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Oligonucleotide, Therapy, and Applications

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pharmacology".

Deadline for manuscript submissions: closed (27 February 2021) | Viewed by 22929

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Special Issue Editors

Pharmacogenetics Platform, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain
Interests: pharmacogenetics; genetic variants; non-viral vector; transfection; hydrodynamic transfection; ex vivo organ; therapeutic oligonucleotides; antisense oligonucleotides
Special Issues, Collections and Topics in MDPI journals
Pharmacogenetic Unit, Instituto de Investigación Sanitaria La Fe, Gene Therapy Group, Universitat de València, Valencia, Spain
Interests: gene therapy; non viral; oligonucleotides; vaccines; pharmacogenetic
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Oligonucleotides (ON) constitute a new group of molecular agents, the object of significant interest due to their potential value as drugs for diagnostic and therapeutic applications. Their special interest derives from the intrinsic characteristics of ONs: a) ONs are informative agents, a property that derives from the order in which the nucleotides of each particular ON are arranged; b) ONs can act as ligands (ASO, TFO, aptamers, G-quadruplex, etc.) of complementary nucleic acid sequences (DNA or RNA) due to their high capacity to hybridize (by means of Watson and Crick or Hoogsteen links) with other nucleotide sequences, resulting in specific gene modulatory effects. However, nonspecific sequences may also be of interest, as is the case with repetitive nucleotide sequences (CpG) with adjuvant effects of vaccines; c) ONs can also rapidly evolve to achieve specific advantages of utility (targeting, stability, efficacy, toxicity, etc.) or high-sensitivity diagnostic technology (markers, analyzes, biosensors, FISH, microarrays, etc.), by chemical modification of nucleotides in any of their atoms. These properties show that ONs are first-order molecules due to their potential usefulness in practice.

In this collection of research articles and review papers, we aim to highlight their therapeutic, but also diagnostic and technological utility as drugs.

Prof. Salvador F. Aliño
Dr. Luis Sendra
Guest Editors

Manuscript Submission Information

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Keywords

  • antisene
  • silencing
  • splicing
  • aptamer
  • gapmer
  • siRNA
  • CpG
  • editing
  • targeting
  • delivery
  • release
  • analysis
  • bioassay
  • biosensor
  • array
  • disease
  • diagnostics
  • FISH
  • beacons
  • vaccine
  • adjuvant
  • immunomodulation

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Published Papers (8 papers)

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Research

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16 pages, 18583 KiB  
Article
An Antisense Oligonucleotide against a Splicing Enhancer Sequence within Exon 1 of the MSTN Gene Inhibits Pre-mRNA Maturation to Act as a Novel Myostatin Inhibitor
by Kazuhiro Maeta, Manal Farea, Hisahide Nishio and Masafumi Matsuo
Int. J. Mol. Sci. 2022, 23(9), 5016; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23095016 - 30 Apr 2022
Cited by 2 | Viewed by 2354
Abstract
Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 [...] Read more.
Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in rhabdomyosarcoma cells. Remarkably, it did not decrease GDF11 mRNA levels, indicating myostatin-specific inhibition. As expected, KMM001 enhanced the proliferation of human myoblasts. We conclude that KMM001 is a novel myostatin inhibitor that inhibits pre-mRNA maturation. KMM001 has great promise for clinical applications and should be examined for its ability to treat various muscle-wasting conditions. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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16 pages, 1965 KiB  
Article
Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52
by Jessica M. Cale, Kane Greer, Sue Fletcher and Steve D. Wilton
Int. J. Mol. Sci. 2021, 22(7), 3479; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073479 - 27 Mar 2021
Cited by 4 | Viewed by 2703
Abstract
Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 individuals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different [...] Read more.
Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 individuals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different fibrillin-1 monomers unable to interact to form functional microfibrils. Here, we describe in vitro evaluation of antisense oligonucleotides designed to mediate exclusion of FBN1 exon 52 during pre-mRNA splicing to restore monomer homology. Antisense oligonucleotide sequences were screened in healthy control fibroblasts. The most effective sequence was synthesised as a phosphorodiamidate morpholino oligomer, a chemistry shown to be safe and effective clinically. We show that exon 52 can be excluded in up to 100% of FBN1 transcripts in healthy control fibroblasts transfected with PMO52. Immunofluorescent staining revealed the loss of fibrillin 1 fibres with ~50% skipping and the subsequent re-appearance of fibres with >80% skipping. However, the effect of exon skipping on the function of the induced fibrillin-1 isoform remains to be explored. Therefore, these findings demonstrate proof-of-concept that exclusion of an exon from FBN1 pre-mRNA can result in internally truncated but identical monomers capable of forming fibres and lay a foundation for further investigation to determine the effect of exon skipping on fibrillin-1 function. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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13 pages, 2422 KiB  
Article
Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii
by Alexander Batista-Duharte, Luis Sendra, Maria José Herrero, Deivys Leandro Portuondo, Damiana Téllez-Martínez, Gladys Olivera, Manuel Fernández-Delgado, Beatriz Javega, Guadalupe Herrera, Alicia Martínez, Paulo Inacio Costa, Iracilda Zeppone Carlos and Salvador Francisco Aliño
Int. J. Mol. Sci. 2021, 22(7), 3470; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073470 - 27 Mar 2021
Cited by 5 | Viewed by 2474
Abstract
Background: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of [...] Read more.
Background: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2′OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. Methods: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. Conclusion: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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20 pages, 6699 KiB  
Article
Covalent Bi-Modular Parallel and Antiparallel G-Quadruplex DNA Nanocostructs Reduce Viability of Patient Glioma Primary Cell Cultures
by Valeria Legatova, Nadezhda Samoylenkova, Alexander Arutyunyan, Vadim Tashlitsky, Elena Zavyalova, Dmitry Usachev, Galina Pavlova and Alexey Kopylov
Int. J. Mol. Sci. 2021, 22(7), 3372; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073372 - 25 Mar 2021
Cited by 4 | Viewed by 1773
Abstract
G-quadruplex oligonucleotides (GQs) exhibit specific anti-proliferative activity in human cancer cell lines, and they can selectively inhibit the viability/proliferation of cancer cell lines vs. non-cancer ones. This ability could be translated into a cancer treatment, in particular for glioblastoma multiform (GBM), which currently [...] Read more.
G-quadruplex oligonucleotides (GQs) exhibit specific anti-proliferative activity in human cancer cell lines, and they can selectively inhibit the viability/proliferation of cancer cell lines vs. non-cancer ones. This ability could be translated into a cancer treatment, in particular for glioblastoma multiform (GBM), which currently has a poor prognosis and low-efficiency therapeutic treatments. A novel bi-modular GQ, bi-(AID-1-T), a twin of the previously described three-quartet AID-1-T, was designed and studied in terms of both its structure and function. A covalent conjugation of two AID-1-Ts via three thymidine link, TTT, did not interfere with its initial GQ structure. A comparison of bi-(AID-1-T) with its mono-modular AID-1-T, mono-modular two-quartet HD1, and bi-modular bi-HD1, as well as conventional two-quartet AS1411, was made. Among the five GQs studied, bi-(AID-1-T) had the highest anti-proliferative activity for the neural cancer cell line U87, while not affecting the control cell line, human embryonic fibroblasts. GQs, for the first time, were tested on several primary glioma cultures from patient surgical samples. It turned out that the sensitivity of the patient primary glioma cultures toward GQs varied, with an apparent IC50 of less than 1 μM for bi-(AID-1-T) toward the most sensitive G11 cell culture (glioma, Grade III). Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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20 pages, 2846 KiB  
Article
Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice
by Rubén Pavia-Collado, Valentín Cóppola-Segovia, Lluís Miquel-Rio, Diana Alarcón-Aris, Raquel Rodríguez-Aller, María Torres-López, Verónica Paz, Esther Ruiz-Bronchal, Leticia Campa, Francesc Artigas, Andrés Montefeltro, Raquel Revilla and Analia Bortolozzi
Int. J. Mol. Sci. 2021, 22(6), 2939; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22062939 - 13 Mar 2021
Cited by 15 | Viewed by 4303
Abstract
α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson’s disease (PD). Point mutations and multiplications of the α-Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if [...] Read more.
α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson’s disease (PD). Point mutations and multiplications of the α-Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α-Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α-Syn (A30P*A53T*α-Syn). We found that A30P*A53T*α-Syn mice at 4–5 months of age showed 3.5-fold increases in human α-Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α-Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND-1233-ASO administration (100 µg/day, 28 days) prevented the α-Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α-Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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13 pages, 2919 KiB  
Article
SERS-Based Colloidal Aptasensors for Quantitative Determination of Influenza Virus
by Dmitry Gribanyov, Gleb Zhdanov, Andrei Olenin, Georgii Lisichkin, Alexandra Gambaryan, Vladimir Kukushkin and Elena Zavyalova
Int. J. Mol. Sci. 2021, 22(4), 1842; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22041842 - 12 Feb 2021
Cited by 23 | Viewed by 3000
Abstract
Development of sensitive techniques for rapid detection of viruses is on a high demand. Surface-enhanced Raman spectroscopy (SERS) is an appropriate tool for new techniques due to its high sensitivity. DNA aptamers are short structured oligonucleotides that can provide specificity for SERS biosensors. [...] Read more.
Development of sensitive techniques for rapid detection of viruses is on a high demand. Surface-enhanced Raman spectroscopy (SERS) is an appropriate tool for new techniques due to its high sensitivity. DNA aptamers are short structured oligonucleotides that can provide specificity for SERS biosensors. Existing SERS-based aptasensors for rapid virus detection had several disadvantages. Some of them lacked possibility of quantitative determination, while others had sophisticated and expensive implementation. In this paper, we provide a new approach that combines rapid specific detection and the possibility of quantitative determination of viruses using the example of influenza A virus. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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14 pages, 3455 KiB  
Article
Light-Induced Oxidase Activity of DNAzyme-Modified Quantum Dots
by Krzysztof Żukowski, Joanna Kosman and Bernard Juskowiak
Int. J. Mol. Sci. 2020, 21(21), 8190; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218190 - 01 Nov 2020
Cited by 2 | Viewed by 2494
Abstract
Here, we report the synthesis of a quantum dot (QD)-DNA covalent conjugate to be used as an H2O2-free DNAzyme system with oxidase activity. Amino-coupling conjugation was carried out between amino-modified oligonucleotides (CatG4-NH2) and carboxylated quantum dots (CdTe@COOH [...] Read more.
Here, we report the synthesis of a quantum dot (QD)-DNA covalent conjugate to be used as an H2O2-free DNAzyme system with oxidase activity. Amino-coupling conjugation was carried out between amino-modified oligonucleotides (CatG4-NH2) and carboxylated quantum dots (CdTe@COOH QDs). The obtained products were characterized by spectroscopic methods (UV-Vis, fluorescence, circular dichroizm (CD), and IR) and the transmission electron microscopy (TEM) technique. A QD-DNA system with a low polydispersity and high stability in aqueous solutions was successfully obtained. The catalytic activity of the QD-DNA conjugate was examined with Amplex Red and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) indicators using reactive oxygen species (ROS) generated by visible light irradiation. The synthesized QD-DNAzyme exhibited enhanced catalytic activity compared with the reference system (a mixture of QDs and DNAzyme). This proved the assumption that the covalent attachment of DNAzyme to the surface of QD resulted in a beneficial effect on its catalytic activity. The results proved that the QD-DNAzyme system can be used for generation of the signal by light irradiation. The light-induced oxidase activity of the conjugate was demonstrated, proving that the QD-DNAzyme system can be useful for the development of new cellular bioassays, e.g., for the determination of oxygen radical scavengers. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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Review

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17 pages, 1684 KiB  
Review
Epigenetic Targets for Oligonucleotide Therapies of Pulmonary Arterial Hypertension
by William Gerthoffer
Int. J. Mol. Sci. 2020, 21(23), 9222; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21239222 - 03 Dec 2020
Cited by 8 | Viewed by 2894
Abstract
Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverse established arterial wall thickening, stiffening, and hypercontractility. Therefore, new approaches are needed to achieve [...] Read more.
Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverse established arterial wall thickening, stiffening, and hypercontractility. Therefore, new approaches are needed to achieve long-acting prevention and reversal of occlusive pulmonary vascular remodeling. Several promising new drug classes are emerging from a better understanding of pulmonary vascular gene expression programs. In this review, potential epigenetic targets for small molecules and oligonucleotides will be described. Most are in preclinical studies aimed at modifying the growth of vascular wall cells in vitro or normalizing vascular remodeling in PAH animal models. Initial success with lung-directed delivery of oligonucleotides targeting microRNAs suggests other epigenetic mechanisms might also be suitable drug targets. Those targets include DNA methylation, proteins of the chromatin remodeling machinery, and long noncoding RNAs, all of which act as epigenetic regulators of vascular wall structure and function. The progress in testing small molecules and oligonucleotide-based drugs in PAH models is summarized. Full article
(This article belongs to the Special Issue Oligonucleotide, Therapy, and Applications)
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