Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

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.

Reduction of Off-Target Effects of Gapmer Antisense Oligonucleotides by Oligonucleotide Extension.

AIM: Antisense oligonucleotide (ASO) has the potential to induce off-target effects by inadvertent binding of ASOs to unintended RNAs that have a sequence similar to the target RNA. In the present study, we focused on the association between oligonucleotide length and off-target effects. Oligonucleotide extension is assumed to have bilateral effects on hybridization-dependent changes in gene expression, i.e., one is the decrease of off-target effects based on the reduced number of off-target candidate genes with perfect matches, and the other is the increase of off-target effects based on the increased binding affinity between the ASO and the complementary RNAs that leads to better tolerability for mismatches. METHODS: To determine the effects of oligonucleotide extension of gapmer ASOs on off-target effects, an extensive microarray analysis was performed using human cells treated with a 14-mer gapmer ASO and the extended 18-mer derivatives with the same core 14-mer region. RESULTS AND DISCUSSION: Our data indicated that change in gene expression in the cells treated with 18-mer ASOs was significantly smaller than those with a 14-mer ASO, showing the decrease of off-target effects by oligonucleotide extension.

Evaluation of off-target effects of gapmer antisense oligonucleotides using human cells.

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.

Estimated number of off-target candidate sites for antisense oligonucleotides in human mRNA sequences.

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.

[A Case of Rectal Cancer with Portal Vein Tumor Thrombosis Successfully Treated with First-Line Panitumumab Monotherapy].

The patient was a 66-year-old man with progressive rectal cancer and portal vein tumor thrombosis near the porta hepatis. A subileus further complicated the situation. Therefore, an abdominoperineal resection was performed. As the KRAS gene was the wild-type gene, we administered panitumumab monotherapy every 2 weeks after the operation. Two months after the operation, the tumor marker levels were normal and the tumor size was reduced. Hence, oral capecitabine, with 1-week administration followed by 1-week of rest, was added to the therapy. In the 12 months after the operation, panitumumab was stopped once, and treatment with capecitabine alone was continued in the same schedule. As of 26 months, the disease showed a partial response using capecitabine alone. We report our experience of a case of rectal cancer with portal vein tumor thrombosis that was successfully treated with panitumumab.

Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH.

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.

Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice.

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'-BNA(NC) 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'-BNA(NC)-AON showed an earlier LDL-C-lowering effect and was more tolerable in mice. Our results validate the optimization of 2',4'-BNA(NC)-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH.

Due to the 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 gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.

Interrupted 2'-o,4'-C-aminomethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition.

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(NC)) 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(NC) 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(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-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(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) 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.

Promotion of triplex formation by 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification.

We examined the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid (3'-amino-2',4'-BNA) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 3'-amino-2',4'-BNA modified TFO was significantly higher than that observed with unmodified TFO. The 3'-amino-2',4'-BNA modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 3'-amino-2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Thermodynamic and kinetic effects of morpholino modification on pyrimidine motif triplex nucleic acid formation under physiological condition.

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.

Promotion of triplex formation by 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNA NC) modification.

We examined the effect of 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNA(NC)) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 2',4'-BNA(NC) modified TFO was significantly higher than that observed with unmodified TFO. The 2',4'-BNA(NC) modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 2',4'-BNA(NC) backbone modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

DX DNAs as templates for multiple arrangement of zinc fingers.

The present paper reveals that double crossover-DNAs (DX) serve as scaffolds for the multiple arrangement of a [Ru(bpy)(3)](2+)-bound zinc finger (ZF) protein, Ru-ZF. This series of results would lead to the realization of the two-dimensional arrangement of functional molecules and nanomaterials on DX-tiles.

Food antigen causes TH2-dependent enteropathy followed by tissue repair in T-cell receptor transgenic mice.

BACKGROUND: Clarification of the mechanisms underlying the development of food-sensitive intestinal inflammation will provide an important clue to combating food allergies. OBJECTIVE: To establish a model of intestinal inflammation caused by oral administration of antigen without additional treatments, we focused on the ovalbumin (OVA) 23-3 T-cell receptor transgenic mouse, which had been reported to have high serum antigen-specific IgE responses to the feeding of an egg white diet. METHODS: Changes in body weight of mice fed an egg white diet were monitored throughout the 28-day experimental period. After the 28-day feeding, intestinal tissues were harvested for histologic examination. Endogenous production of cytokines and histamine in the jejunum, and production of cytokines secreted by OVA-specific CD4+ T cells purified from mesenteric lymph nodes, were analyzed. RESULTS: Egg white diet-fed OVA23-3 mice developed weight loss and inflammation with villous atrophy and goblet cell hyperplasia, especially in the jejunum. A further characteristic feature was evidence of weight recovery and tissue repair. Jejunal inflammation was also observed in egg white diet-fed recombination activating gene (RAG)-2-deficient OVA23-3 mice. In addition, tissue sections revealed significant infiltration of specific IgE-positive cells and IgE-positive degranulating mast cells. Higher levels of IL-4 and significant levels of histamine were detected in the tissues. In the supernatant of OVA-stimulated T cells, IL-10 levels were also markedly elevated. CONCLUSION: We report that high-dose and continuous intake of primitive OVA alone induces enteropathy containing regions under repair in OVA23-3 mice. Antigen-specific T cells and inflammatory cells primed by T(H)2 responses play important roles in regulation of development and improvement of the disease. CLINICAL IMPLICATIONS: Long-term antigen intake causes T(H)2-dependent and food-sensitive enteropathy followed by tissue repair.

A unique mechanism for cyclic adenosine 3',5'-monophosphate-induced increase of 32-kDa tyrosine-phosphorylated protein in boar spermatozoa.

A cAMP-induced increase of tyrosine-phosphorylated proteins is involved in the expression of fertilizing ability in mammalian spermatozoa. We (Harayama, 2003: J Androl 24:831-842) reported that incubation of boar spermatozoa with a cell-permeable cAMP analog (cBiMPS) increased a 32-kDa tyrosine-phosphorylated protein (TyrP32). The purpose of this study is to characterize the signaling cascades that regulate the cAMP-induced increase of TyrP32. We examined effects of tyrosine kinase inhibitor (lavendustin A), tyrosine phosphatase inhibitor (Na3VO4), cell-permeable calcium chelator (BAPTA-AM), and cholesterol acceptor (methyl-beta-cyclodextrin: MBC) on the increase of TyrP32 and the change and loss of acrosomes in boar spermatozoa. The spermatozoa were used for detection of tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence and for examination of acrosomal integrity by Giemsa staining. At least eight tyrosine-phosphorylated proteins including TyrP32 exhibited the cAMP-dependent increase during incubation with cBiMPS. In many proteins of them, this increase was reduced by lavendustin A but was enhanced by Na3VO4. In contrast, the cAMP-induced increase of TyrP32 was abolished by Na3VO4 but was hardly affected by lavendustin A. Giemsa staining showed that the increase of spermatozoa with weakly Giemsa-stained acrosomes (severely damaged acrosomes) or without acrosomes was correlative to the cAMP-induced increase of TyrP32. Moreover, the lack of calcium chloride in the incubation medium or pretreatment of spermatozoa with BAPTA-AM blocked the change and loss of acrosomes and the increase of TyrP32, suggesting these events are dependent on the extracellular and intracellular calcium. On the other hand, incubation of spermatozoa with MBC in the absence of cBiMPS could mimic the change and loss of acrosomes and increase of TyrP32 without increase of other tyrosine-phosphorylated proteins. Based on these results, we conclude that the cAMP-induced increase of TyrP32 is regulated by a unique mechanism that may be linked to the calcium-dependent change and loss of acrosomes.