Favorable efficacy and reduced acute neurotoxicity by antisense oligonucleotides with 2',4'-BNA/LNA with 9-(aminoethoxy)phenoxazine.

An increasing number of antisense oligonucleotides (ASOs) have been approved for clinical use. However, improvements of both efficacy and safety in the central nervous system (CNS) are crucial for the treatment with CNS diseases. We aimed to overcome the crucial issues by our development of various gapmer ASOs with a novel nucleoside derivative including a 2',4'-BNA/LNA with 9-(aminoethoxy)phenoxazine (BNAP-AEO). The various gapmer ASOs with BNAP-AEO were evaluated for thermal stability, in vitro and in vivo efficacy, and acute CNS toxicity. Thermal stability analysis of the duplexes with their complementary RNAs showed that ASOs with BNAP-AEO had a higher binding affinity than those without BNAP-AEO. In vitro assays, when transfected into neuroblastoma cell lines, demonstrated that ASOs with BNAP-AEO, had a more efficient gene silencing effect than those without BNAP-AEO. In vivo assays, involving intracerebroventricular injections into mice, revealed ASOs with BNAP-AEO potently suppressed gene expression in the brain. Surprisingly, the acute CNS toxicity in mice, as assessed through open field tests and scoring systems, was significantly lower for ASOs with BNAP-AEO than for those without BNAP-AEO. This study underscores the efficient gene-silencing effect and low acute CNS toxicity of ASOs incorporating BNAP-AEO, indicating the potential for future therapeutic applications.

Separation and Characterization of Therapeutic Oligonucleotide Isomer Impurities by Cyclic Ion Mobility Mass Spectrometry.

Therapeutic oligonucleotides such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA) are among the most remarkable modalities in modern medicine. ASOs and siRNA are composed of single- or double-stranded 15-25 mer synthesized oligonucleotides, which can be used to modulate gene expression. Liquid chromatography-mass spectrometry (LC/MS) is a necessary technique for the quality control of therapeutic oligonucleotides; it is used to evaluate the quantities of target oligonucleotides and their impurities. The widely applied oligonucleotide therapeutic quantitation method uses both ultraviolet (UV) absorbance and the MS signal intensity. Peaks separated from the main peak, which contains full-length product, are generally quantitated by UV. However, coeluting impurities, such as n - 1 shortmers, abasic oligonucleotides, and PS → PO (phosphorothiate to phosphodiester) oligonucleotides, are quantitated by MS. These coeluting impurities can also be comprised of various isomers with the same modification, thus increasing the difficulty in their separation and relative quantitation by LC/MS. It is possible that a specific isomer with a certain structural form induces toxicities. Therefore, characterization of each isomer separation is in high demand. In this study, we separated and characterized oligonucleotide isomers by employing a cyclic ion mobility mass spectrometry (cyclic IMS) system, which allows the separation of ions with the same m/z ratio based on their structural differences. Patisiran antisense and sense strands and their n - 1 and abasic isomers were used as sample sequences, and their ratio characterization was achieved by cyclic IMS. In addition, we evaluated the PS → PO conversion isomers of the antisense strand of givosiran, which originally contained four PS modification sites. The PS → PO isomers exhibited specific and distinguishable mobiligram patterns. We believe that cyclic IMS is a promising method for evaluating therapeutic oligonucleotide isomers.

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.

Erratum: Favorable efficacy and reduced acute neurotoxicity by antisense oligonucleotides with 2',4' -BNA/LNA with 9-(aminoethoxy)phenoxazine.

[This corrects the article DOI: 10.1016/j.omtn.2024.102161.].

Separation of the diastereomers of phosphorothioated siRNAs by anion-exchange chromatography under non-denaturing conditions.

In recent years, several small interfering RNA (siRNA) therapeutics have been approved, and most of them are phosphorothioate (PS)-modified for improving nuclease resistance. This chemical modification induces chirality in the phosphorus atom, leading to the formation of diastereomers. Recent studies have revealed that Sp and Rp configurations of PS modifications of siRNAs have different biological properties, such as nuclease resistance and RNA-induced silencing complex (RISC) loading. These results highlight the importance of determining diastereomeric distribution in quality control. Although various analytical approaches have been used to separate diastereomers (mainly single-stranded oligonucleotides), it becomes more difficult to separate all of them as the number of PS modifications increases. Despite siRNA exhibits efficacy in the double-stranded form, few reports have examined the separation of diastereomers in the double-stranded form. In this study, we investigated the applicability of non-denaturing anion-exchange chromatography (AEX) for the separation of PS-modified siRNA diastereomers. Separation of the four isomers of the two PS bonds tended to improve in the double-stranded form compared to the single-stranded form. In addition, the effects of the analytical conditions and PS-modified position on the separation were evaluated. Moreover, the elution order of the Sp and Rp configurations was confirmed, and the steric difference between them, i.e., the direction of the anionic sulfur atom, appeared to be important for the separation mechanism in non-denaturing AEX. Consequently, all 16 peak tops of the four PS modifications were detected in one sequence, and approximately 30 peak tops were detected out of 64 isomers of six PS bonds, indicating that non-denaturing AEX is a useful technique for the quality control of PS-modified siRNA therapeutics.

Applicability of supercritical fluid chromatography for oligonucleotide analysis: A proof-of-concept study.

We evaluated the suitability of supercritical fluid chromatography (SFC) for oligonucleotide analysis using 4-mer oligonucleotides with various phosphorothioate (PS) contents as model compounds. Column screening showed that the diol-modified column was able to separate sequences with different PS contents. Optimization of the column body and additives allowed us to analyze polar oligonucleotides using SFC. Various sequences were also analyzed using the optimized method. A good peak shape was obtained when the guanine plus cytosine content of the analyte was two or less in the 4-mer oligonucleotides. Furthermore, we found that the retention times of the selected sequences were positively correlated with polar surface areas, indicating that oligonucleotides interact with polar stationary phases. In contrast, more hydrophobic full PS sequences were retained more strongly in the diol column than the full phosphodiester (PO) sequences. This suggests that the diol column has unique selectivity for PO and PS linkages. These results indicate that SFC is potentially applicable to oligonucleotide analysis with a separation mechanism that is different from that of ion-pair reversed-phase liquid chromatography.

Development of 8-17 XNAzymes that are functional in cells.

DNA enzymes (DNAzymes), which cleave target RNA with high specificity, have been widely investigated as potential oligonucleotide-based therapeutics. Recently, xeno-nucleic acid (XNA)-modified DNAzymes (XNAzymes), exhibiting cleavage activity in cultured cells, have been developed. However, a versatile approach to modify XNAzymes that function in cells has not yet been established. Here, we report an X-ray crystal structure-based approach to modify 8-17 DNAzymes; this approach enables us to effectively locate suitable XNAs to modify. Our approach, combined with a modification strategy used in designing antisense oligonucleotides, rationally designed 8-17 XNAzyme ("X8-17") that achieved high potency in terms of RNA cleavage and biostability against nucleases. X8-17, modified with 2'-O-methyl RNA, locked nucleic acid and phosphorothioate, successfully induced endogenous MALAT-1 and SRB1 RNA knockdown in cells. This approach may help in developing XNAzyme-based novel therapeutic agents.

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

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.

Synthesis of multivalent fatty acid-conjugated antisense oligonucleotides: Cell internalization, physical properties, and in vitro and in vivo activities.

Herein, we describe the design and synthesis of multi-conjugatable fatty acid monomer phosphoramidites and their conjugation to antisense oligonucleotides (ASOs). Multivalent long-chain fatty acid conjugation improved the cellular uptake of ASOs but decreased in vitro activity due to alterations in physical properties and cellular localization. In addition, multivalently fatty acid-conjugated ASOs showed different organ specificity compared with that of unconjugated ASO in in vivo experiment. Although optimization of the linker structure between the fatty acid moiety and the ASO may be required, divalent long-chain fatty acid conjugation provides a new approach to increase endocytosis, thereby potentially improving the activity of therapeutic ASOs.

Retention behavior of short double-stranded oligonucleotide and its potential impurities by anion-exchange chromatography under non-denaturing conditions.

Small interfering RNA (siRNA), consisting of two complementary single-stranded RNAs with overhanging bases, is being adopted as a potent and specific inhibitor of target gene expression. However, non-duplexed single strands and undesired double strands composed of impurities (e.g., n-1 mer) could be produced in addition to the target double strand in the siRNA manufacturing process. Compared to the liquid chromatography analysis of single strands, the analysis of the duplexes under non-denaturing conditions is challenging, since restricted chromatographic conditions are required to maintain the Watson-Crick hydrogen bonds. This study reports the analysis of double-stranded oligomers having approximately 20 base pairs with some overhanging bases as non-denatured forms by anion-exchange chromatography (AEX). Optimization of the chromatographic conditions could potentially achieve the adequate separation of excess single strands from the double strand. Non-optimal duplexes, such as duplexes with long overhangs or bulge structures, were prepared by intentionally deleting terminal or middle nucleotide(s) of either the sense or the antisense strand, and these non-optimal duplexes were eluted at different retention times in most of the cases. Interestingly, under alkaline chromatographic conditions (pH 9.0), non-optimal duplexes containing a shortmer tended to exhibit a stronger retention than their parent duplexes, although they possessed a less negative charge. This study demonstrated some retention behavior of double strands with overhangs by AEX under non-denaturing conditions.

Synthesis and Properties of Nucleobase-Sugar Dual Modified Nucleic Acids: 2'-OMe-RNA and scpBNA Bearing a 5-Hydroxycytosine Nucleobase.

Naturally occurring 5-hydroxycytosine (5-OHCyt), which is associated with DNA damage, was recently found to reduce the hepatotoxicity of antisense oligonucleotides (ASOs) without compromising its antisense activity when used as a replacement for cytosine (Cyt). Additionally, sugar-modified nucleic acids, such as 2'-O-methylribonucleic acid (2'-OMe-RNA) and 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA), have emerged as useful antisense materials. Herein, we aimed to combine these two advantages by designing dual modified nucleic acids 2'-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt bearing the 5-OHCyt nucleobase to develop efficient and safe ASOs. We describe the synthesis of 2'-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt phosphoramidites and their incorporation into oligonucleotides (ONs). The duplex-forming ability and base discrimination properties of 2'-OMe-RNA-5-OHCyt- and scpBNA-5-OHCyt-modified ONs were similar to those of 2'-OMe-RNA-Cyt- and scpBNA-mCyt-modified ONs, respectively. We also synthesized two 2'-OMe-RNA-5-OHCyt-modified ASOs, and one of the two was found to exhibit reduced hepatotoxicity while retaining target mRNA knockdown activity in in vivo experiments.

Synthesis and physical and biological properties of 1,3-diaza-2-oxophenoxazine-conjugated oligonucleotides.

The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tCO) and its derivative G-clamp strongly bind to guanine and, when incorporated into double-stranded DNA, significantly increase the stability of the latter. As the phenoxazine skeleton is a constituent of major pharmaceuticals, we hypothesized that oligonucleotides (ONs) containing phenoxazine bases would induce property changes related to intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker antisense oligonucleotide (ASO) in which a G-clamp base with a flexible linker was introduced into the 5'-end of an ASO targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (mMALAT1). Compared to unconjugated ASO, the G-clamp-linker ASO induced significantly more effective knockdown of mMALAT1 in mouse skeletal muscle. The ASOs conjugated with 2'-deoxyribonucleotide(s) bearing a tCO nucleobase at the 5'-end exhibited a similar knockdown effect in skeletal muscle. Thus, it may be possible to improve therapeutic effects against skeletal muscle diseases, such as muscular dystrophy, by using ONs with incorporated phenoxazine nucleobases.

Physicochemical property evaluation of modified oligonucleotides by traveling-wave ion mobility mass spectrometry.

RATIONALE: Therapeutic oligonucleotides have molecular weights of more than 6000 Da. They typically contain chemically modified structures such as phosphorothioate (PS) and a locked nucleic acid (LNA). To determine the effect of the length and chemical modification on the physicochemical properties, various nucleic acids with different lengths and modified structures were analyzed using traveling-wave ion mobility mass spectrometry (TWIMS). METHODS: The physicochemical characteristics of the modified oligonucleotides were determined using IM-MS. Each oligonucleotide was evaluated by confirming the multivalent charge state drift times, collision cross-section (CCS) values, and CCS widths. RESULTS: By plotting the m/z for oligonucleotides of different lengths and the CCS values at each charge state, a bottoming-out shape plot at one charge per 4.0-3.5 bases was confirmed. Moreover, significant differences were observed in the CCS values between the PS-modified and unmodified oligonucleotides. The PS-modified oligonucleotide showed a wider CCS range that was proportional to the PS modification ratio of the oligonucleotide sequence. CONCLUSIONS: The TWIMS results showed a correlation between the length and modification of oligonucleotides and the CCS values. In addition, it suggested that each charge state of the oligonucleotide ion has different physicochemical properties.

Identification of Potent and Selective Inhibitors of Fat Mass Obesity-Associated Protein Using a Fragment-Merging Approach.

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO-selective inhibitors.

Synthesis and duplex-forming ability of oligonucleotides modified with 4'-C,5'-C-methylene-bridged nucleic acid (4',5'-BNA).

We describe herein the design and synthesis of 4'-C,5'-C-methylene-bridged nucleic acid (4',5'-BNA), a novel artificial nucleic acid with the torsion angle γ in a non-canonical +ac range. The 4',5'-BNA phosphoramidite bearing a thymine nucleobase was synthesized from a commercially available thymidine analog in 11 steps and successfully incorporated into oligonucleotides. The resulting oligonucleotides were evaluated for their duplex-forming ability toward single-stranded DNA and RNA.

Synthesis and properties of oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing adenine, guanine, or 5-methylcytosine nucleobases.

Chemical modifications have been extensively used for therapeutic oligonucleotides because they strongly enhance the stability against nucleases, binding affinity to the targets, and efficacy. We previously reported that oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing the thymine (T) nucleobase show excellent biophysical properties for applications in antisense technology. In this paper, we describe the synthesis of GuNA[Me] phosphoramidites bearing other typical nucleobases including adenine (A), guanine (G), and 5-methylcytosine (mC). The phosphoramidites were successfully incorporated into oligonucleotides following the method previously developed for the GuNA[Me]-T-modified oligonucleotides. The binding affinity of the oligonucleotides modified with GuNA[Me]-A, -G, or -mC toward the complementary single-stranded DNAs or RNAs was systematically evaluated. All of the GuNA[Me]-modified oligonucleotides were found to have a strong affinity for RNAs. These data indicate that GuNA[Me] could be a useful modification for therapeutic antisense oligonucleotides.

Synthesis and properties of GuNA purine/pyrimidine nucleosides and oligonucleotides.

We recently designed guanidine-bridged nucleic acids (GuNA), and GuNA bearing a thymine (T) nucleobase was synthesized and successfully incorporated into oligonucleotides. The GuNA-T-modified oligonucleotides possessed high duplex-forming ability towards their complementary single-stranded RNAs and were highly stable against 3'-exonuclease. Therefore, GuNA is a promissing artificial nucleic acid for therapeutic antisense oligonucleotides. We herein report the facile synthesis of GuNA phosphoramidites bearing adenine (A), guanine (G), and 5-methylcytosine (mC) nucleobases and a robust method for the preparation of GuNA-modified oligonucleotides, even with sequences having acid-sensitive purine nucleobases. Oligonucleotides modified with GuNA-A, -G, or -mC possessed high duplex-forming ability, similar to those modified with GuNA-T. Moreover, some of the GuNA-modified oligonucleotides were revealed to have high base discriminating ability compared with that of their natural counterparts. GuNA nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. Thus, all GuNAs (GuNA-T, -A, -G, and -mC) are now available to be examined in therapeutic applications.

Different reactivity of Sp and Rp isomers of phosphorothioate-modified oligonucleotides in a duplex structure.

The presence of a stereoisomeric center at the phosphorus atom in phosphorothioate-modified oligonucleotides (PS-ONs) has been recognized as an important feature since the early stages of their development. Therefore, several studies have been conducted on the chirality of PS-ONs. In this study, we evaluated the stereo-biased chemistry of PS-ON duplexes. Depending on their absolute configurations, PS-ON duplexes were found to have significantly different and stereospecific reactivities towards simple alkylating reagent.

Synthesis and Evaluation of Artificial Nucleic Acid Bearing an Oxanorbornane Scaffold.

Natural oligonucleotides have many rotatable single bonds, and thus their structures are inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form a duplex with single-stranded DNA or RNA. An effective approach to reduce such entropic loss in the duplex-formation is the conformational restriction of the flexible phosphodiester linkage and/or sugar moiety. We here report the synthesis and biophysical properties of a novel artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA), where the adamant oxanorbornane was expected to rigidify the structures of both the linkage and sugar parts of nucleic acid. OxNorNA phosphoramidite with a uracil (U) nucleobase was successfully synthesized over 15 steps from a known sugar-derived cyclopentene. Thereafter, the given phosphoramidite was incorporated into the designed oligonucleotides. Thermal denaturation experiments revealed that oligonucleotides modified with the conformationally restricted OxNorNA-U properly form a duplex with the complementally DNA or RNA strands, although the Tm values of OxNorNA-U-modified oligonucleotides were lower than those of the corresponding natural oligonucleotides. As we had designed, entropic loss during the duplex-formation was reduced by the OxNorNA modification. Moreover, the OxNorNA-U-modified oligonucleotide was confirmed to have extremely high stability against 3'-exonuclease activity, and its stability was even higher than those of the phosphorothioate-modified counterparts (Sp and Rp). With the overall biophysical properties of OxNorNA-U, we expect that OxNorNA could be used for specialized applications, such as conformational fixation and/or bio-stability enhancement of therapeutic oligonucleotides (e.g., aptamers).

Structure-Activity Relationship for the Oxadiazole Class of Antibacterials.

A structure-activity relationship (SAR) for the oxadiazole class of antibacterials was evaluated by syntheses of 72 analogs and determination of the minimal-inhibitory concentrations (MICs) against the ESKAPE panel of bacteria. Selected compounds were further evaluated for in vitro toxicity, plasma protein binding, pharmacokinetics (PK), and a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) infection. Oxadiazole 72c shows potent in vitro antibacterial activity, exhibits low clearance, a high volume of distribution, and 41% oral bioavailability, and shows efficacy in mouse models of MRSA infection.