Publications by authors named "Jesper Wengel"

246 Publications

Chirality transmission in macromolecular domains.

Nat Commun 2022 01 10;13(1):76. Epub 2022 Jan 10.

Department of Chemistry and Biochemistry, Kent State University, Kent, OH, 44242, USA.

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.
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http://dx.doi.org/10.1038/s41467-021-27708-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8748818PMC
January 2022

Intramuscular Evaluation of Chimeric Locked Nucleic Acid/2'Methyl-Modified Antisense Oligonucleotides for Targeted Exon 23 Skipping in Mdx Mice.

Pharmaceuticals (Basel) 2021 Oct 30;14(11). Epub 2021 Oct 30.

Molecular Genetics, Function & Therapy Department, The Cyprus Institute of Neurology & Genetics, Nicosia 2371, Cyprus.

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2'-Methyl (2'Me)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2'Me AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2'Me AON and LNA/2'Me chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2'Me AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.
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http://dx.doi.org/10.3390/ph14111113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8622172PMC
October 2021

Antisense locked nucleic acid gapmers to control Candida albicans filamentation.

Nanomedicine 2022 Jan 2;39:102469. Epub 2021 Oct 2.

LIBRO-Laboratório de Investigação em Biofilmes Rosário Oliveira, CEB-Centre of Biological Engineering, University of Minho, Braga, Portugal; National Institute for Agrarian and Veterinary Research, Vairão, Vila do Conde, Portugal. Electronic address:

Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2'-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.
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http://dx.doi.org/10.1016/j.nano.2021.102469DOI Listing
January 2022

Oligonucleotides Targeting DNA Repeats Downregulate Gene Expression in Huntington's Patient-Derived Neural Model System.

Nucleic Acid Ther 2021 12 13;31(6):443-456. Epub 2021 Sep 13.

Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Huddinge, Sweden.

Huntington's disease (HD) is one of the most common, dominantly inherited neurodegenerative disorders. It affects the striatum, cerebral cortex, and other subcortical structures leading to involuntary movement abnormalities, emotional disturbances, and cognitive impairments. HD is caused by a CAG•CTG trinucleotide-repeat expansion in exon 1 of the () gene leading to the formation of mutant HTT (mtHTT) protein aggregates. Besides the toxicity of the mutated protein, there is also evidence that mt transcripts contribute to the disease. Thus, the reduction of both mutated mRNA and protein would be most beneficial as a treatment. Previously, we designed a novel anti-gene oligonucleotide (AGO)-based strategy directly targeting the trinucleotide-repeats in DNA and reported downregulation of mRNA and protein in HD patient fibroblasts. In this study, we differentiate HD patient-derived induced pluripotent stem cells to investigate the efficacy of the AGO, a DNA/Locked Nucleic Acid mixmer with phosphorothioate backbone, to modulate transcription during neural development. For the first time, we demonstrate downregulation of mRNA following both naked and magnetofected delivery into neural stem cells (NSCs) and show that neither emergence of neural rosette structures nor self-renewal of NSCs is compromised. Furthermore, the inhibition potency of both mRNA and protein without off-target effects is confirmed in neurons. These results further validate an anti-gene approach for the treatment of HD.
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http://dx.doi.org/10.1089/nat.2021.0021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8713517PMC
December 2021

Cationic oligonucleotide derivatives and conjugates: A favorable approach for enhanced DNA and RNA targeting oligonucleotides.

Beilstein J Org Chem 2021 29;17:1828-1848. Epub 2021 Jul 29.

Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.

Antisense oligonucleotides (ASOs) have the ability of binding to endogenous nucleic acid targets, thereby inhibiting the gene expression. Although ASOs have great potential in the treatment of many diseases, the search for favorable toxicity profiles and distribution has been challenging and consequently impeded the widespread use of ASOs as conventional medicine. One strategy that has been employed to optimize the delivery profile of ASOs, is the functionalization of ASOs with cationic amine groups, either by direct conjugation onto the sugar, nucleobase or internucleotide linkage. The introduction of these positively charged groups has improved properties like nuclease resistance, increased binding to the nucleic acid target and improved cell uptake for oligonucleotides (ONs) and ASOs. The modifications highlighted in this review are some of the most prevalent cationic amine groups which have been attached as single modifications onto ONs/ASOs. The review has been separated into three sections, nucleobase, sugar and backbone modifications, highlighting what impact the cationic amine groups have on the ONs/ASOs physiochemical and biological properties. Finally, a concluding section has been added, summarizing the important knowledge from the three chapters, and examining the future design for ASOs.
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http://dx.doi.org/10.3762/bjoc.17.125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329367PMC
July 2021

Dual role of the miR-146 family in rhinovirus-induced airway inflammation and allergic asthma exacerbation.

Clin Transl Med 2021 06;11(6):e427

Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia.

Rhinovirus (RV) infections are associated with asthma exacerbations. MicroRNA-146a and microRNA-146b (miR-146a/b) are anti-inflammatory miRNAs that suppress signaling through the nuclear factor kappa B (NF-κB) pathway and inhibit pro-inflammatory chemokine production in primary human bronchial epithelial cells (HBECs). In the current study, we aimed to explore whether miR-146a/b could regulate cellular responses to RVs in HBECs and airways during RV-induced asthma exacerbation. We demonstrated that expression of miR-146a/b and pro-inflammatory chemokines was increased in HBECs and mouse airways during RV infection. However, transfection with cell-penetrating peptide (CPP)-miR-146a nanocomplexes before infection with RV significantly reduced the expression of the pro-inflammatory chemokines CCL5, IL-8 and CXCL1, increased interferon-λ production, and attenuated infection with the green fluorescent protein (GFP)-expressing RV-A16 in HBECs. Concordantly, compared to wild-type (wt) mice, Mir146a/b mice exhibited more severe airway neutrophilia and increased T helper (Th)1 and Th17 cell infiltration in response to RV-A1b infection and a stronger Th17 response with a less prominent Th2 response in house dust mite extract (HDM)-induced allergic airway inflammation and RV-induced exacerbation models. Interestingly, intranasal administration of CPP-miR-146a nanocomplexes reduced HDM-induced allergic airway inflammation without a significant effect on the Th2/Th1/Th17 balance in wild-type mice. In conclusion, the overexpression of miR-146a has a strong anti-inflammatory effect on RV infection in HBECs and a mouse model of allergic airway inflammation, while a lack of miR-146a/b leads to attenuated type 2 cell responses in mouse models of allergic airway inflammation and RV-induced exacerbation of allergic airway inflammation. Furthermore, our data indicate that the application of CPP-miR-146a nanocomplexes has therapeutic potential for targeting airway inflammation.
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http://dx.doi.org/10.1002/ctm2.427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8161513PMC
June 2021

Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?

Antibiotics (Basel) 2021 Apr 3;10(4). Epub 2021 Apr 3.

Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE), Faculty of Engineering, University of Porto, R. Dr. Roberto Frias, 4200-465 Porto, Portugal.

The emergence of bacterial resistance to traditional small-molecule antibiotics is fueling the search for innovative strategies to treat infections. Inhibiting the expression of essential bacterial genes using antisense oligonucleotides (ASOs), particularly composed of nucleic acid mimics (NAMs), has emerged as a promising strategy. However, their efficiency depends on their association with vectors that can translocate the bacterial envelope. Vitamin B is among the largest molecules known to be taken up by bacteria and has very recently started to gain interest as a trojan-horse vector. Gapmers and steric blockers were evaluated as ASOs against (). Both ASOs were successfully conjugated to B by copper-free azide-alkyne click-chemistry. The biological effect of the two conjugates was evaluated together with their intracellular localization in . Although not only B but also both B-ASO conjugates interacted strongly with , they were mostly colocalized with the outer membrane. Only 6-9% were detected in the cytosol, which showed to be insufficient for bacterial growth inhibition. These results suggest that the internalization of B-ASO conjugates is strongly affected by the low uptake rate of the B in and that further studies are needed before considering this strategy against biofilms in vivo.
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http://dx.doi.org/10.3390/antibiotics10040379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065541PMC
April 2021

Improved Cancer Targeting by Multimerizing Aptamers on Nanoscaffolds.

Mol Ther Nucleic Acids 2020 Dec 15;22:994-1003. Epub 2020 Oct 15.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.

Aptamers are short single-stranded oligonucleotides selected to bind with high affinity and specificity to a target. In contrast to antibodies, aptamers can be produced in large-scale systems without the need for any biological agents, making them highly attractive as targeting ligands for bioimaging and drug delivery. For applications it is often desirable to multimerize the aptamers in order to increase their binding strength and overall specificity. Additional functionalities, such as imaging and therapeutic agents, as well as pharmacokinetic modifiers, need to be attached in a stoichiometric fashion. Herein, we present a robust method for assembly of up to three aptamers and a fluorophore in a single well-defined nanostructure. The process is entirely modular and can be applied to any aptamer requiring only a single reactive "click handle." Multimerization of two aptamers, A9g and GL21.T, previously shown to target cancer cells, led to a strong increase in cell uptake. A similar effect was observed for the prostate-specific membrane antigen (PSMA)-targeting A9g aptamer in mice where multivalent aptamer binding led to increased tumor specificity. Altogether, this method provides a platform for multimerization of aptamers with advantages in terms of combinatorial screening capacity and multifunctional design of nanomedicine.
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http://dx.doi.org/10.1016/j.omtn.2020.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679244PMC
December 2020

Polyamine-Functionalized 2'-Amino-LNA in Oligonucleotides: Facile Synthesis of New Monomers and High-Affinity Binding towards ssDNA and dsDNA.

Chemistry 2021 Jan 16;27(4):1416-1422. Epub 2020 Dec 16.

Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.

Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2'-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2'-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson-Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3'-nucleolytic digestion relative to the non-functionalized controls.
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http://dx.doi.org/10.1002/chem.202004495DOI Listing
January 2021

Carbazole modified oligonucleotides: synthesis, hybridization studies and fluorescence properties.

Org Biomol Chem 2020 Sep 2;18(35):6935-6948. Epub 2020 Sep 2.

Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.

Synthesis of the novel thiophenyl carbazole phosphoramidite DNA building block 5 was accomplished in four steps using a Suzuki-Miyaura cross-coupling reaction from the core carbazole and it was seamlessly accommodated into a 9-mer DNA-based oligonucleotide by incorporation at the flanking 5'-end in combination with a central insertion of an LNA-T nucleotide. The carbazole-containing oligonucleotide was combined in different duplex hybrids, which were characterized by thermal denaturation, circular dichroism and fluorescence studies. The carbazole monomer modulates the duplex stability in various ways. Thus, monomer Z increased the thermal stability of the 9-mer towards the complementary 9-mer/15-mer DNA duplex by 4.2 °C. Furthermore, indications of its intercalation into the duplex were obtained by modeling studies and robust decreases in fluorescence emission intensities upon duplex formation. In contrast, no clear intercalating tendency was corroborated for monomer Z within the DNA/RNA hybrid duplex as indicated by moderate quenching of the fluorescence and similar duplex thermal stabilities relative to the corresponding control duplex. The recognition efficiencies of the carbazole modified oligonucleotide toward single nucleotide mismatches were studied with two 15-mer model targets (DNA and RNA). For both systems, mismatches positioned at the juxtaposition of the carbazole monomer showed pronounced deceases in thermal denaturation temperature. Steady-state fluorescence emission studies of all mismatched duplexes with incorporation of Z monomer typically displayed efficient fluorescence quenching.
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http://dx.doi.org/10.1039/d0ob01553aDOI Listing
September 2020

NickFect type of cell-penetrating peptides present enhanced efficiency for microRNA-146a delivery into dendritic cells and during skin inflammation.

Biomaterials 2020 12 29;262:120316. Epub 2020 Aug 29.

Institute of Biomedicine and Translational Medicine, University of Tartu, Estonia. Electronic address:

MicroRNAs (miRNAs) are post-transcriptional gene expression regulators with potential therapeutic applications. miR-146a is a negative regulator of inflammatory processes in both tissue-resident and specialized immune cells and may therefore have therapeutic effect in inflammatory skin diseases. PepFect (PF) and NickFect (NF) type of cell-penetrating peptides (CPPs) have previously been shown to deliver miRNA mimics and/or siRNAs into cell cultures and in vivo. Here, we first demonstrate that selected PF- and NF-type of CPPs support delivery of fluorescent labelled miRNA mimics into keratinocytes (KCs) and dendritic cells (DCs). Second, we show that both PF- and NF-miR-146a nanocomplexes were equally effective in KCs, while NFs were more efficient in DCs as assessed by downregulation of miR-146a-influenced genes. None of miRNA nanocomplexes with the tested CPPs influenced the viability of KCs and DCs nor caused activation of DCs according to CD86 and CD83 markers. Transmission electron microscopy analysis with Nanogold-labelled miR-146a mimics and assessment of endocytic trafficking pathways revealed endocytosis as an active route of delivery in both KCs and DCs for all tested CPPs. However, consistent with the higher efficiency, NF-delivered miR-146a was detected more often outside endosomes in DCs. Finally, pre-injection of NF71:miR-146a nanocomplexes was confirmed to suppress inflammatory responses in a mouse model of irritant contact dermatitis as shown by reduced ear swelling response and downregulation of pro-inflammatory cytokines, including IL-6, IL-1β, IL-33 and TNF-α. In conclusion, NF71 efficiently delivers miRNA mimics into KCs as well as DCs, and therefore may have advantage in therapeutic delivery of miRNAs in case of inflammatory skin diseases.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120316DOI Listing
December 2020

Albumin-Binding Fatty Acid-Modified Gapmer Antisense Oligonucleotides for Modulation of Pharmacokinetics.

Methods Mol Biol 2020 ;2176:163-174

The Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.

Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASOs. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.
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http://dx.doi.org/10.1007/978-1-0716-0771-8_12DOI Listing
March 2021

Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro.

Int J Mol Sci 2020 Mar 31;21(7). Epub 2020 Mar 31.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150 Australia.

Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing exon-23 skipping in mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2'-O-methyl phosphorothioate (2'-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2'-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2'-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.
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http://dx.doi.org/10.3390/ijms21072434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177859PMC
March 2020

Self-Assembly of DNA-Peptide Supermolecules: Coiled-Coil Peptide Structures Templated by d-DNA and l-DNA Triplexes Exhibit Chirality-Independent but Orientation-Dependent Stabilizing Cooperativity.

Chemistry 2020 May 17;26(25):5676-5684. Epub 2020 Apr 17.

Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.

DNA nanostructures have been designed and used in many different applications. However, the use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimics is only starting to emerge. Herein five coiled-coil peptide structures were templated by the hybridization of a d-DNA triplex or its mirror-image counterpart, an l-DNA triplex. The self-assembly of the desired trimeric structures in solution was confirmed by gel electrophoresis and small-angle X-ray scattering, and the stabilizing synergy between the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l-DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-based nanotechnology field by adding two extra dimensions, that is, chirality and polarity, to provide innovative molecular tools for rational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.
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http://dx.doi.org/10.1002/chem.201905636DOI Listing
May 2020

Systemic Evaluation of Chimeric LNA/2'-O-Methyl Steric Blockers for Myotonic Dystrophy Type 1 Therapy.

Nucleic Acid Ther 2020 04 23;30(2):80-93. Epub 2019 Dec 23.

Department of Molecular Genetics, Function & Therapy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited, multisystemic disorder characterized clinically by delayed muscle relaxation and weakness. The disease is caused by a CTG repeat expansion in the 3' untranslated region (3' UTR) of the gene, which leads to the expression of a toxic gain-of-function mRNA. The expanded CUG repeat mRNA sequesters the MBNL1 splicing regulator in nuclear-retained foci structures, resulting in loss of protein function and disruption of alternative splicing homeostasis. In this study, we used CAG repeat antisense oligonucleotides (ASOs), composed of locked nucleic acid (LNA)- and 2'--methyl (2'Me)-modified bases in a chimeric design, to alleviate CUG-mediated toxicity. Chimeric 14-18mer LNA/2'Me oligonucleotides, exhibiting an LNA incorporation of ∼33%, significantly ameliorated the misregulated alternative splicing of Mbnl1-dependent exons in primary DM1 mouse myoblasts and tibialis anterior muscles of DM1 mice. Subcutaneous delivery of 14mer and 18mer LNA/2'Me chimeras in DM1 mice resulted in high levels of accumulation in all tested skeletal muscles, as well as in the diaphragm and heart tissue. Despite the efficient delivery, chimeric LNA/2'Me oligonucleotides were not able, even at a high-dosage regimen (400 mg/kg/week), to correct the misregulated splicing of exon 22 in skeletal muscles. Nevertheless, oligonucleotide doses were well-tolerated as determined by histological and plasma biochemistry analyses. Our results provide proof of concept that inhibition of MBNL1 sequestration by systemic delivery of a steric-blocking ASO is extremely challenging, considering the large number of target sites that need to be occupied per RNA molecule. Although not suitable for DM1 therapy, chimeric LNA/2'Me oligonucleotides could prove to be highly beneficial for other diseases, such as Duchenne muscular dystrophy, that require inhibition of a single target site per RNA molecule.
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http://dx.doi.org/10.1089/nat.2019.0811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7133450PMC
April 2020

In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3' and 5' thiophosphate linkages.

Nucleic Acids Res 2020 01;48(1):63-74

RNA Therapeutics, Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.
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http://dx.doi.org/10.1093/nar/gkz1099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943131PMC
January 2020

Gapmer Antisense Oligonucleotides Containing 2',3'-Dideoxy-2'-fluoro-3'-C-hydroxymethyl-β-d-lyxofuranosyl Nucleotides Display Site-Specific RNase H Cleavage and Induce Gene Silencing.

Chemistry 2020 Jan 20;26(6):1368-1379. Epub 2020 Jan 20.

Biomolecular Nanonscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-β-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNase H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.
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http://dx.doi.org/10.1002/chem.201904540DOI Listing
January 2020

Novel assemblies based on oligonucleotides containing intercalating nucleic acid monomers.

Nucleosides Nucleotides Nucleic Acids 2020 1;39(1-3):82-96. Epub 2019 Nov 1.

Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark.

This is the first report exploring the capability of twisted intercalating nucleic acid (TINA) and naphthalene-functionalized non-nucleosidic linkers to stabilize and engage in double-helical structures. Four designs were studied with respect to the formation of duplexes and/or other types of self-assemblies. One of the constructs involving TINA provides a thermostable duplex. The biophysical properties of the individual constructs were investigated by UV thermal melting experiments, circular dichroism, and fluorescence emission spectroscopy. Molecular modeling studies were performed in attempts of explaining the biophysical measurements for the duplex based on the TINA-containing oligonucleotide strands.
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http://dx.doi.org/10.1080/15257770.2019.1683188DOI Listing
September 2020

Synthetic, Structural, and Anticancer Activity Evaluation Studies on Novel Pyrazolylnucleosides.

Molecules 2019 Oct 30;24(21). Epub 2019 Oct 30.

Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India.

The synthesis of novel pyrazolylnucleosides -, -, -, and - are described. The structures of the regioisomers were elucidated by using extensive NMR studies. The pyrazolylnucleosides - and - were screened for anticancer activities on sixty human tumor cell lines. The compound showed good activity against 39 cancer cell lines. In particular, it showed significant inhibition against the lung cancer cell line Hop-92 (GI 9.3 µM) and breast cancer cell line HS 578T (GI 3.0 µM).
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http://dx.doi.org/10.3390/molecules24213922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864788PMC
October 2019

A self-assembled, modular nucleic acid-based nanoscaffold for multivalent theranostic medicine.

Theranostics 2019 13;9(9):2662-2677. Epub 2019 Apr 13.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.

: Within the field of personalized medicine there is an increasing focus on designing flexible, multifunctional drug delivery systems that combine high efficacy with minimal side effects, by tailoring treatment to the individual. : We synthesized a chemically stabilized ~4 nm nucleic acid nanoscaffold, and characterized its assembly, stability and functional properties and . We tested its flexibility towards multifunctionalization by conjugating various biomolecules to the four modules of the system. The pharmacokinetics, targeting capability and bioimaging properties of the structure were investigated in mice. The role of avidity in targeted liver cell internalization was investigated by flow cytometry, confocal microscopy and by fluorescent scanning of the blood and organs of the animals. : We have developed a nanoscaffold that rapidly and with high efficiency can self-assemble four chemically conjugated functionalities into a stable, -applicable system with complete control of stoichiometry and site specificity. The circulation time of the nanoscaffold could be tuned by functionalization with various numbers of polyethylene glycol polymers or with albumin-binding fatty acids. Highly effective hepatocyte-specific internalization was achieved with increasing valencies of tri-antennary galactosamine (triGalNAc) and . : With its facile functionalization, stoichiometric control, small size and high serum- and thermostability, the nanoscaffold presented here constitutes a novel and flexible platform technology for theranostics.
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http://dx.doi.org/10.7150/thno.32060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525989PMC
May 2020

Molecular determinants for agonist recognition and discrimination in P2X2 receptors.

J Gen Physiol 2019 07 24;151(7):898-911. Epub 2019 May 24.

Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark

P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.
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http://dx.doi.org/10.1085/jgp.201912347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605687PMC
July 2019

microRNA-155 inhibition restores Fibroblast Growth Factor 7 expression in diabetic skin and decreases wound inflammation.

Sci Rep 2019 04 9;9(1):5836. Epub 2019 Apr 9.

Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Treatment for chronic diabetic foot ulcers is limited by the inability to simultaneously address the excessive inflammation and impaired re-epithelization and remodeling. Impaired re-epithelization leads to significantly delayed wound closure and excessive inflammation causes tissue destruction, both enhancing wound pathogen colonization. Among many differentially expressed microRNAs, miR-155 is significantly upregulated and fibroblast growth factor 7 (FGF7) mRNA (target of miR-155) and protein are suppressed in diabetic skin, when compared to controls, leading us to hypothesize that topical miR-155 inhibition would improve diabetic wound healing by restoring FGF7 expression. In vitro inhibition of miR-155 increased human keratinocyte scratch closure and topical inhibition of miR-155 in vivo in wounds increased murine FGF7 protein expression and significantly enhanced diabetic wound healing. Moreover, we show that miR-155 inhibition leads to a reduction in wound inflammation, in accordance with known pro-inflammatory actions of miR-155. Our results demonstrate, for the first time, that topical miR-155 inhibition increases diabetic wound fibroblast growth factor 7 expression in diabetic wounds, which, in turn, increases re-epithelization and, consequently, accelerates wound closure. Topical miR-155 inhibition targets both excessive inflammation and impaired re-epithelization and remodeling, being a potentially new and effective treatment for chronic diabetic foot ulcers.
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http://dx.doi.org/10.1038/s41598-019-42309-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456606PMC
April 2019

Oligonucleotide Binding to Non-B-DNA in .

Molecules 2019 Mar 12;24(5). Epub 2019 Mar 12.

Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Huddinge, Sweden.

, originally named c-, is an oncogene deregulated in many different forms of cancer. Translocation of the gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt's lymphoma (BL). Sporadic BL constitutes one subgroup where one of the translocation sites is located at the 5'-vicinity of the two major promoters P₁ and P₂. A non-B-DNA forming sequence within this region has been reported with the ability to form an intramolecular triplex (H-DNA) or a G-quadruplex. We have examined triplex formation at this site first by using a 17 bp triplex-forming oligonucleotide (TFO) and a double strand DNA (dsDNA) target corresponding to the sequence. An antiparallel purine-motif triplex was detected using electrophoretic mobility shift assay. Furthermore, we probed for H-DNA formation using the BQQ-OP based triplex-specific cleavage assay, which indicated the formation of the structure in the supercoiled plasmid containing the corresponding region of the promoter. Targeting non-B-DNA structures has therapeutic potential; therefore, we investigated their influence on strand-invasion of anti-gene oligonucleotides (ON)s. We show that in vitro, non-B-DNA formation at the vicinity of the ON target site facilitates dsDNA strand-invasion of the anti-gene ONs.
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http://dx.doi.org/10.3390/molecules24051000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429085PMC
March 2019

The ability of locked nucleic acid oligonucleotides to pre-structure the double helix: A molecular simulation and binding study.

PLoS One 2019 12;14(2):e0211651. Epub 2019 Feb 12.

Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.

Locked nucleic acid (LNA) oligonucleotides bind DNA target sequences forming Watson-Crick and Hoogsteen base pairs, and are therefore of interest for medical applications. To be biologically active, such an oligonucleotide has to efficiently bind the target sequence. Here we used molecular dynamics simulations and electrophoresis mobility shift assays to elucidate the relation between helical structure and affinity for LNA-containing oligonucleotides. In particular, we have studied how LNA substitutions in the polypyrimidine strand of a duplex (thus forming a hetero duplex, i.e. a duplex with a DNA polypurine strand and an LNA/DNA polypyrimidine strand) enhance triplex formation. Based on seven polypyrimidine single strand oligonucleotides, having LNAs in different positions and quantities, we show that alternating LNA with one or more non-modified DNA nucleotides pre-organizes the hetero duplex toward a triple-helical-like conformation. This in turn promotes triplex formation, while consecutive LNAs distort the duplex structure disfavoring triplex formation. The results support the hypothesis that a pre-organization in the hetero duplex structure enhances the binding of triplex forming oligonucleotides. Our findings may serve as a criterion in the design of new tools for efficient oligonucleotide hybridization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372149PMC
November 2019

Improved RE31 Analogues Containing Modified Nucleic Acid Monomers: Thermodynamic, Structural, and Biological Effects.

J Med Chem 2019 03 22;62(5):2499-2507. Epub 2019 Feb 22.

Department of Nucleic Acids Bioengineering, Institute of Bioorganic Chemistry , Polish Academy of Sciences , Noskowskiego 12/14 , 61-704 Poznan , Poland.

RE31 is a 31-nt DNA aptamer, consisting of the G-quadruplex and a duplex domain, which is able to effectively prolong thrombin time. This article reports on the influence of certain modified nucleotide residues on thermodynamic and biological properties as well as the folding topology of RE31. Particularly, the effect of the presence of nucleosides in unlocked nucleic acid (UNA), locked nucleic acid (LNA), or β-l-RNA series was evaluated. The studies presented herein show that all modified residues can influence thermal and biological stabilities of G-quadruplex in a position-dependent manner. The aptamers modified simultaneously with UNA at the T position and LNAs in the duplex part possess the highest value of melting temperature and a 2-fold higher anticoagulant effect. Importantly, RE31 variants modified with nucleosides in UNA, LNA, or β-l-RNA series exhibit unchanged G-quadruplex folding topology. Crucially, introduction of any of the modified residues into RE31 causes prolongation of aptamer stability in human serum.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01806DOI Listing
March 2019

Fibrin-hyaluronic acid hydrogel-based delivery of antisense oligonucleotides for ADAMTS5 inhibition in co-delivered and resident joint cells in osteoarthritis.

J Control Release 2019 01 18;294:247-258. Epub 2018 Dec 18.

Department of Orthopedics, University Medical Center Utrecht, the Netherlands. Electronic address:

To date no disease-modifying drugs for osteoarthritis (OA) are available, with treatment limited to the use of pain killers and prosthetic replacement. The ADAMTS (A Disintegrin and Metallo Proteinase with Thrombospondin Motifs) enzyme family is thought to be instrumental in the loss of proteoglycans during cartilage degeneration in OA, and their inhibition was shown to reverse osteoarthritic cartilage degeneration. Locked Nucleic Acid (LNA)-modified antisense oligonucleotides (gapmers) released from biomaterial scaffolds for specific and prolonged ADAMTS inhibition in co-delivered and resident chondrocytes, is an attractive therapeutic strategy. Here, a gapmer sequence identified from a gapmer screen showed 90% ADAMTS5 silencing in a monolayer culture of human OA chondrocytes. Incorporation of the gapmer in a fibrin-hyaluronic acid hydrogel exhibited a sustained release profile up to 14 days. Gapmers loaded in hydrogels were able to transfect both co-embedded chondrocytes and chondrocytes in a neighboring gapmer-free hydrogel, as demonstrated by flow cytometry and confocal microscopy. Efficient knockdown of ADAMTS5 was shown up to 14 days in both cell populations, i.e. the gapmer-loaded and gapmer-free hydrogel. This work demonstrates the use applicability of a hydrogel as a platform for combined local delivery of chondrocytes and an ADAMTS-targeting gapmer for catabolic gene modulation in OA.
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http://dx.doi.org/10.1016/j.jconrel.2018.12.030DOI Listing
January 2019

MicroRNA therapeutics: design of single-stranded miR-216b mimics to target KRAS in pancreatic cancer cells.

RNA Biol 2018 11;15(10):1273-1285. Epub 2018 Oct 11.

a Department of Medicine, Laboratory of Biochemistry , University of Udine , Italy.

Datasets reporting microRNA expression profiles in normal and cancer cells show that miR-216b is aberrantly downregulated in pancreatic ductal adenocarcinoma (PDAC). We found that KRAS, whose mutant G12D allele drives the pathogenesis of PDAC, is a target of miR-216b. To suppress oncogenic KRAS in PDAC cells, we designed single-stranded (ss) miR-216b mimics with unlocked nucleic acid (UNA) modifications to enhance their nuclease resistance. We prepared variants of ss-miR-216b mimics with and without a 5' phosphate group. Both variants strongly suppressed oncogenic KRAS in PDAC cells and inhibited colony formation in pancreatic cancer cells. We observed that the designed ss-miR-216b mimics engaged AGO2 to promote the silencing of KRAS. We also tested a new delivery strategy based on the use of palmityl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with ss-miR-216b conjugated with two palmityl chains and a lipid-modified cell penetrating peptide (TAT). These versatile nanoparticles suppressed oncogenic KRAS in PDAC cells.
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http://dx.doi.org/10.1080/15476286.2018.1526536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284578PMC
January 2019

Modification of oligodeoxynucleotides by on-column Suzuki cross-coupling reactions.

Chem Commun (Camb) 2018 Jul;54(57):8003-8006

Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, DK-5230 Odense M, Denmark.

The on-column functionalization of oligodeoxynucleotides via base-free Suzuki cross-coupling reactions is reported herein. These cross-coupling reactions were carried out with various boronic acids and either full-length modified oligonucleotides containing one or more 2'-deoxy-5-iodouridine (5IdU) monomer(s) or on oligonucleotide fragments immediately after incorporation of 5IdU. Five different functionalities were coupled to oligonucleotides containing one or three attachment points.
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http://dx.doi.org/10.1039/c8cc01360hDOI Listing
July 2018

Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides.

BMC Mol Biol 2018 06 7;19(1). Epub 2018 Jun 7.

Department of Chemical Engineering, LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering of the University of Porto, Rua Dr. Roberto Frias, 4200-465, Porto, Portugal.

Background: Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to influence the expression of E-cadherin-encoding gene, triggering cell motility and invasiveness.

Results: In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2 h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using different LNA anti-miRNA oligonucleotide designs, and to observe its subsequent effect on E-cadherin expression.

Conclusions: The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and influenced the expression of E-cadherin by significantly increasing its levels.
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http://dx.doi.org/10.1186/s12867-018-0107-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992815PMC
June 2018

Hydrogel-Assisted Antisense LNA Gapmer Delivery for In Situ Gene Silencing in Spinal Cord Injury.

Mol Ther Nucleic Acids 2018 Jun 20;11:393-406. Epub 2018 Mar 20.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal; INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 4200-135 Porto, Portugal; Faculdade de Engenharia da Universidade do Porto, 4200-465 Porto, Portugal; Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, 4050-313 Porto, Portugal. Electronic address:

After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favorable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration-RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture system and in vivo at a SCI lesion site, achieving around 75% downregulation 5 days after hydrogel injection. Our results show that local implantation of a AON-gapmer-loaded hydrogel matrix mediated efficient gene silencing in the lesioned spinal cord and is an innovative platform that can potentially combine gene regulation with regenerative permissive substrates aiming at SCI therapeutics and nerve regeneration.
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http://dx.doi.org/10.1016/j.omtn.2018.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992461PMC
June 2018
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