Inês Fial, Seth A Farrier, David P Chimento, Carl A Ascoli, Xiao Wan, Peter L Oliver
{"title":"Characterizing Antibodies Targeting Antisense Oligonucleotide Phosphorothioate and 2'-<i>O</i>-Methoxyethyl Modifications for Intracellular Trafficking and Biodistribution Studies.","authors":"Inês Fial, Seth A Farrier, David P Chimento, Carl A Ascoli, Xiao Wan, Peter L Oliver","doi":"10.1177/21593337251361396","DOIUrl":"https://doi.org/10.1177/21593337251361396","url":null,"abstract":"<p><p>The efficacy of nucleic acid therapeutics (NATs) such as antisense oligonucleotides (ASOs) and small interfering RNAs relies on multiple stages of extra- and intracellular trafficking. Assessing uptake and efficacy often relies on fluorescent tagging of the NAT for imaging, although the exogenous tag undoubtedly influences the kinetics of intracellular transport and does not represent the compound used clinically. Therefore, better methods to assess the cellular and tissue distribution of NATs are needed. Here, we have validated new panels of antibody reagents that target clinically relevant nucleic acid modifications for visualizing ASOs both <i>in vitro</i> and <i>in vivo</i>. Using the ModDetect™ library of antibodies, we have tested ASOs <i>in vitro</i> for intracellular localization by immunocytochemistry and for biodistribution in mouse tissues by immunohistochemistry. Antibodies specific for the commonly used phosphorothioate (PS) or 2'-<i>O</i>-methoxyethyl (2'-MOE) modifications successfully detected gapmer ASOs, facilitating colocalization studies with endosomal markers in 2D and 3D cell models. In addition, we assessed colocalization of anti-PS signals with fluorescently tagged ASOs. Our data demonstrate the utility of these reagents for the NAT field, where modified nucleic acids can be detected irrespective of the nucleotide sequence, rendering the system amenable for multiple clinical and preclinical workflows and quantitative immunoassays.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Molecular Modeling of Antisense Oligonucleotide Analogs.","authors":"Rodrigo Galindo-Murillo","doi":"10.1089/nat.2025.0014","DOIUrl":"https://doi.org/10.1089/nat.2025.0014","url":null,"abstract":"<p><p>Antisense oligonucleotides (ASOs) are short, synthetic nucleic acids designed to specifically bind to complementary sequences of RNA. They have become powerful tools in research and medicine due to their ability to modulate gene expression through RNase H-mediated target reduction as well as splice modulation. Molecular dynamics simulations and molecular modeling play critical roles in the study, design, and optimization of ASOs. These computational techniques provide detailed insights into the structure, behavior, and interactions of ASOs at the molecular level. Here, we present a summary of the applications of computational chemistry tools in the study of ASOs and discuss the strengths and disadvantages of each approach.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144560644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Exosome-Mediated Mitochondrial Delivery of Antisense Oligonucleotides.","authors":"Dora von Trentini, Ivan J Dmochowski","doi":"10.1089/nat.2024.0067","DOIUrl":"https://doi.org/10.1089/nat.2024.0067","url":null,"abstract":"<p><p>We present a general method for in-cellulo delivery of 2'-<i>O</i>-methyl (2'-OMe) RNA oligonucleotides (oligos) to mitochondria for antisense applications, with potential for implementation in other mitochondrial DNA (mtDNA)-targeted therapies. Exosomes, which are nanoscale, naturally occurring extracellular vesicles (EVs), have been employed for biotechnology applications in oligonucleotide delivery in recent years. We discovered that exosomes from fetal bovine serum (FBS) can be used as a simple and biologically compatible delivery agent of 2'-OMe RNA antisense oligonucleotides to cellular mitochondria, leading to target protein knockdown. While most RNA interference and antisense mechanisms occur in the cytoplasm or nucleus, the need for mitochondrial targeting has become increasingly apparent. Mitochondrial disease describes a variety of currently incurable syndromes that especially affect organs requiring significant energy including the muscles, heart, and brain. Many of these syndromes result from mutations in mtDNA, which codes for the 13 proteins of the oxidative phosphorylation system and are thus often implicated in inherited metabolic disorders.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144485293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jurriën Prins, Janneke Kouwenberg, Anouk Spruit, Lizanne Daleman, Iris van Wissen, Bianca Matthee, Angela Koudijs, Caroline de Brouwer, Ellen Lievers, Franca Witjas, Vita Dauksaite, Carla van Alem, Roel Bijkerk, Annemieke Aartsma-Rus, Jessica Sipkens, Ton Rabelink, Mariet Feltkamp, Tamara Pfaff, Holger Zimmermann, Anton Jan van Zonneveld, Peter Lischka, Eric van der Veer
{"title":"Design and <i>In Vitro</i> Evaluation of a Novel Antisense Oligonucleotide for Treatment of BK Virus Infection.","authors":"Jurriën Prins, Janneke Kouwenberg, Anouk Spruit, Lizanne Daleman, Iris van Wissen, Bianca Matthee, Angela Koudijs, Caroline de Brouwer, Ellen Lievers, Franca Witjas, Vita Dauksaite, Carla van Alem, Roel Bijkerk, Annemieke Aartsma-Rus, Jessica Sipkens, Ton Rabelink, Mariet Feltkamp, Tamara Pfaff, Holger Zimmermann, Anton Jan van Zonneveld, Peter Lischka, Eric van der Veer","doi":"10.1089/nat.2025.0010","DOIUrl":"10.1089/nat.2025.0010","url":null,"abstract":"<p><p>BK polyomavirus, a virus that latently resides in tubular epithelial cells of human kidneys, represents a major clinical problem for immunocompromised patients undergoing kidney transplantation. No proven effective specific antiviral therapies other than reduction of immunosuppressants exist. We exploited splice-modulating antisense oligonucleotides (ASOs) to block expression of the viral \"gatekeeper protein\" T antigen to specifically inhibit viral replication. Candidate oligonucleotides were screened for inhibitory activity in BK virus-infected cells, resulting in up to 97% reduction of viral T antigen mRNA and virus-encapsulating protein. The most promising ASO candidates were validated in BK virus-infected human kidney epithelial cells, showing sequence-specific activity that was exacerbated by chemical modifications. Administration of the candidate oligonucleotide in mice revealed long-lasting uptake in proximal tubules of the kidney, where BK virus resides. A final optimization round yielded an oligonucleotide displaying superior activity. Studies in transformed cells revealed a discernible shift in T antigen splicing not observed with a scrambled control, indicating that targeting of the donor splice site was responsible for the observed effects. Here, we demonstrate proof-of-principle for a direct-acting, splice-modulating, universal ASO that distributes to sites where BK viral replication occurs, warranting further development of this novel therapeutic to combat BK virus replication in kidney transplant patients.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144216439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Hepatotoxicity Reduction Profiles of Antisense Oligonucleotides Containing Amido-Bridged Nucleic Acid and 2'-O,4'-C-Spirocyclopropylene Bridged Nucleic Acid.","authors":"Takaaki Kawanobe, Shinya Asano, Hitoshi Kandori, Masami Aoki, Ajaya Ram Shrestha, Kazuo Sekiguchi, Kotaro Yokoyama, Ryo Fukuda, Tadashi Umemoto","doi":"10.1089/nat.2024.0047","DOIUrl":"10.1089/nat.2024.0047","url":null,"abstract":"<p><p>Amido-bridged nucleic acid (AmNA) and a 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (scpBNA) are bridged nucleic acid analogs with high binding affinity toward complementary strands along with high nuclease resistance. AmNA and scpBNA have been developed to overcome phosphorothioate modified gapmer hepatotoxicity, while the mechanism of reducing hepatotoxicity still remains unknown. Here, we found that antisense oligonucleotides (ASOs) with combination of AmNA, scpBNA, and phosphodiester (PO) bonds could significantly reduce hepatotoxicity in mice. Histopathological findings of the periportal spaces of the liver were observed only in the locked nucleic acid and AmNA-scpBNA groups, but not in the AmNA-scpBNA-PO group. Furthermore, bioinformatics and histopathological analysis revealed that the reduced hepatotoxicity might be related to mitochondrial abnormalities, such as decreased expression levels of Atp5o and Sdhb genes. Taken together, the results of this study demonstrated that AmNA, scpBNA, and PO modification are able to reduce hepatotoxicity for improving the potential of ASOs.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":"114-124"},"PeriodicalIF":4.0,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143710655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bethany R Hannas, Sara M Bender, Eileen Blasi, Christopher J Bowman, Joy A Cavagnaro, Connie L Chen, Pragati S Coder, Britt Duijndam, Alan Hoberman, Tae-Won Kim, Isabelle Leconte, Kazushige Maki, Mineo Matsumoto, Fumito Mikashima, Dinah L Misner, Lutz Mueller, Nicola Powles-Glover, Stephanie Rayhon, Camelia Saffarini, Christine Siezen, Jennifer Sisler, Ronald L Wange, Tacey White, Michael V Templin
{"title":"Developmental and Reproductive Toxicity Testing Strategies for Oligonucleotides: A Workshop Proceedings.","authors":"Bethany R Hannas, Sara M Bender, Eileen Blasi, Christopher J Bowman, Joy A Cavagnaro, Connie L Chen, Pragati S Coder, Britt Duijndam, Alan Hoberman, Tae-Won Kim, Isabelle Leconte, Kazushige Maki, Mineo Matsumoto, Fumito Mikashima, Dinah L Misner, Lutz Mueller, Nicola Powles-Glover, Stephanie Rayhon, Camelia Saffarini, Christine Siezen, Jennifer Sisler, Ronald L Wange, Tacey White, Michael V Templin","doi":"10.1089/nat.2025.0013","DOIUrl":"10.1089/nat.2025.0013","url":null,"abstract":"<p><p>A 2023 workshop brought together stakeholders involved in the development and safety assessment of oligonucleotide (ONT) therapeutics. The purpose was to discuss potential strategies and opportunities for enhancing developmental and reproductive toxicity (DART) assessment of ONTs. The workshop was timely, bringing together regulators, industry representatives, consultants, and contract research organization partners interested in the ongoing development of internationally harmonized guidance for nonclinical safety assessment of ONTs. Given DART's importance in nonclinical safety assessment and the unique attributes of ONTs, the forum discussed case studies, consensus approaches, and areas needing further development to optimize DART strategies. This report covers the workshop proceedings, highlighting methods to achieve a robust DART assessment for ONTs. It includes case studies that described strategies for dose level selection, dosing frequency, species selection, and alternative animal model approaches. Topics also cover surrogate ONT use, exposure of the placenta and embryo/fetal compartment, and weight of evidence approaches. A goal of these workshop proceedings is to describe example approaches to hopefully inform the DART strategy expectations in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidance currently under development for nonclinical safety assessment of ONTs.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":"93-113"},"PeriodicalIF":4.0,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Saket Agarwal, Elizabeth Taft, Micah Gauthier, Justin Darcy, Kira Buckowing, Daniel Berman, Wendell P Davis, Arlin B Rogers, Maja M Janas
{"title":"Mechanistic Insights into Hybridization-Based Off-Target Activity of GalNAc-siRNA Conjugates.","authors":"Saket Agarwal, Elizabeth Taft, Micah Gauthier, Justin Darcy, Kira Buckowing, Daniel Berman, Wendell P Davis, Arlin B Rogers, Maja M Janas","doi":"10.1089/nat.2024.0090","DOIUrl":"10.1089/nat.2024.0090","url":null,"abstract":"<p><p>Nonclinical safety screening of small interfering RNAs (siRNAs) conjugated to a trivalent <i>N</i>-acetylgalactosamine (GalNAc) ligand is typically carried out in rats at exaggerated exposures in a repeat-dose regimen. We have previously shown that at these suprapharmacological doses, hepatotoxicity observed with a subset of GalNAc-siRNAs is largely driven by undesired RNA-induced silencing complex (RISC)-mediated antisense strand seed-based off-target activity, similar to microRNA-like regulation. However, the RISC component requirements for off-target activity of siRNAs have not been evaluated. Here, we evaluate the roles of major RISC components, AGO and TNRC6 (or GW182) proteins, in driving on- and off-target activity of GalNAc-siRNAs in hepatocytes, <i>in vitro</i> and <i>in vivo</i>. We demonstrate that knocking down AGO2, but not AGO1 or AGO4, is protective against GalNAc-siRNA-driven off-target activity and hepatotoxicity. As expected, knocking down AGO2, but not AGO1 or AGO4, reduces the on-target activity of GalNAc-siRNA. Similarly, knocking down TNRC6 paralogs, TNRC6A or TNRC6B, but not TNRC6C, is protective against off-target activity and hepatotoxicity while having minimal impact on the on-target activity of GalNAc-siRNA. These data indicate that while AGO2 is the only RISC component required for the on-target activity of GalNAc-siRNAs, the undesired off-target activity and hepatotoxicity of a subset of GalNAc-siRNAs are mediated via the RISC composed predominantly of AGO2 and TNRC6 paralogs TNRC6A and/or TNRC6B.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":"125-136"},"PeriodicalIF":4.0,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143710661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Daniel O'Reilly, Raymond Furgal, Vignesh Hariharan, Clemens Lochmann, David Cooper, Dimas Echeverria, Anastasia Khvorova
{"title":"Systematic Evaluation of Position-Specific Tolerability of Seven Backbone and Ribose Modifications in Fully Chemically Stabilized siRNAs.","authors":"Daniel O'Reilly, Raymond Furgal, Vignesh Hariharan, Clemens Lochmann, David Cooper, Dimas Echeverria, Anastasia Khvorova","doi":"10.1089/nat.2024.0077","DOIUrl":"10.1089/nat.2024.0077","url":null,"abstract":"<p><p>Chemically modified short interfering RNAs (siRNAs) unequivocally represent a groundbreaking class of drugs. The deliberate chemical modification of the natural structure has been pivotal to their resounding success. Specific modifications at certain positions bolster their potency, safety, stability, and specificity. In clinical research, 2'-<i>O</i>-methyl and 2'-fluoro are the most used modifications. The effects of a wide range of chemical changes in fully modified siRNAs have not been thoroughly evaluated for tolerability. In this study, we utilized two sequences in a fully modified siRNA to systematically assess the tolerability of single nucleotide backbone and sugar modifications, including deoxyribonucleic acid, 2'-<i>O</i>-(2-methoxyethyl), locked nucleic acid, unlocked nucleic acid, mismatches, butane diol substitution, and butane diol insertion. We synthesized 522 siRNA variants and evaluated their efficacy <i>in vitro</i>. Our findings demonstrate that individual tolerability is significantly influenced by the modification's sequence, pattern, and position, with limited universal principles identifiable from this dataset. The efficacy results are probably driven by the thermodynamic balance defined by a combination of parameters. The framework presented here will serve as a reference dataset to facilitate the expansion of chemical diversity in therapeutic siRNAs.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":"137-149"},"PeriodicalIF":4.0,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12167845/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144143193","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Joel D Parry, Tod A Harper, Patrik Andersson, Joanne M Elloway, Natalie S Holman, William E Achanzar, Anthony Lynch, Yann Tessier, Meredith Crosby, Eike Floettmann, Marie Coeffet, Melanie Guérard, Nicole H P Cnubben, Onyi N Irrechukwu, Olivier Wattrelos, Yi Yang
{"title":"Opportunities for More Tailored Approaches to Genotoxicity Testing and Carcinogenicity Strategy for Oligonucleotide Therapeutics: Outcome of an Industry Survey.","authors":"Joel D Parry, Tod A Harper, Patrik Andersson, Joanne M Elloway, Natalie S Holman, William E Achanzar, Anthony Lynch, Yann Tessier, Meredith Crosby, Eike Floettmann, Marie Coeffet, Melanie Guérard, Nicole H P Cnubben, Onyi N Irrechukwu, Olivier Wattrelos, Yi Yang","doi":"10.1089/nat.2024.0075","DOIUrl":"10.1089/nat.2024.0075","url":null,"abstract":"<p><p>The Oligonucleotide Nonclinical Working Group (WG) of the European Federation of Pharmaceutical Industries and Associations conducted an industry survey to understand current practices and regulatory expectations for genotoxicity and carcinogenicity assessment of oligonucleotide therapeutics (ONTs), along with historical genotoxicity testing results. The survey, involving 29 pharmaceutical and biotechnology companies, revealed a consistent absence of genotoxicity across a diverse range of oligonucleotide classes and chemistries, consistent with previous observations. Despite the lack of genotoxicity, companies continue to follow standard testing guidelines, with only limited divergence. The survey data support the view that well-established ONT modifications can be considered \"precedented,\" in terms of negligible genotoxic risk. As such, further testing of new ONT candidates containing only precedented modifications is unwarranted, when defined criteria are met. Further, we propose a pathway for novel ONT chemical modifications to achieve precedented status. The survey results also indicate that alternative strategies for carcinogenicity assessment (e.g., single-species testing) can be accepted by regulatory agencies under certain circumstances. Overall, the survey findings underscore the need for a more tailored approach to the nonclinical safety assessment of ONTs, and the WG proposes development of supplementary questions for International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S2(R1) guidance to reflect this broad industry experience.</p>","PeriodicalId":19412,"journal":{"name":"Nucleic acid therapeutics","volume":" ","pages":"34-48"},"PeriodicalIF":4.0,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143024165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}