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Bioconjugate Chemistry Nov 2023Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration...
Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood-brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising activity and pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.
Topics: Oligonucleotides, Antisense; Antibodies; Oligonucleotides; Peptides
PubMed: 37916986
DOI: 10.1021/acs.bioconjchem.3c00393 -
International Journal of Oncology Jun 2022Cancer‑related deaths remain a challenging and devastating obstacle to defeat despite the tremendous advances in cancer treatment. Cancer metastasis is the major cause... (Review)
Review
Cancer‑related deaths remain a challenging and devastating obstacle to defeat despite the tremendous advances in cancer treatment. Cancer metastasis is the major cause of these cancer‑related deaths. Metastasis involves sequential steps during cancer cells' journey to a new site. These steps are coordinately regulated by specific intracellular regulators and cellular interactions between the cancer cells and the supporting microenvironment of the different organs. The development of aptamer‑based therapeutics is a promising strategy to fight cancer metastasis as it holds potential advantages. Oligonucleotide and peptide aptamers are short sequences of single‑stranded nucleic acids or amino acids, respectively, that target proteins, genetic materials, and cells. Antimetastatic aptamer‑based therapeutics exert their pharmacological effect by direct interaction with the signaling pathways inside the cancer cells or the communications between cancer cells and the tumor microenvironment. In addition, aptamers have been utilized as a guiding ligand to deliver a therapeutic moiety to cancer cells or the supporting microenvironment. The selected aptamer possesses high specificity since it is designed to recognize and interact with its target. This review summarizes recent advances in the development of aptamer‑based therapeutics targeting mediators of cancer metastasis. In addition, potential opportunities are discussed to inspire researchers in the field to develop novel aptamer‑based antimetastatic treatments.
Topics: Aptamers, Nucleotide; Drug Delivery Systems; Humans; Neoplasms; Tumor Microenvironment
PubMed: 35425991
DOI: 10.3892/ijo.2022.5355 -
Biomedical Chromatography : BMC Jan 2018Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, owing to their complex structure and... (Review)
Review
Phosphorothioate (PS) oligonucleotides are a rapidly rising class of drugs with significant therapeutic applications. However, owing to their complex structure and multistep synthesis and purification processes, generation of low-level impurities and degradation products are common. Therefore, they require significant investment in quality control and impurity identification. This requires the development of advanced methods for analysis, characterization and quantitation. In addition, the presence of the PS linkage leads to the formation of chiral centers which can affect their biological properties and therapeutic efficiency. In this review, the different types of oligonucleotide impurities and degradation products, with an emphasis on their origin, mechanism of formation and methods to reduce, prevent or even eliminate their production, will be extensively discussed. This review will focus mainly on the application of chromatographic techniques to determine these impurities but will also discuss other approaches such as mass spectrometry, capillary electrophoresis and nuclear magnetic resonance spectroscopy. Finally, the chirality and formation of diastereomer mixtures of PS oligonucleotides will be covered as well as approaches used for their characterization and the application for the development of stereochemically-controlled PS oligonucleotides.
Topics: Chromatography, High Pressure Liquid; Electrophoresis; Humans; Magnetic Resonance Spectroscopy; Mass Spectrometry; Oligonucleotides; Quality Control; Stereoisomerism
PubMed: 28869310
DOI: 10.1002/bmc.4088 -
Bioanalysis 2016Technical advances and demands for high-throughput accurate quantification of oligonucleotide therapeutics and biomarkers in pharmaceutical research and clinical... (Review)
Review
Technical advances and demands for high-throughput accurate quantification of oligonucleotide therapeutics and biomarkers in pharmaceutical research and clinical diagnosis have aided evolution in quantitative bioanalysis of oligonucleotides. Many bioanalytical methods are available for absolute quantification of oligonucleotides in biological matrices. They can be broadly classified into two categories: hybridization-based assays commonly used by molecular biologists and chromatographic assays routinely used by chemists. Each category has its own advantages and disadvantages for specific applications. This review summarizes the mechanisms and applications of some of the current most commonly used techniques in each category.
Topics: Animals; Biomarkers; Chemistry Techniques, Analytical; Genetic Techniques; Humans; Oligonucleotides
PubMed: 26652713
DOI: 10.4155/bio.15.234 -
Molecular Diagnosis & Therapy Jun 2019Aptamers are synthetic DNA or RNA oligonucleotide ligands with great potential for therapeutic applications. A vast number of disease-related targets have been used to... (Review)
Review
Aptamers are synthetic DNA or RNA oligonucleotide ligands with great potential for therapeutic applications. A vast number of disease-related targets have been used to identify agonistic, antagonistic, or inhibitory aptamers, or aptamer-based targeting ligands. However, only a few aptamers have reached late-stage clinical trials so far and the commercial infrastructure is still far behind that of other therapeutic agents such as monoclonal antibodies. The desirable properties of aptamers such as selectivity, chemical flexibility, or cost-efficiency are faced by challenges, including a short half-life in vivo, immunogenicity, and entrapment in cellular organelles. Aptamer research is still in an early stage, and a deeper understanding of their structure, target interactions, and pharmacokinetics is necessary to catch up to the clinical market. In this review, we will discuss the benefits and limitations in the development of therapeutic aptamers, as well as the advances and future directions of aptamer research. The progress towards effective therapies seems to be slow, but it has not stopped and the best is yet to come.
Topics: Animals; Aptamers, Nucleotide; Biological Transport; Clinical Trials as Topic; Drug Evaluation, Preclinical; Humans; SELEX Aptamer Technique
PubMed: 31037641
DOI: 10.1007/s40291-019-00400-6 -
Investigational New Drugs Feb 2023One of the features that differentiate cancer cells is their increased proliferation rate, which creates an opportunity for general anti-tumor therapy directed against...
One of the features that differentiate cancer cells is their increased proliferation rate, which creates an opportunity for general anti-tumor therapy directed against the elevated activity of replicative apparatus in tumor cells. Besides DNA synthesis, successful genome replication requires the reparation of the newly synthesized DNA. Malfunctions in reparation can cause fatal injuries in the genome and cell death. Recently we have found that the ultra-short single-stranded deoxyribose polynucleotides of random sequence (ssDNA) effectively inhibit the catalytic activity of DNA polymerase [Formula: see text]. This effect allowed considering these substances as potential anti-tumor drugs, which was confirmed experimentally both in vitro (using cancer cell cultures) and in vivo (using cancer models in mice). According to the obtained results, ssDNA significantly suppresses cancer development and tumor growth, allowing consideration of them as novel candidates for anti-cancer drugs.
Topics: Animals; Mice; Polydeoxyribonucleotides; DNA; DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins
PubMed: 36749469
DOI: 10.1007/s10637-023-01333-y -
Talanta Dec 2021The design and fabrication of high sensitive and selective biosensing platforms areessential goals to precisely recognize biomaterials in biological assays. In... (Review)
Review
The design and fabrication of high sensitive and selective biosensing platforms areessential goals to precisely recognize biomaterials in biological assays. In particular, determination of adenosine triphosphate (ATP) as the main energy currency of the cells and one of the most important biomolecules in living organisms is a pressing need in advanced biological detection. Recently, aptamer-based biosensors are introduced as a new direct strategy in which the aptamers (Apts) directly bind to the different targets and detect them on the basis of conformational changes and physical interactions. They can also be conjugated to optical and electronic probes such as quantum dot (QD) nanomaterials and provide unique QD aptasensing platforms. Currently, these Apt-based biosensors with excellent recognition features have attracted extensive attention due to the high specificity, rapid response and facile construction. Therefore, in this review article, recent achievements and advances in aptasensing detection of ATP based on different detection methods and types of QDs are discussed. In this regard, the optical and electrochemical aptasensors have been categorized based on detection methods; fluorescence (FL), electrochemiluminescence (ECL) and photoelectrochemical (PEC) and they have been also divided to two main groups based on QDs; metal-based (M-based) and carbon-based (C-based) materials. Then, their advantages and limitations have been highlighted, compared and discussed in detail.
Topics: Adenosine Triphosphate; Aptamers, Nucleotide; Biosensing Techniques; Electrochemical Techniques; Oligonucleotides; Quantum Dots
PubMed: 34517621
DOI: 10.1016/j.talanta.2021.122753 -
Small (Weinheim An Der Bergstrasse,... Jan 2022Currently, nucleic acid aptamers are exploited as robust targeting ligands in the biomedical field, due to their specific molecular recognition, little immunogenicity,... (Review)
Review
Currently, nucleic acid aptamers are exploited as robust targeting ligands in the biomedical field, due to their specific molecular recognition, little immunogenicity, low cost, ect. Thanks to the facile chemical modification and high hydrophilicity, aptamers can be site-specifically linked with hydrophobic moieties to prepare aptamer-organic amphiphiles (AOAs), which spontaneously assemble into aptamer-organic amphiphile self-assemblies (AOASs). These polyvalent self-assemblies feature with enhanced target-binding ability, increased resistance to nuclease, and efficient cargo-loading, making them powerful platforms for bioapplications, including targeted drug delivery, cell-based cancer therapy, biosensing, and bioimaging. Besides, the morphology of AOASs can be elaborately manipulated for smarter biomedical functions, by regulating the hydrophilicity/hydrophobicity ratio of AOAs. Benefiting from the boom in DNA synthesis technology and nanotechnology, various types of AOASs, including aptamer-polymer amphiphile self-assemblies, aptamer-lipid amphiphile self-assemblies, aptamer-cell self-assemblies, ect, have been constructed with great biomedical potential. Particularly, stimuli-responsive AOASs with transformable structure can realize site-specific drug release, enhanced tumor penetration, and specific target molecule detection. Herein, the general synthesis methods of oligonucleotide-organic amphiphiles are firstly summarized. Then recent progress in different types of AOASs for bioapplications and strategies for morphology control are systematically reviewed. The present challenges and future perspectives of this field are also discussed.
Topics: Aptamers, Nucleotide; Drug Delivery Systems; Humans; Hydrophobic and Hydrophilic Interactions; Nanotechnology; Neoplasms; Oligonucleotides
PubMed: 34622570
DOI: 10.1002/smll.202104341 -
International Journal of Molecular... Jul 2023Osteoarthritis (OA) is characterized by degeneration of the joint cartilage, inflammation, and a change in the chondrocyte phenotype. Inflammation also promotes cell...
Osteoarthritis (OA) is characterized by degeneration of the joint cartilage, inflammation, and a change in the chondrocyte phenotype. Inflammation also promotes cell hypertrophy in human articular chondrocytes (HC-a) by activating the NF-κB pathway. Chondrocyte hypertrophy and inflammation promote extracellular matrix degradation (ECM). Chondrocytes depend on Smad signaling to control and regulate cell hypertrophy as well as to maintain the ECM. The involvement of these two pathways is crucial for preserving the homeostasis of articular cartilage. In recent years, Polynucleotides Highly Purified Technology (PN-HPT) has emerged as a promising area of research for the treatment of OA. PN-HPT involves the use of polynucleotide-based agents with controlled natural origins and high purification levels. In this study, we focused on evaluating the efficacy of a specific polynucleotide sodium agent, known as CONJURAN, which is derived from fish sperm. Polynucleotides (PN), which are physiologically present in the matrix and function as water-soluble nucleic acids with a gel-like property, have been used to treat patients with OA. However, the specific mechanisms underlying the effect remain unclear. Therefore, we investigated the effect of PN in an OA cell model in which HC-a cells were stimulated with interleukin-1β (IL-1β) with or without PN treatment. The CCK-8 assay was used to assess the cytotoxic effects of PN. Furthermore, the enzyme-linked immunosorbent assay was utilized to detect MMP13 levels, and the nitric oxide assay was utilized to determine the effect of PN on inflammation. The anti-inflammatory effects of PN and related mechanisms were investigated using quantitative PCR, Western blot analysis, and immunofluorescence to examine and analyze relative markers. PN inhibited IL-1β induced destruction of genes and proteins by downregulating the expression of MMP3, MMP13, iNOS, and COX-2 while increasing the expression of aggrecan (ACAN) and collagen II (COL2A1). This study demonstrates, for the first time, that PN exerted anti-inflammatory effects by partially inhibiting the NF-κB pathway and increasing the Smad2/3 pathway. Based on our findings, PN can potentially serve as a treatment for OA.
Topics: Animals; Humans; Male; NF-kappa B; Matrix Metalloproteinase 13; Polynucleotides; Cells, Cultured; Semen; Inflammation; Osteoarthritis; Chondrocytes; Anti-Inflammatory Agents; Hypertrophy; Interleukin-1beta
PubMed: 37569659
DOI: 10.3390/ijms241512282 -
Clinical Chemistry Oct 2021Chemically synthesized oligonucleotides are vital to most nucleic acids-based technologies and several applications are sensitive to oligonucleotide sequence errors....
BACKGROUND
Chemically synthesized oligonucleotides are vital to most nucleic acids-based technologies and several applications are sensitive to oligonucleotide sequence errors. However, it is challenging to identify and quantify the types and amount of errors in synthetic oligonucleotides.
METHODS
We applied a digital sequencing approach using unique molecular identifiers to quantify errors in chemically synthesized oligonucleotides from multiple manufacturers with different synthesis strategies, purity grades, batches, and sequence context.
RESULTS
We detected both deletions and substitutions in chemical oligonucleotide synthesis, but deletions were 7 times more common. We found that 97.2% of all analyzed oligonucleotide molecules were intact across all manufacturers and purity grades, although the number of oligonucleotide molecules with deletions ranged between 0.2% and 11.7% for different types. Different batches of otherwise identical oligonucleotide types also varied significantly, and batch effect can impact oligonucleotide quality more than purification. We observed a bias of increased deletion rates in chemically synthesized oligonucleotides toward the 5'-end for 1 out of 2 sequence configurations. We also demonstrated that the performance of sequencing assays depends on oligonucleotide quality.
CONCLUSIONS
Our data demonstrate that manufacturer, synthesis strategy, purity, batch, and sequence context all contribute to errors in chemically synthesized oligonucleotides and need to be considered when choosing and evaluating oligonucleotides. High-performance oligonucleotides are essential in numerous molecular applications, including clinical diagnostics.
Topics: Humans; Oligonucleotides
PubMed: 34459892
DOI: 10.1093/clinchem/hvab136