2PS-: m/z 94.936) was formed efficiently upon collisional activation. Based on this fragment an accurate determination of metabolites of oligonucleotides was achieved, independent of their sequence or conjugation in an untargeted but highly selective manner. The method was effectively applied to investigate uptake and metabolism of GalNAc-conjugated oligonucleotides in incubations of primary rat hepatocytes; the elucidation of expected and unexpected degradation products was achieved in subnanomolar range.

NOX2 Antisense Attenuates Hypoxia-Induced Oxidative Stress and Apoptosis in Cardiomyocyte.

Heart ischemia is a hypoxia related disease. NOX2 and HIF-1α proteins were increased in cardiomyocytes after acute myocardial infarction. However, the relationship of the hypoxia-induced HIF-1α. NOX2-derived oxidative stress and apoptosis in cardiomyocyte remains unclear. In the current study, we use NOX2 antisense strategy to investigate the role of NOX2 in hypoxia-induced oxidative stress and apoptosis in rat cardiomyocytes. Here, we show that transduction of ADV-NOX2-AS induces potent silencing of NOX2 in cardiomyocytes, and resulting in attenuation of hypoxia-induced oxidative stress and apoptosis. This study indicates the potential of antisense-based therapies and validates NOX2 as a potent therapeutic candidate for heart ischemia.

TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells.

Phosphorothioate (PS) antisense oligonucleotides (ASOs) have been successfully developed as drugs to reduce the expression of disease-causing genes. PS-ASOs can be designed to induce degradation of complementary RNAs via the RNase H pathway and much is understood about that process. However, interactions of PS-ASOs with other cellular proteins are not well characterized. Here we report that in cells transfected with PS-ASOs, the chaperonin T-complex 1 (TCP1) proteins interact with PS-ASOs and enhance antisense activity. The TCP1-ß subunit co-localizes with PS-ASOs in distinct nuclear structures, termed phosphorothioate bodies or PS-bodies. Upon Ras-related nuclear protein (RAN) depletion, cytoplasmic PS-body-like structures were observed and nuclear concentrations of PS-ASOs were reduced, suggesting that TCP1-ß can interact with PS-ASOs in the cytoplasm and that the nuclear import of PS-ASOs is at least partially through the RAN-mediated pathway. Upon free uptake, PS-ASOs co-localize with TCP1 proteins in cytoplasmic foci related to endosomes/lysosomes. Together, our results indicate that the TCP1 complex binds oligonucleotides with TCP1-ß subunit being a nuclear PS-body component and suggest that the TCP1 complex may facilitate PS-ASO uptake and/or release from the endocytosis pathway.

Antisense phosphorothioate oligodeoxyribonucleotide targeted against ICAM-1: synthetic and biological characterization of a process-related impurity formed during oligonucleotide synthesis.

A phosphorothioate-linked oligonucleotide bearing a 3'-terminal phosphorothioate monoester has been synthesized and characterized. This oligonucleotide has been identified as a process-related impurity formed during synthesis of ISIS 2302. Biological properties of the compound have been determined. Based on these data, it can be concluded that this species (3'-TPT) has biological properties similar to parent drug.

Synthesis and characterization of chimeric 2-5A-DNA oligonucleotides.

This unit provides protocols for the synthesis and characterization of 2-5A-antisense nucleic acids. These chimeric oligonucleotides consist of 2',5'-phosphodiester-linked oligoadenylates ligated to 3',5'-deoxyribonucleotides and are readily prepared using phosphoramidite chemistry on CPG solid supports. The 3',5'-deoxyribonucleotide functions as the antisense domain to target a given mRNA sequence, while the 2',5'-phosphodiester-linked oligoadenylate serves to locally activate 2-5A-dependent RNase L, causing the targeted sequence to be cleaved.

Large-scale purification of antisense oligonucleotides by high-performance membrane adsorber chromatography.

Very high flux ion-exchange membranes were utilized for a novel purification of antisense oligonucleotides (20-mer). Strong anion-exchange membranes were produced by attaching polymeric ligands onto a microporous cellulosic matrix. The oligonucleotides purified were therapeutic single-stranded phosphorothioates deoxyribonucleotides. Although small-scale membrane devices (15 cm2) had similar resolution to traditional chromatographic columns; their throughputs were superior. Greater than a 1300-fold scale-up produced very similar purity and yields of the phosphorothionate product. Scale-up experiments were conducted with a 2 m2 surface area membrane module. These modules were easily capable of very high throughputs of 0.5 to 2 l/min. High purity and yields were achieved by both step and linear gradient elution.

Purification of antisense oligonucleotides.

Chromatography is an effective tool for obtaining high-purity synthetic oligonucleotides for a variety of end uses, including antisense drug therapy. Reversed-phase and anion-exchange chromatographies are widely used techniques for this application. While selectivity of these techniques can be modified by methods such as ion-pair RP-HPLC or affinity chromatography, these are presently used only at small scales. RP chromatography makes use of terminal hydrophobic-protecting groups to increase retention and selectivity. The main advantages of the RP method are its utility for the purification of a wide variety of modified oligonucleotide structures, its applicability across a range of terminal hydrophobic groups, such as fluorescein, and its ready use from small scale to very large scale with a minimal requirement for process development. AX-HPLC can also give high-purity products at generally higher media capacities. A more extensive method development effort is typically required for the AX-HPLC purification of AO. The AX yield per unit operation can be lower, but the isolated yield of DMT-off desalted oligonucleotide can be equal to or higher than that from RP-HPLC. As additional AO drugs enter and mature in the market, there will be a potential need for ton-scale purification processes. AX provides a way to scale up production on somewhat less expensive equipment with reduced organic solvent requirements.

Electrochemical method for entrapment of oligonucleotides in polymer-coated electrodes.

Oligonucleotides were incorporated into conducting films of poly(3, 4-ethylene dioxythiophene) by an electrochemical method that involves two steps. The electrode is first coated with the polymer film by electropolymerization followed by electrooxidation of the formed polymer in the presence of the oligonucleotide. Neutral water soluble polymers such as poly(vinylpyrrolidone) or poly(ethylene glycol) were added to the polymerizing medium resulting in higher incorporation yields of oligonucleotides. The results showed that a payload of about 1.5 nmol/cm2 can be achieved in an 8 micron thick film for an oligomer concentration of about 70 microM in the working solution. Various physical methods were used to analyze the surface of the polymer-coated electrodes. Results showed the presence of domains of varying sizes at the film surface, corresponding to the insertion of the hydrophilic polymer within the matrix of the conductive polymer. The release of entrapped oligonucleotides from the coated film exhibited a three-step profile: a "burst" period during the first 5 min followed by an intermediate and a very slow release period lasting several days. Such polymer films could be exploited as useful reservoirs of biologically active substances for in vivo delivery to targeted tissues. For example, coated stents that can release antiproliferative agents such as antisense oligonucleotides at the site of balloon dilatation may be of interest in the treatment of postangioplasty restenosis.

Synthesis of an antisense oligonucleotide targeted against C-raf kinase: efficient oligonucleotide synthesis without chlorinated solvents.

It is demonstrated that a solution of dichloroacetic acid in toluene removes dimethoxytrityl groups from the 5'-terminus of an antisense phosphorothioate oligodeoxyribonucleotide (ISIS 5132/CGP69846A) during synthesis on solid support cleanly and efficiently. It is therefore suggested to replace health hazardous dichloromethane which is typically used in oligonucleotide synthesis as solvent for DMTr-removal by toluene.

Highly loaded nanoparticulate carrier using an hydrophobic antisense oligonucleotide complex.

Antisense oligonucleotides, and particularly those with phosphorothioate backbones, have emerged as potential gene specific therapeutic agents and are currently undergoing evaluation in clinical trials for a variety of diseases. In the area of HIV-1 therapeutics, targeting of oligonucleotides to infected cells, such as macrophages, would be highly desirable. The present study was designed to prepare and characterize oligonucleotide-loaded nanoparticles for this purpose. Due to their hydrophilic characteristics, oligonucleotides are difficult to entrap in polymeric particles. Here, the oligonucleotides were first complexed with cetyltrimethylammonium bromide. The oligonucleotide-loaded nanoparticles were prepared by the emulsification-diffusion method and subsequently purified. In comparison with previous studies, a high oligonucleotide-loading was achieved; 2.5, 5 and 10% oligonucleotide loading were assessed. If the initial oligonucleotide content was 4%, this method produced a final oligonucleotide loading of 1.9% with an entrapment efficiency of 47%. The integrity of the oligonucleotide and of the polymer, in the final freeze-dried product, was retained.

Analysis of an oligonucleotide N3'-->P5' phosphoramidate/phosphorothioate chimera with capillary gel electrophoresis.

N3'-->P5' phosphoramidate/phosphorothioate chimeric oligonucleotides (ODNs) are presently under investigation as potential antisense drugs. Within the field of antisense research, "second generation" chimeric ODNs have exhibited improved characteristics relative to oligonucleotides with uniformly modified backbones. The ODN of interest for this study consisted of a chemically synthesized 18-mer of mixed nucleotide base sequence with a backbone consisting of eight central phosphorothioate linkages flanked by four N3'-->P5' phosphoramidate (amidate) linkages on the 5'-end and five amidate linkages ont he 3'-end. This chimera presents analytical challenges due to the central phosphorothioate region. Here, we present a capillary gel electrophoresis (CGE) method for the analysis of the above N3'-->P5' phosphoramidate/phosphorothioate chimera. CGE was used to analyze the product prior to its purification by reversed phase - high performance liquid chromatography (RP-HPLC), and each fraction collected from the purification was similarly analyzed. An internal standard was utilized to determine the relative mobility of our product, and polyacrylamide gel electrophoresis (PAGE) analysis was used to verify CGE results.

An improved synthesis of oligodeoxynucleotide N3'-->P5' phosphoramidates and their chimera using hindered phosphoramidite monomers and a novel handle for reverse phase purification.

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.

Preconcentration and separation of antisense oligonucleotides by on-column isotachophoresis and capillary electrophoresis in polymer-filled capillaries.

Small, single-stranded, chemically modified oligonucleotides, complementary to a specific gene section, commonly referred to as antisense compounds, are being investigated as potential therapeutic drugs. A number of modified oligonucleotides, in particular phosphorothioates, are in clinical development. Shorter fragments are found as metabolic products. Isotachophoresis (ITP) allows the introduction of large, diluted sample plugs into the separation capillary. In this work, ITP and capillary electrophoresis (CE) in polymer solutions were successfully coupled in a single capillary in a commercial instrument to increase sensitivity with UV detection and to shorten the time for sample pretreatment. It was shown that ITP-CE can be used as a preconcentration and clean-up method for phosphodiester- and phosphorothioate-containing samples. Up to 3 microL sample could be injected into the capillary without significantly disturbing the separation performance. ITP-CE of phosphodiesters directly out of salt- and protein-containing samples could be demonstrated. For phosphorothioates in serum samples an additional sample clean-up was necessary, due to oligonucleotide-protein binding. An optimized replaceable polymer solution was developed to increase the separation performance for heterogeneous phosphorothioates. A dextran-based sieving medium showed a good separation performance in ITP-CE of phosphorothioates. A concentration detection limit of 8.10(-9) mol/L for the 20-mer phosphorothioate ISIS5132, isolated from rat serum, was found.