Oligonucleotide Agents Research Articles

Calcium Influx of Mast Cells Is Inhibited by Aptamers Targeting the First Extracellular Domain of Orai1

Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we identified oligonucleotides that bind to the first extracellular domain of the Orai1 protein with high affinities and high specificities. These ligands were isolated from a random single-strand DNA (ssDNA) library with 40 randomized sequence positions, using synthesized peptides with amino acid sequences identical to the first extracellular domain of the Orai1 protein as the targets for SELEX selection. Seven aptamers were obtained after 12 rounds of SELEX. An enzyme-linked oligonucleotide assay (ELONA) was performed to determine the affinities of the aptamers. Aptamer Y1 had the highest affinity (Kd = 1.72×10−8 mol/L) and was selected for functional experiments in mast cells. Using LAD2 cells with the human high-affinity IgE receptor and Ca2+ release activation channel (CRAC), we demonstrated that Aptamer Y1 blocked IgE-mediated β-hexosaminidase release from cells triggered by biotin-IgE and streptavidin. A specific binding assay showed that Aptamer Y1 not only bound the Orai1 peptide specifically but also that the Orai1 peptide did not bind significantly to other random oligonucleotide molecules. Furthermore, Aptamer Y1 regulation of intracellular Ca2+ mobilization was investigated by probing intracellular Ca2+ with a Fluo-4-AM fluorescent probe. We found that Aptamer Y1 inhibits Ca2+ influx into antigen-activated mast cells. These results indicate that the target of Aptamer Y1 in the degranulation pathway is upstream of Ca2+ influx. Therefore, these oligonucleotide agents represent a novel class of CRAC inhibitors that may be useful in the fight against allergic diseases.

Preparation of Targeted Anionic Lipid-Coated Polyplexes for MicroRNA Delivery.

As nonviral nucleic acid delivery vehicles, lipid nanoparticles (LNPs) have been widely used. Here we describe the synthesis and evaluation of LNPs based on targeted anionic lipid-coated polyplexs for therapeutic delivery of microRNA (miRNA) mimics. These LNPs are particularly suited for therapeutic delivery of oligonucleotide agents to leukemia cells.

Oligonucleotide-Based Drug Development: Considerations for Clinical Pharmacology and Immunogenicity.

The field of oligonucleotide (OGN)-based therapeutics has been growing dramatically in the past decade, providing innovative platforms to develop agents for the treatment of a wide variety of clinical conditions. OGN agents have unique physicochemical properties and pharmacokinetic/pharmacodynamic characteristics. This review considers findings from the literature and information on new molecular entities submitted to the US Food and Drug Administration as OGN-based therapeutics. In addition, the article discusses several challenging issues from the perspective of clinical pharmacology, emphasizing the potential of immunogenicity, the effect of renal impairment on OGN exposure, drug-drug interactions, and the utility of pharmacokinetic/pharmacodynamic modeling. The field of OGN-based therapeutics is in evolution and will benefit from further studies as well as clinical experience to formulate guidelines and promote the development of this class of agents.

Oligonucleotide Conjugates for Therapeutic Applications

Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake mechanisms and pharmacokinetic properties.

PNA-based artificial nucleases as antisense and anti-miRNA oligonucleotide agents

Because of its interesting chemical, physical and biological properties, Peptide Nucleic Acid (PNA) has attracted major attention in molecular biology, for diagnostics purposes and development of biosensors. PNAs have become candidates for gene therapeutic drugs in ANTISENSE (AO) strategy with favorable in vivo biochemical properties. Recently, antisense PNA oligonucleotides have been described in anti-miRNA approach (AMO). We propose PNA-based nucleases as AO and AMO agents. We report the design, synthesis and characterization of two kinds of artificial nucleases composed of a PEG-PNA-PEG domain conjugated to HGG·Cu (A) and DETA (B) as well known cleavage sites. Qualitative (MALDI-TOF) and quantitative (HTS) assays were planned to study nuclease activity of constructs A and B on RNA-3'-FAM target sequence. The results have highlighted the best performance of nuclease B and the relevance of the PEG spacer, in particular for conjugate A, in terms of efficiency of the cleavage, suggesting that conjugates A and B also act as potential antisense and anti-miRNA agents.

Fine-Tuning of ENA®Gapmers as Antisense Oligonucleotides for Sequence-Specific Inhibition

For gene validation and the development of oligonucleotide agents, 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) antisense gapmers are widely available. An in vitro Escherichia coli RNase H reaction analysis using ENA gapmers and an RNA oligonucleotide with mouse peptidylarginine deiminase 4 (PADI4) gene sequences revealed that the RNA oligonucleotide was specifically cleaved in the only reported case of the use of an ENA gapmer with an antisense sequence. On the other hand, duplexes of the full-length transcripts of PADI4 mRNA and ENA gapmers with a wide DNA window were cleaved not only at the target site, but also at nontarget sites by RNase H derived from partial base-pairing between the transcript and the ENA gapmer. When the DNA window region of the ENA gapmer was shortened to 5 or 6 nucleotides, the nontarget cleavage was effectively diminished. Moreover, the specific inhibition of PADI4 mRNA expression was observed in the cotransfection of PADI4 cDNA and ENA gapmers containing a short DNA region into NIH3T3 cells. These results demonstrated that ENA gapmers with a short DNA region improved the sequence-specificity of mRNA downregulation. These optimized ENA gapmers could reduce the "off-target" effect and be applicable to gene validation and oligonucleotide therapeutics.

Update:Aptamers as Novel Radiopharmaceuticals: Their Applications and Future Prospects in Diagnosis and Therapy

The production of biomaterials with the capacity to bind tightly and specifically to cell surface receptors of malignant cells can greatly benefit cancer diagnosis and treatment. Whereas antibodies have the ability to specifically recognize some tumor cell makers, their large size and immunogenecity markedly limit their value. The development of nuclease-resistant oligonucleotide agents, termed aptamers, offers an alternative to antibodies as targeting, diagnostic, and delivery agents. Using the systematic evolution of ligands by exponential enrichment (SELEX) methodology or other variations, one can select specific sequences that have appropriate binding affinities and specificities against clinically relevant markers from large libraries of oligonucleotide ligands. Aptamers have been found to bind their targets with high specificity and with dissociation constants in the subnanomolar or picomolar range. However, the possibility for the selected aptamers to be developed as targeting agents for diagnostic imaging or targeted radiotherapy purposes has yet to be realized. Peptide-coupling reactions between amino and carboxylic groups offer the possibility of labeling the aptamers with a number of chelators that, coupled with appropriate radionuclides, would generate novel targeted radiopharmaceuticals for the diagnosis and therapy of disease. The unparalleled combinatorial chemical diversity, small size, and modification ability of aptamers is expected to meet the criteria for robust, generic drug discovery technology and open new horizons for the development of future radiopharmaceuticals.

Delivery of double-stranded DNA thioaptamers into HIV-1 infected cells for antiviral activity

Oligonucleotide agents (ODN) are emerging as attractive alternatives to chemical drugs. However, the clinical use of ODNs as therapeutics has been hindered by their susceptibility to degradation by cellular enzymes and their limited ability to penetrate intact cells. We have used various liposome-mediated transfection agents, for the in vitro delivery of DNA thioaptamers into U373-MAGI-CCR5 cells. Our lead thioaptamer, R12-2, targets the RNase H domain of the HIV-1 reverse transcriptase (RT) and inhibits viral infection in U373-MAGI-CCR5 cells. R12-2, a 62-base-pair, double-stranded DNA molecule with a monothio-phosphate modified backbone, was selected through a novel combinatorial selection method. We studied the use of oligofectamine (OF), TFX-20, Transmessenger (TM), and Gene Jammer (GJ) for transfection of the thio-modified DNA aptamers. OF-transfected U373-MAGI-CCR5 cells resulted in 68% inhibition of HIV infection in the treated cells compared to the untreated control. Inhibition was observed in a dose-dependent manner with maximal inhibition of 83%. In this report, we demonstrate that monothioate-modified DNA duplex oligonucleotides can be efficiently delivered into cells by liposome-based transfection agents to inhibit HIV replication.

Reversal of allergic airway inflammation and airway hyperresponsiveness by second-generation immunomodulatory oligonucleotide agents containing novel RpG motifs

The immunomodulatory oligonucleotide (IMO) agents containing novel RpG motif have been shown to potently modulate immune responses. Importantly, this novel motif has been shown to override species-specific receptor recognition observed with natural CpG motif. We have recently reported that second-generation IMOs containing a different stimulatory motif, CpR (R=2'-deoxy-7-deazaguanosine), effectively prevent key parameters of allergic asthma. Since most asthmatics are treated after the development of asthma, in the present study we examined whether the IMOs containing novel RpG motif can reverse established allergic airway inflammation. Mice sensitized and challenged with ovalbumin (OVA) were evaluated for airway hyperresponsiveness (AHR) to methacholine. Following this, sensitized mice were randomized and treated with placebo or two doses (30 μg and 60 μg/dose, s.c.) of RpG IMO during the following 10 days. Two days after the final treatment, mice were rechallenged with OVA and pulmonary functions were recorded. Mice treated with RpG IMO were significantly protected from both early (EAR) and late (LAR) allergic response, AHR to methacholine, BAL and peribronchial eosinophilia, BAL and serum IL-5, and total serum IgE as compared to vehicle-treated OVA-sensitized and challenged animals. There was a significant increase in immature lung dendritic cells (CD11c+CD45R+) together with increase in serum IL-10 levels and significant decrease in lung DC2 type cells (CD11c+ CD11b+CD8α-) following treatment with RpG IMO. These data suggest that the second-generation IMOs containing novel RpG motif are effective and potent in attenuating established allergic airway inflammation in bronchial asthma, and this effect could be mediated via decreasing lung DC2 cells and increasing immature DCs.

Cyclodextrins in oligonucleotide delivery

The aim of this contribution is to summarize recent findings on the potential use of cyclodextrins and their derivatives as carriers for oligonucleotide agents. Their peculiar properties could be exploited in such an emerging therapeutic area by virtue of their capability of interacting with cellular membranes, thus giving rise to improved cellular uptake. In particular, some specific derivatives could be considered as promising future excipients for the delivery of ‘naked’ antisense and/or decoy oligonucleotides which are difficult to formulate with existing pharmaceutical excipients.

Inhibition of the Human Immunodeficiency Virus Type 1 DNA Integration by Modified Oligonucleotides

Oligonucleotide inhibitors of the HIV-1 DNA integration identified to date are reviewed. Two basic strategies of blocking the integration are considered: shielding the integrase-binding sites on the viral DNA by triplex-forming oligonucleotides, and directly inhibiting the enzyme with oligonucleotide agents.

2D 1H and 31P NMR spectra and distorted A-DNA-like duplex structure of a phosphorodithioate oligonucleotide.

Assignment of the 1H and 31P NMR spectra of a phosphorodithioate modified oligonucleotide decamer duplex, d(CGCTTpS2-AAGCG)2 (10-mer-S; a site of dithioate substitution is designated with the symbols pS2-), was achieved by two-dimensional homonuclear TOCSY, NOESY and 1H-31P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. In contrast to the parent palindromic decamer sequence (1) which has been shown to exist entirely in the duplex B-DNA conformation under comparable conditions (100 mM KCl), the dithiophosphate analogue forms a hairpin loop. However, the duplex form of the dithioate oligonucleotide can be stabilized at lower temperatures, higher salt and strand concentration. The solution structure of the decamer duplex was calculated by an iterative hybrid relaxation matrix method (MORASS) combined with 2D NOESY-distance restrained molecular dynamics. These backbone modified compounds, potentially attractive antisense oligonucleotide agents, are often assumed to possess similar structure as the parent nucleic acid complex. Importantly, the refined structure of the phosphorodithioate duplex shows a significant deviation from the parent unmodified, phosphoryl duplex. An overall bend and unwinding in the phosphorodithioate duplex is observed. The structural distortion of the phosphorodithioate duplex was confirmed by comparison of helicoidal parameters and groove dimensions. Especially, the helical twists of the phosphorodithioate decamer deviate significantly from the parent phosphoryl decamer. The minor groove width of phosphorodithioate duplex 10-mer-S varies between 8.4 and 13.3 A which is much wider than those of the parent phosphoryl decamer d(CGCTTAAGCG)2 (4.2 approximately 9.4 A). The larger minor groove width of 10-mer-S duplex contributes to the unwinding of the backbone and indicates that the duplex has an overall A-DNA-like conformation in the region surrounding the dithiophosphate modification.

Antisense oligonucleotides for therapeutic intervention.

Advances have been made in defining the best target sequences for use in antisense oligonucleotide technology, and new chemical derivatives of oligonucleotides are being investigated. Although the potential use of antisense oligonucleotide agents in the treatment of neoplastic, viral and parasitic diseases continues to be explored, they are not yet suitable for administration to humans for reasons that are discussed.