Antisense oligonucleotide (ASO) has the potential to induce off‐target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional ...animal studies cannot be used to assess toxicity induced by off‐target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off‐target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed.
Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off‐target effects at the cellular level is not clear. In this study, we evaluated off‐target effects of gapmer ASOs, which cleave the target RNA in an RNase H‐dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off‐target effects depending on the degree of complementarity between the ASO and off‐target candidate genes. Based on our results, we also propose a scheme for the assessment of off‐target effects of gapmer ASOs.
We showed that gapmer oligonucleotide (ASO) induce off‐target effects depending on the degree of complementarity between the ASO and off‐target candidate genes. Based on our results, we also proposed a scheme for the assessment of off‐target effects of gapmer ASOs.
Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various ...human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.
Antisense oligonucleotide (ASO) therapeutics are single‐stranded oligonucleotides which bind to RNA through sequence‐specific Watson–Crick base pairings. A unique mechanism of toxicity for ASOs is ...hybridization‐dependent off‐target effects that can potentially occur due to the binding of ASOs to complementary regions of unintended RNAs. To reduce the off‐target effects of ASOs, it would be useful to know the approximate number of complementary regions of ASOs, or off‐target candidate sites of ASOs, of a given oligonucleotide length and complementarity with their target RNAs. However, the theoretical number of complementary regions with mismatches has not been reported to date. In this study, we estimated the general number of complementary regions of ASOs with mismatches in human mRNA sequences by mathematical calculation and in silico analysis using several thousand hypothetical ASOs. By comparing the theoretical number of complementary regions estimated by mathematical calculation to the actual number obtained by in silico analysis, we found that the number of complementary regions of ASOs could be broadly estimated by the theoretical number calculated mathematically. Our analysis showed that the number of complementary regions increases dramatically as the number of tolerated mismatches increases, highlighting the need for expression analysis of such genes to assess the safety of ASOs.
In this study, we estimated the general number of complementary regions of ASOs with mismatches in human mRNA sequences by mathematical calculation and in silico analysis using several thousand hypothetical ASOs. By comparing the theoretical number of complementary regions estimated by mathematical calculation to the actual number obtained by in silico analysis, we found that the number of complementary regions of ASOs could be broadly estimated by the theoretical number calculated mathematically. Our analysis showed that the number of complementary regions increases dramatically as the number of tolerated mismatches increases, highlighting the need for expression analysis of such genes to assess the safety of ASOs.
Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of ...genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3′-amino-2′-O,4′-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2′-O and 4′-C of the sugar moiety were bridged with the methylene chain and 3′-O was replaced by 3′-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.
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► 3′-amino-2′,4′-BNA modified TFO significantly stabilized triplex at physiological pH. ► 3′-amino-2′,4′-BNA modified TFO promoted triplex formation at physiological pH. ► 3′-amino-2′,4′-BNA modification increased nuclease resistance of TFO in human serum. ► Number and position of TFO modification did not affect degree of triplex promotion. ► Design of TFO with increased rigidity and hydration promoted triplex formation.
Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The ...cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2′,4′-BNA (also called as locked nucleic acid (LNA)) and 2′,4′-BNANC chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2′,4′-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2′,4′-BNA-AON, 2′,4′-BNANC-AON showed an earlier LDL-C–lowering effect and was more tolerable in mice. Our results validate the optimization of 2′,4′-BNANC-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.
Due to instability of pyrimidine motif triplex nucleic acid under physiological pH and low magnesium ion concentration, stabilization of the triplex under the physiological condition is crucial in ...improving its therapeutic potential to artificially control gene expression in vivo. To this end, we investigated the thermodynamic and kinetic effects of morpholino (MOR) modification of triplex-forming oligonucleotide (TFO) on the triplex formation under the physiological condition. The thermodynamic analyses indicated that the MOR modification of TFO not only significantly increased the thermal stability of the triplex but also increased the binding constant for the triplex formation by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the MOR-modified TFO in the free state relative to the corresponding unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. This information will be valuable for designing novel chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, leading to progress in therapeutic applications of the antigene strategy in vivo.
Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies ...in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′- BNA
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) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2′,4′-BNA
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modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2′,4′-BNA
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modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2′,4′-BNA
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modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2′,4′-BNA
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-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2′,4′-BNA
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-modified positions in TFO. We conclude that the interrupted 2′,4′-BNA
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modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.
Oligonucleotide therapeutics are single- or double-stranded DNA or RNA-based molecules consisting of short strand of nucleotides (generally 10-50 nucleotides). They are manufactured by chemical ...synthesis and act directly on RNA or protein. In recent years, oligonucleotide therapeutics, such as antisense oligonucleotides (ASOs), siRNAs, miRNAs, aptamers or CpG-motif oligodeoxynucleotides (CpG ODN), are active areas of drug development designed to treat a variety of genetic and/or intractable diseases. So far, eight oligonucleotide therapeutics have achieved marketing authorization in USA or Europe, i.e. fomivirsen (Vitravene®), pegaptanib (Macugen®), mipomersen (Kynamro®), eteplirsen (Exondys 51®), nusinersen (Spinraza®), inotersen (Tegsedi®), patisiran (Onpattro®), and hepatitis B vaccine containing CpG ODN (CpG1018) as adjuvants (HEPLISAV-B®), and more than 150 oligonucleotide therapeutics are currently in clinical development. In this review, I outline classification, modifications and mechanism of actions of oligonucleotide therapeutics, and introduce the current status of development of RNA-targeted oligonucleotide therapeutics (ASOs, siRNAs and miRNAs).
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