Altered Biodistribution and Hepatic Safety Profile of a Gapmer Antisense Oligonucleotide Bearing Guanidine-Bridged Nucleic Acids

Sasaki, Takashi; Hirakawa, Yoko; Yamairi, Fumiko; Kurita, Takashi; Murahashi, Karin; Nishimura, Haruki; Iwazaki, Norihiko; Yasuhara, Hidenori; Tateoka, Takashi; Ohta, Tetsuya; Obika, Satoshi; Kotera, Jun

doi:10.1089/nat.2021.0034

Cited by 7 publications

(6 citation statements)

References 27 publications

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“…A major challenge in developing ASO therapeutics, including LNA gapmers, is to find an efficient way to deliver agents to the target brain crossing the blood-brain barrier (BBB). Although LNA gapmers can be distributed to various tissues, an issue is that they have a limited ability to reach the brain [56]. An intrathecal injection may be an option to overcome this biological barrier, as an ASO drug called nusinersen, for the neuromuscular disease spinal muscular atrophy, is in clinical application via this local administration [57].…”

Section: Discussionmentioning

confidence: 99%

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus

Okamoto,

Echigoya,

Tago

et al. 2023

IJMS

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RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3′ untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

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Section: Discussionmentioning

confidence: 99%

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus

Okamoto,

Echigoya,

Tago

et al. 2023

IJMS

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“…Although the MENβ triple helix is less studied than its counterpart in MALAT1, compounds like aurintricarboxylic acid, emodin, GW5074, mitoxantrone and rottlerin bind to the MENβ triple helix near the micromolar range, and the kinase inhibitor PIK-75 abolishes paraspeckles in the neuroblastoma cell line SH-SY5Y [30]. ASO therapeutics have been used to target and regulate the MALAT1 lncRNA in various cancer types [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. A variety of ASOs, typically 16-20 nucleotides in length, have been designed, such as small interfering RNA (siRNA) [35][36][37]; gapmers with two to three locked nucleic acids (LNAs) [31,33,34,[38][39][40]; 2 ′ -O-methylethyl groups; 2 ′ -O,4 ′ -C-ethylene-bridged nucleic acid [41] or guanidine-bridged nucleic acid [42] at the end(s); PNA-DNA chimeras [43]; and the conjugation of ASO to single-wall carbon nanotubes [44], gold nanoparticles [32], TAT peptides [32], fatty acids [45] or membrane protein-binding aptamers [46] to improve delivery and biodistribution.…”

Section: Of 19mentioning

confidence: 99%

“…ASO therapeutics have been used to target and regulate the MALAT1 lncRNA in various cancer types [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. A variety of ASOs, typically 16-20 nucleotides in length, have been designed, such as small interfering RNA (siRNA) [35][36][37]; gapmers with two to three locked nucleic acids (LNAs) [31,33,34,[38][39][40]; 2 ′ -O-methylethyl groups; 2 ′ -O,4 ′ -C-ethylene-bridged nucleic acid [41] or guanidine-bridged nucleic acid [42] at the end(s); PNA-DNA chimeras [43]; and the conjugation of ASO to single-wall carbon nanotubes [44], gold nanoparticles [32], TAT peptides [32], fatty acids [45] or membrane protein-binding aptamers [46] to improve delivery and biodistribution. Most ASO gapmers target unstructured regions of MALAT1, leading to RNase H-mediated knockdown from 2-50-fold [31,34,39,40].…”

Section: Of 19mentioning

confidence: 99%

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Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels

Shivakumar,

Mahendran,

Brown

2024

IJMS

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Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) are two long noncoding RNAs upregulated in multiple cancers, marking these RNAs as therapeutic targets. While traditional small-molecule and antisense-based approaches are effective, we report a locked nucleic acid (LNA)-based approach that targets the MALAT1 and MENβ triple helices, structures comprised of a U-rich internal stem-loop and an A-rich tract. Two LNA oligonucleotides resembling the A-rich tract (i.e., A9GCA4) were examined: an LNA (L15) and a phosphorothioate LNA (PS-L15). L15 binds tighter than PS-L15 to the MALAT1 and MENβ stem loops, although both L15 and PS-L15 enable RNA•LNA-RNA triple-helix formation. Based on UV thermal denaturation assays, both LNAs selectively stabilize the Hoogsteen interface by 5–13 °C more than the Watson–Crick interface. Furthermore, we show that L15 and PS-L15 displace the A-rich tract from the MALAT1 and MENβ stem loop and methyltransferase-like protein 16 (METTL16) from the METTL16-MALAT1 triple-helix complex. Human colorectal carcinoma (HCT116) cells transfected with LNAs have 2-fold less MALAT1 and MENβ. This LNA-based approach represents a potential therapeutic strategy for the dual targeting of MALAT1 and MENβ.

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“…with the 2′,4′-BNA/LNA-modified ones. We recently demonstrated that GuNA[H]-modified ASOs are well tolerated and have altered organ-specificity, as well as a preference for skeletal muscle, compared with their 2′,4′-BNA/LNA-modified counterparts ( 33 ). Moreover, GuNA[H]-modified anti-miRNA oligonucleotides were shown to have high activity, possibly due to the additional interactions between GuNA[H] and miRISC ( 34 ).…”

Section: Introductionmentioning

confidence: 99%

Mechanism of the extremely high duplex-forming ability of oligonucleotides modified with N-tert-butylguanidine- or N-tert-butyl-N′- methylguanidine-bridged nucleic acids

Yamaguchi

Horie

Aoyama

et al. 2023

Nucleic Acids Research

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Antisense oligonucleotides (ASOs) are becoming a promising class of drugs for treating various diseases. Over the past few decades, many modified nucleic acids have been developed for application to ASOs, aiming to enhance their duplex-forming ability toward cognate mRNA and improve their stability against enzymatic degradations. Modulating the sugar conformation of nucleic acids by substituting an electron-withdrawing group at the 2′-position or incorporating a 2′,4′-bridging structure is a common approach for enhancing duplex-forming ability. Here, we report on incorporating an N-tert-butylguanidinium group at the 2′,4′-bridging structure, which greatly enhances duplex-forming ability because of its interactions with the minor groove. Our results indicated that hydrophobic substituents fitting the grooves of duplexes also have great potential to increase duplex-forming ability.

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Altered Biodistribution and Hepatic Safety Profile of a Gapmer Antisense Oligonucleotide Bearing Guanidine-Bridged Nucleic Acids

Cited by 7 publications

References 27 publications

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus

Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels

Mechanism of the extremely high duplex-forming ability of oligonucleotides modified with N-tert-butylguanidine- or N-tert-butyl-N′- methylguanidine-bridged nucleic acids

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