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Background: The objective of this study was to develop an innovative treatment strategy utilizing antisense oligonucleotides (ASOs) that target the gene encoding protocadherin alpha 11 (PCDHA11) and to elucidate the role of PCDHA11 in gastric cancer cells., Methods: We designed and screened 54 amido-bridged nucleic acid (AmNA)-modified ASOs, selecting them based on PCDHA11-knockdown efficacy, in vitro and in vivo activity, and off-target effects. We assessed the impact of AmNA-modified anti-PCDHA11 ASOs on cellular functions and signaling pathways, and investigated the effects of Pcdha11 deficiency in mice., Results: AmNA-modified anti-PCDHA11 ASOs significantly reduced the proliferation of gastric cancer cells and other solid tumors, whereas overexpression of PCDHA11 enhanced cell proliferation. The selected ASOs inhibited cellular functions related to the metastatic potential of gastric cancer cells, including migration, invasiveness, spheroid formation, and cancer stemness. Our findings revealed that AmNA-modified anti-PCDHA11 ASOs disrupted the AKT/mTOR, Wnt/β-catenin, and JAK/STAT signaling pathways. In mouse models of peritoneal metastasis (gastric and pancreatic cancer), systemic metastasis, and established subcutaneous tumors, administration of AmNA-modified anti-PCDHA11 ASOs inhibited tumor growth. ASO treatment induced reversible, dose- and sequence-dependent liver damage. Pcdha11-deficient mice demonstrated normal reproductive, organ, and motor functions., Conclusions: AmNA-modified anti-PCDHA11 ASOs offer a promising therapeutic strategy for the treatment of gastric cancer and other solid malignancies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Peritoneal metastasis of gastric cancer is closely associated with dismal prognosis. In previous preclinical proof-of-concept studies, an amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotide (ASO), designated ASO-4733 that targets the gene encoding synaptotagmin XIII (SYT13), inhibited cellular functions required for the formation of peritoneal metastasis of gastric cancer cells. ASO-4733 achieved therapeutic effects when intra-abdominally administered to mouse xenograft models. Here, we conducted an analysis of Syt13-deficient mice to determine the pharmacokinetics and toxicity of intra-abdominal administration of ASO-4733., Methods: The effects of Syt13-deficiency in mice were determined. Good Laboratory Practice toxicity tests and the toxicokinetics of intra-abdominal administration of ASO-4733 were conducted in cynomolgus monkeys and rats. The pharmacokinetics of ASO-4733 administered intravenously or intra-abdominally to rats were investigated., Results: Syt13-deficient mice exhibited normal reproduction, organ functions, and motor functions. Weekly intra-abdominal administration of ASO-4733 (125 mg/kg), corresponding to a 50-fold increase of the estimated clinical dose for 4 weeks, was well tolerated by cynomolgus monkeys. In rats, off-target toxicity (not attributable to hybridization) was observed after weekly intra-abdominal administration of ASO-4733. Blood concentrations of ASO-4733 were lower and rose more slowly after intra-abdominal administration compared with intravenous administration., Conclusions: The preclinical profile of intra-abdominal administration of ASO-4733 demonstrated its suitability for entry into clinical trials of patients with peritoneal metastasis of gastric cancer., (© 2024. The Author(s) under exclusive licence to The International Gastric Cancer Association and The Japanese Gastric Cancer Association.)

Antisense oligonucleotides (ASOs) are generally obtained via chemical synthesis on a solid support and phosphorothioate (PS) modification with a phosphate backbone to increase their in vivo stability and activity. However, desulphurised products, in which PS is partially replaced by phosphodiesters, are generally formed during the chemical synthesis of ASO and are difficult to separate from the desired PS-modified ASO by chromatography. Therefore, revealing the unknown factors that cause the formation of desulphurised products and proposing methods to inhibit their formation are highly desirable. In this study, it was found that peroxides in THF, which is used as a solvent for the acetyl capping agent, oxidise phosphite triesters to produce desulphurisation products. The use of THF with antioxidants effectively suppresses the oxidation caused by THF peroxides. Moreover, THF peroxide was found to oxidise phosphoramidites, which are the building blocks of oligonucleotide chemical syntheses, indicating that caution should be taken with the organic solvents used during the synthesis and purification of phosphoramidites., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

RNA splicing regulation has revolutionized the treatment of challenging diseases. Neuroendocrine cancers, including small cell lung cancer (SCLC) and neuroendocrine prostate cancer (PCa), are highly aggressive, with metastatic neuroendocrine phenotypes, leading to poor patient outcomes. We investigated amido-bridged nucleic acid (AmNA)-based splice-switching oligonucleotides (SSOs) targeting RE1-silencing transcription factor (REST) splicing as a novel therapy. We designed AmNA-based SSOs to alter REST splicing. Tumor xenografts were generated by subcutaneously implanting SCLC or PCa cells into mice. SSOs or saline were intraperitoneally administered and tumor growth was monitored. Blood samples were collected from mice after SSO administration, and serum alanine aminotransferase and aspartate aminotransferase levels were measured to assess hepatotoxicity using a biochemical analyser.  In vitro , REST_SSO reduced cancer cell viability. In a tumor xenograft model, it exhibited significant antitumor effects. It repressed REST-controlled RE1-harboring genes and upregulated miR-4516, an SCLC biomarker. Our findings suggest that REST_SSO suppresses tumorigenesis in neuroendocrine cancers by restoring REST function. This novel therapeutic approach holds promise for intractable neuroendocrine cancers such as SCLC and neuroendocrine PCa., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2024. The Author(s).)

The enzymatic synthesis of xenobiotic nucleic acids (XNA), which are artificially sugar-modified nucleic acids, is essential for the preparation of XNA libraries. XNA libraries are used in the in vitro selection of XNA aptamers and enzymes (XNAzymes). Efficient enzymatic synthesis of various XNAs can enable the screening of high-quality XNA aptamers and XNAzymes by expanding the diversity of XNA libraries and adding a variety of properties to XNA aptamers and XNAzymes. However, XNAs that form unstable duplexes with DNA, such as arabino nucleic acid (ANA), may dissociate during enzyme synthesis at temperatures suitable for thermophilic polymerases. Thus, such XNAs are not efficiently synthesised by the thermophilic polymerase mutants at the end of the sequence. This undesirable bias reduces the possibility of generating high-quality XNA aptamers and XNAzymes. Here, we demonstrate that polyamine-induced DNA/ANA duplex stabilisation promotes ANA synthesis that is catalysed by thermophilic polymerase mutants. Several polyamines, including spermine, spermidine, cadaverine, and putrescine promote ANA synthesis. The negative effect of polyamines on the fidelity of ANA synthesis was negligible. We also showed that polyamines promote the synthesis of other XNAs, including 2'-amino-RNA/2'-fluoro-RNA mixture and 2'- O -methyl-RNA. In addition, we found that polyamine promotes DNA synthesis from the 2'- O -methyl-RNA template. Polyamines, with the use of thermophilic polymerase mutants, may allow further development of XNA aptamers and XNAzymes by promoting the transcription and reverse transcription of XNAs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., Competing Interests: T.Yokota collaborates with Takeda Pharmaceuticals Co., Ltd., Daiichi Sankyo Co., Ltd., Rena Therapeutics Co., Ltd., Toray Industries Co., Ltd., Eisai Co., Ltd., and Sumitomo Pharma Co., Ltd., and serves as the academic adviser for Rena Therapeutics, Inc., and Braizon Therapeutics, Inc. A provisional patent application (PCT/JP2023/024244) describing the work and chemistry used in this study has been submitted (authors: T.Yokota, K.Y., T.M., S.O., and O.N.)., (© 2024 The Authors.)

Replacement of a phosphodiester linkage with an amide linkage can improve the binding affinity of oligonucleotides to complementary RNA and their stability toward nucleases. In addition, restricting the conformation of the sugar moiety and the phosphate backbone in oligonucleotides effectively improves duplex stability. In this study, we designed amide-linked dinucleotides containing a 3',4'-tetrahydropyran-bridged nucleic acid (3',4'-tpBNA) with a constrained sugar conformation as well as a torsion angle ε. Phosphoramidites of the designed dinucleotides were synthesized and incorporated into oligonucleotides. Conformational analysis of the synthesized dinucleotides showed that the sugar conformation of the S -isomer of the amide-linked dinucleotide containing 3',4'-tpBNA was N-type, which has the same conformation as that of the RNA duplex, while that of another R -isomer was S-type. T m analysis indicated that the oligonucleotides containing the synthesized S -isomer showed RNA-selective hybridizing ability, although their duplex-forming ability was slightly inferior to that of natural oligonucleotides. Interestingly, the stability of the oligonucleotides toward endonucleases was significantly improved by modification with the two types of amide-linked dinucleotides developed in this study.

DNA-encoded libraries (DELs) are attracting attention as a screening tool in the early stages of drug discovery. In the development of DELs, drug candidate compounds are chemically synthesized on barcode DNA. Therefore, it is important to perform the synthesis under mild conditions so as to not damage the DNA. On the other hand, coumarins are gaining increasing research focus not only because they possess excellent fluorescence properties, but also because many medicines contain a coumarin skeleton. Among the various reactions developed for the synthesis of coumarins thus far, Knoevenagel condensation followed by intramolecular cyclization under mild conditions can yield coumarins. In this study, we developed a new synthetic method for preparing a coumarin-conjugated oligonucleotide library via Knoevenagel condensation. The results showed that coumarins substituted at the 5-, 6-, 7-, or 8-positions could be constructed on DNA to afford a total of 26 coumarin-conjugated DNAs. Moreover, this method was compatible with enzymatic ligation, demonstrating its utility in DEL synthesis. The developed strategy for the construction of coumarin scaffolds based on Knoevenagel condensation may contribute to the use of DELs in drug discovery and medicinal chemistry.

Enzymatic oligonucleotide synthesis is used for the development of functional oligonucleotides selected by in vitro selection. Expanding available sugar modifications for in vitro selection helps the functional oligonucleotides to be used as therapeutics reagents. We previously developed a KOD DNA polymerase mutant, KOD DGLNK, that enzymatically synthesized fully-LNA- or 2'- O -methyl-modified oligonucleotides. Here, we report a further expansion of the available 2'- O -alkyl-modified nucleotide for enzymatic synthesis by KOD DGLNK. We chemically synthesized five 2'- O -alkyl-5-methyluridine triphosphates and incorporated them into the oligonucleotides. We also enzymatically synthesized a 2'- O -alkyl-modified oligonucleotide with a random region (oligonucleotide libraries). The 2'- O -alkyl-modified oligonucleotide libraries showed high nuclease resistance and a wide range of hydrophobicity. Our synthesized 2'- O -alkyl-modified oligonucleotide libraries provide novel possibilities that can promote the development of functional molecules for therapeutic use.

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2',4'-BNA-based VASH2-ASO. When systemically administered, naked 2',4'-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2',4'-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2',4'-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: The authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH., (© 2023 Miyagawa et al.)

Background: Antisense oligonucleotide (ASO) medicine for clinical applications has been becoming a reality. We previously developed a gapmer ASO targeting Ser/Arg repetitive matrix 4 (SRRM4) that is abnormally expressed in small cell lung cancer (SCLC). However the detailed mechanism of ASO through repressing SRRM4 has not been completely elucidated. Further, effectiveness of SRRM4 ASO to prostate cancer (PCa) cells expressing SRRM4 similar to SCLC remains to be elucidated. RE1-silencing transcription factor (REST) is a tumor suppressor, and its splicing isoform (sREST) is abnormally expressed by SRRM4 and causes carcinogenesis with neuroendocrine phenotype in SCLC. The present study aimed to understand the contribution of REST splicing by SRRM4 ASO administration., Methods: SRRM4 expression and REST splicing were analyzed by RT-qPCR and conventional RT-PCR after treating SRRM4 ASO, and cell viability was analyzed in vitro. Exogenous reconstitution of Flag-tagged REST plasmid in SCLC cells and the splice-switching oligonucleotide (SSO) specific for REST was analyzed for cell viability. Furthermore, we expanded the application of SRRM4 ASO in PCa cells abnormally expressing SRRM4 mRNA in vitro., Results: SRRM4 ASO successfully downregulated SRRM4 expression, followed by repressed cell viability of SCLC and PCa cells in a dose-dependent manner. Administration of SRRM4 ASO then modified the alternative splicing of REST, resulting reduced cell viability. REST SSO specifically modified REST splicing increased REST expression, resulting in reduced cell viability., Conclusions: Our data demonstrate that a gapmer ASO targeting SRRM4 (SRRM4 ASO) reduces cell viability through splicing changes of REST, followed by affecting REST-controlled genes in recalcitrant tumors SCLC and PCa cells., (© 2023. The Author(s).)

Naturally occurring 5-hydroxycytosine ( 5-OH Cyt), 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-OH Cyt and scpBNA- 5-OH Cyt bearing the 5-OH Cyt nucleobase to develop efficient and safe ASOs. We describe the synthesis of 2'-OMe-RNA- 5-OH Cyt and scpBNA- 5-OH Cyt phosphoramidites and their incorporation into oligonucleotides (ONs). The duplex-forming ability and base discrimination properties of 2'-OMe-RNA- 5-OH Cyt- and scpBNA- 5-OH Cyt-modified ONs were similar to those of 2'-OMe-RNA-Cyt- and scpBNA- m Cyt-modified ONs, respectively. We also synthesized two 2'-OMe-RNA- 5-OH Cyt-modified ASOs, and one of the two was found to exhibit reduced hepatotoxicity while retaining target mRNA knockdown activity in in vivo experiments.