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To date, approved therapies remain available for only a small proportion of rare genetic diseases. Oligonucleotide-based RNA therapeutics, particularly antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), offer a promising therapeutic avenue for rare genetic diseases with sequence-level precision. However, traditional preclinical paths may mis-predict human outcomes when disease biology diverges from animal models. New approach methodologies (NAMs), including patient-derived induced pluripotent stem cells (iPSCs), organoid models, and clinical-trials-in-a-dish (CTiD), aim to bring human biology earlier into the translational pipeline. NAMs enable variant-to-function studies, efficacy screening, and safety triage at clinically relevant speed and scale. While critics argue that NAMs are unvalidated and cannot replace preclinical animal models, proponents report that they are increasingly able to recapitulate human phenotypes and predict clinical liabilities, although their predictive validity remains context-dependent. Here, a front-loaded human filter refers to the use of human-derived systems early in development to support mechanistic interpretation, candidate prioritization, and early liability assessment before broader nonclinical evaluation. Recent studies pairing NAMs with ASOs support rapid, patient-specific preclinical screening in selected settings, while also showing the need for broader evidence on delivery, pharmacology, safety, and clinical relevance. This review places these developments within the translational realities of oligonucleotide-based therapeutics, including model fidelity, ASO chemistry and optimization, delivery challenges, pharmacology, regulatory pathways for individualized ASOs, and accessibility. We also propose a pragmatic validation framework to assess the scientific and translational credibility of NAMs across rare genetic diseases.<\/jats:p>","DOI":"10.3390\/genes17070780","type":"journal-article","created":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T14:33:06Z","timestamp":1783089186000},"page":"780","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Molecular Diagnosis to Individualized Therapies in Rare Genetic Diseases: New Approach Methodologies, RNA Therapeutics, and the Case for a Human-First Filter"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3688-5923","authenticated-orcid":false,"given":"Saeed","family":"Anwar","sequence":"first","affiliation":[{"name":"Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshifumi","family":"Yokota","sequence":"additional","affiliation":[{"name":"Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada"},{"name":"Neuroscience and Mental Health Institute, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2E1, Canada"},{"name":"Cardiovascular Research Institute, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 1C9, Canada"},{"name":"Women and Children\u2019s Health Research Institute, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 1C9, Canada"},{"name":"The Friends of Garrett Cumming Research and Muscular Dystrophy Canada Endowed Research, University of Alberta, Edmonton, AB T6G 2H7, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1186\/s13023-023-02790-7","article-title":"A Comprehensive Study of the Rare Diseases and Conditions Targeted by Orphan Drug Designations and Approvals over the Forty Years of the Orphan Drug Act","volume":"18","author":"Fermaglich","year":"2023","journal-title":"Orphanet J. Rare Dis."},{"key":"ref_2","unstructured":"EURORDIS (2018). What Is a Rare Disease?, EURORDIS-Rare Diseases Europe."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1038\/s41431-019-0508-0","article-title":"Estimating Cumulative Point Prevalence of Rare Diseases: Analysis of the Orphanet Database","volume":"28","author":"Lambert","year":"2020","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1002\/ajmg.a.61124","article-title":"The Burden of Rare Diseases","volume":"179","author":"Ferreira","year":"2019","journal-title":"Am. J. Med. Genet. Part A"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1038\/d41573-019-00180-y","article-title":"How Many Rare Diseases Are There?","volume":"19","author":"Haendel","year":"2020","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1116","DOI":"10.1038\/s41431-024-01604-z","article-title":"Time to Diagnosis and Determinants of Diagnostic Delays of People Living with a Rare Disease: Results of a Rare Barometer Retrospective Patient Survey","volume":"32","author":"Faye","year":"2024","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1038\/s41525-025-00478-4","article-title":"Copy Number Variant Analysis Improves Diagnostic Yield in a Diverse Pediatric Exome Sequencing Cohort","volume":"10","author":"Hahn","year":"2025","journal-title":"npj Genom. Med."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Malmgren, H., Kvarnung, M., Gustafsson, P., Anderlid, B.-M., Arthur, C., Carlsten, J., De Geer, K., Ehn, E., Grigelionien\u00e9, G., and Hammarsj\u00f6, A. (2025). Diagnostic Yield of 1000 Trio Analyses with Exome and Genome Sequencing in a Clinical Setting. Front. Genet., 16.","DOI":"10.3389\/fgene.2025.1580879"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2478","DOI":"10.1056\/NEJMc1812033","article-title":"Reanalysis of Clinical Exome Sequencing Data","volume":"380","author":"Liu","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1038\/s41525-025-00538-9","article-title":"Clinical Utility of Genome Sequencing in Rare Diseases: Lessons from a Single-Center Study of 1,452 Korean Families","volume":"11","author":"Lee","year":"2025","journal-title":"npj Genom. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1502","DOI":"10.1056\/NEJMoa1306555","article-title":"Clinical Whole-Exome Sequencing for the Diagnosis of Mendelian Disorders","volume":"369","author":"Yang","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Anwar, S., Mir, F., and Yokota, T. (2023). Enhancing the Effectiveness of Oligonucleotide Therapeutics Using Cell-Penetrating Peptide Conjugation, Chemical Modification, and Carrier-Based Delivery Strategies. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15041130"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Dhuri, K., Bechtold, C., Quijano, E., Pham, H., Gupta, A., Vikram, A., and Bahal, R. (2020). Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. J. Clin. Med., 9.","DOI":"10.3390\/jcm9062004"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1089\/nat.2016.0656","article-title":"Molecular Mechanisms of Antisense Oligonucleotides","volume":"27","author":"Crooke","year":"2017","journal-title":"Nucleic Acid Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1038\/35078107","article-title":"Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells","volume":"411","author":"Elbashir","year":"2001","journal-title":"Nature"},{"key":"ref_16","unstructured":"FDA, and CDER (2021). Nonclinical Testing of Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Wilton-Clark, H., Yan, E., and Yokota, T. (2024). Preparing for Patient-Customized N-of-1 Antisense Oligonucleotide Therapy to Treat Rare Diseases. Genes, 15.","DOI":"10.20944\/preprints202405.1955.v1"},{"key":"ref_18","unstructured":"US Food and Drug Administration (2021). IND Submissions for Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases: Clinical Recommendations."},{"key":"ref_19","unstructured":"US Food and Drug Administration (2024). Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1258\/jrsm.2008.08k033","article-title":"Why Animal Studies Are Often Poor Predictors of Human Reactions to Exposure","volume":"102","author":"Bracken","year":"2009","journal-title":"J. R. Soc. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1007\/s00204-013-1038-0","article-title":"Inflammatory Findings on Species Extrapolations: Humans Are Definitely No 70-Kg Mice","volume":"87","author":"Leist","year":"2013","journal-title":"Arch. Toxicol."},{"key":"ref_22","first-page":"379","article-title":"LLNA Variability: An Essential Ingredient for a Comprehensive Assessment of Non-Animal Skin Sensitization Test Methods and Strategies","volume":"32","author":"Hoffmann","year":"2015","journal-title":"ALTEX"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1017\/S0963180115000079","article-title":"The Flaws and Human Harms of Animal Experimentation","volume":"24","author":"Akhtar","year":"2015","journal-title":"Camb. Q. Healthc. Ethics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.cotox.2017.04.002","article-title":"The Relevance of Experimental Carcinogenicity Studies to Human Safety","volume":"3","author":"Cohen","year":"2017","journal-title":"Curr. Opin. Toxicol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"235","DOI":"10.14573\/altex.1608281","article-title":"Uncertainties of Testing Methods: What Do We (Want to) Know about Carcinogenicity?","volume":"34","author":"Paparella","year":"2017","journal-title":"ALTEX"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.cotox.2017.05.002","article-title":"The Relevance of Experimental Reproductive Studies in Safety Assessment","volume":"3","author":"Stewart","year":"2017","journal-title":"Curr. Opin. Toxicol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1080\/10408444.2018.1429385","article-title":"Non-Animal Methods to Predict Skin Sensitization (I): The Cosmetics Europe Database","volume":"48","author":"Hoffmann","year":"2018","journal-title":"Crit. Rev. Toxicol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1016\/j.jacbts.2019.10.008","article-title":"Limitations of Animal Studies for Predicting Toxicity in Clinical Trials: Is It Time to Rethink Our Current Approach?","volume":"4","year":"2019","journal-title":"JACC Basic Transl. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1186\/s12967-019-1976-2","article-title":"Animal to Human Translation: A Systematic Scoping Review of Reported Concordance Rates","volume":"17","author":"Leenaars","year":"2019","journal-title":"J. Transl. Med."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"ICCVAM (2024). Validation, Qualification, and Regulatory Acceptance of New Approach Methodologies.","DOI":"10.22427\/NICEATM-2"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1877","DOI":"10.1016\/j.cell.2026.02.012","article-title":"New Approach Methodologies for Drug Discovery","volume":"189","author":"Liu","year":"2026","journal-title":"Cell"},{"key":"ref_32","unstructured":"US Food and Drug Administration (2026). General Considerations for the Use of New Approach Methodologies in Drug Development."},{"key":"ref_33","unstructured":"European Medicines Agency (2026, April 20). Regulatory Acceptance of New Approach Methodologies (NAMs) to Reduce Animal Use Testing. Available online: https:\/\/www.ema.europa.eu\/en\/human-regulatory-overview\/research-development\/ethical-use-animals-medicine-testing\/regulatory-acceptance-new-approach-methodologies-nams-reduce-animal-use-testing."},{"key":"ref_34","unstructured":"Environment and Climate Change Canada, and Health Canada (2025). Strategy to Replace, Reduce or Refine Vertebrate Animal Testing Under the Canadian Environmental Protection Act, 1999 (CEPA)."},{"key":"ref_35","unstructured":"Department for Science Innovation & Technology (2025). Replacing Animals in Science: A Strategy to Support the Development, Validation and Uptake of Alternative Methods."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"104662","DOI":"10.1016\/j.yrtph.2020.104662","article-title":"An FDA\/CDER Perspective on Nonclinical Testing Strategies: Classical Toxicology Approaches and New Approach Methodologies (NAMs)","volume":"114","author":"Avila","year":"2020","journal-title":"Regul. Toxicol. Pharmacol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"105345","DOI":"10.1016\/j.yrtph.2023.105345","article-title":"Gaps and Challenges in Nonclinical Assessments of Pharmaceuticals: An FDA\/CDER Perspective on Considerations for Development of New Approach Methodologies","volume":"139","author":"Avila","year":"2023","journal-title":"Regul. Toxicol. Pharmacol."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Bourcier, T., McGovern, T., Cavaliero, T., Ebere, G., Nishikawa, A., Nishimura, J., Ogawa, K., Pasanen, M., Vespa, A., and Van der Laan, J.W. (2024). ICH S1 Prospective Evaluation Study: Weight of Evidence Approach to Predict Outcome and Value of 2-Year Rat Carcinogenicity Studies. A Report from the Regulatory Authorities Subgroup. Front. Toxicol., 6.","DOI":"10.3389\/ftox.2024.1353783"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1093\/toxsci\/kfac095","article-title":"Use of Human IPSC-CMs in Nonclinical Regulatory Studies for Cardiac Safety Assessment","volume":"190","author":"Ribeiro","year":"2022","journal-title":"Toxicol. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1038\/d41586-025-03344-6","article-title":"Alternatives to Animal Testing Are the Future\u2014It\u2019s Time That Journals, Funders and Scientists Embrace Them","volume":"646","author":"Herron","year":"2025","journal-title":"Nature"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/s41525-024-00410-2","article-title":"Advancing Access to Genome Sequencing for Rare Genetic Disorders: Recent Progress and Call to Action","volume":"9","author":"Jobanputra","year":"2024","journal-title":"npj Genom. Med."},{"key":"ref_42","first-page":"101473","article-title":"The Implementation of Genome Sequencing in Rare Genetic Diseases Diagnosis: A Pilot Study from the Hong Kong Genome Project","volume":"55","author":"Lam","year":"2025","journal-title":"Lancet Reg. Health-West. Pac."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1007\/s40259-024-00644-7","article-title":"Splice-Modulating Antisense Oligonucleotides as Therapeutics for Inherited Metabolic Diseases","volume":"38","author":"Chen","year":"2024","journal-title":"BioDrugs"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1644","DOI":"10.1056\/NEJMoa1813279","article-title":"Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease","volume":"381","author":"Kim","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1038\/s41586-024-08462-1","article-title":"Rapid and Scalable Personalized ASO Screening in Patient-Derived Organoids","volume":"638","author":"Means","year":"2025","journal-title":"Nature"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"572","DOI":"10.4062\/biomolther.2025.022","article-title":"RNA Therapeutics: Focus on Antisense Oligonucleotides in the Nervous System","volume":"33","author":"Ertural","year":"2025","journal-title":"Biomol. Ther."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Collotta, D., Bertocchi, I., Chiapello, E., and Collino, M. (2023). Antisense Oligonucleotides: A Novel Frontier in Pharmacological Strategy. Front. Pharmacol., 14.","DOI":"10.3389\/fphar.2023.1304342"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"102237","DOI":"10.1016\/j.omtn.2024.102237","article-title":"Strategies to Improve the Design of Gapmer Antisense Oligonucleotide on Allele-Specific Silencing","volume":"35","author":"Aguti","year":"2024","journal-title":"Mol. Ther.-Nucleic Acids"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1109","DOI":"10.1038\/s41467-025-67752-y","article-title":"Antisense Oligonucleotides to KIF1A Polymorphisms Expand Targets and Rescue Patient-Derived Neurons in Vitro","volume":"17","author":"Zuccaro","year":"2026","journal-title":"Nat. Commun."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1089\/nat.2023.0070","article-title":"Screening Splice-Switching Antisense Oligonucleotides in Pancreas-Cancer Organoids","volume":"34","author":"Wan","year":"2024","journal-title":"Nucleic Acid Ther."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Clarke, L.A., and Amaral, M.D. (2023). What Can RNA-Based Therapy Do for Monogenic Diseases?. Pharmaceutics, 15.","DOI":"10.3390\/pharmaceutics15010260"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"612","DOI":"10.1038\/s42003-024-06121-9","article-title":"Between Hope and Reality: Treatment of Genetic Diseases through Nucleic Acid-Based Drugs","volume":"7","author":"Baylot","year":"2024","journal-title":"Commun. Biol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"101555","DOI":"10.1016\/j.xcrm.2024.101555","article-title":"Advancements in Clinical RNA Therapeutics: Present Developments and Prospective Outlooks","volume":"5","author":"Saw","year":"2024","journal-title":"Cell Rep. Med."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Rodriguez Carstens, P., Moriyama, H., and Yokota, T. (2026). From Genomic Diagnosis to Personalized RNA Medicine: Advances in Next-Generation Sequencing and N-of-1 Antisense Oligonucleotide Therapies for Rare Genetic Diseases. Genes, 17.","DOI":"10.3390\/genes17030318"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1089\/nat.2018.0736","article-title":"GalNAc-SiRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics","volume":"28","author":"Springer","year":"2018","journal-title":"Nucleic Acid Ther."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1007\/s40265-017-0711-7","article-title":"Nusinersen: First Global Approval","volume":"77","author":"Hoy","year":"2017","journal-title":"Drugs"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1007\/978-1-4939-8651-4_4","article-title":"Nusinersen in the Treatment of Spinal Muscular Atrophy","volume":"1828","author":"Goodkey","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1625","DOI":"10.1007\/s40265-018-0983-6","article-title":"Patisiran: First Global Approval","volume":"78","author":"Hoy","year":"2018","journal-title":"Drugs"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1007\/s40265-020-01463-0","article-title":"Lumasiran: First Approval","volume":"81","author":"Scott","year":"2021","journal-title":"Drugs"},{"key":"ref_60","doi-asserted-by":"crossref","unstructured":"Anwar, S., and Yokota, T. (2025). Progress and Future Directions in Exon Skipping and Inclusion Therapies: The Landscape of Oligonucleotide-Based Genetic Medicine. Antisense RNA Technology: Methods and Protocols, Springer.","DOI":"10.1007\/978-1-0716-4730-1_1"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"533","DOI":"10.2147\/DDDT.S97635","article-title":"Eteplirsen in the Treatment of Duchenne Muscular Dystrophy","volume":"11","author":"Lim","year":"2017","journal-title":"Drug Des. Devel. Ther."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1699","DOI":"10.1007\/s40265-016-0657-1","article-title":"Eteplirsen: First Global Approval","volume":"76","author":"Syed","year":"2016","journal-title":"Drugs"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1007\/s40265-020-01267-2","article-title":"Golodirsen: First Approval","volume":"80","author":"Heo","year":"2020","journal-title":"Drugs"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1358\/dot.2020.56.8.3159186","article-title":"Golodirsen for Duchenne Muscular Dystrophy","volume":"56","author":"Anwar","year":"2020","journal-title":"Drugs Today"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1358\/dot.2021.57.12.3352740","article-title":"Casimersen for Duchenne Muscular Dystrophy","volume":"57","author":"Yokota","year":"2021","journal-title":"Drugs Today"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"875","DOI":"10.1007\/s40265-021-01512-2","article-title":"Casimersen: First Approval","volume":"81","author":"Shirley","year":"2021","journal-title":"Drugs"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1007\/s40265-020-01339-3","article-title":"Viltolarsen: First Approval","volume":"80","author":"Dhillon","year":"2020","journal-title":"Drugs"},{"key":"ref_68","doi-asserted-by":"crossref","unstructured":"Roshmi, R.R., and Yokota, T. (2023). Viltolarsen: From Preclinical Studies to FDA Approval. Muscular Dystrophy Therapeutics, Springer.","DOI":"10.1007\/978-1-0716-2772-3_2"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1038\/s43856-025-00733-w","article-title":"FORCE Platform Overcomes Barriers of Oligonucleotide Delivery to Muscle and Corrects Myotonic Dystrophy Features in Preclinical Models","volume":"5","author":"Weeden","year":"2025","journal-title":"Commun. Med."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"gkaf270","DOI":"10.1093\/nar\/gkaf270","article-title":"Conjugation to a Transferrin Receptor 1-Binding Bicycle Peptide Enhances ASO and SiRNA Potency in Skeletal and Cardiac Muscles","volume":"53","author":"Carrer","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_71","first-page":"1202","article-title":"Antisense Oligonucleotides for the Treatment of Cardiomyopathy in Duchenne Muscular Dystrophy","volume":"11","author":"Nguyen","year":"2019","journal-title":"Am. J. Transl. Res."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1038\/s41573-020-0075-7","article-title":"Advances in Oligonucleotide Drug Delivery","volume":"19","author":"Roberts","year":"2020","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_73","doi-asserted-by":"crossref","unstructured":"Mirlohi, M.S., Yousefi, T., Aref, A.R., and Seyfoori, A. (2025). Integrating New Approach Methodologies (NAMs) into Preclinical Regulatory Evaluation of Oncology Drugs. Biomimetics, 10.","DOI":"10.3390\/biomimetics10120796"},{"key":"ref_74","unstructured":"U.S. Food and Drug Administration (2025, November 18). New Approach Methods (NAMs), Available online: https:\/\/www.fda.gov\/science-research\/science-and-research-special-topics\/new-approach-methodologies-nams."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"tfae044","DOI":"10.1093\/toxres\/tfae044","article-title":"New Approach Methodologies (NAMs): Identifying and Overcoming Hurdles to Accelerated Adoption","volume":"13","author":"Sewell","year":"2024","journal-title":"Toxicol. Res."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.tifs.2023.02.006","article-title":"Implementing New Approach Methodologies (NAMs) in Food Safety Assessments: Strategic Objectives and Actions Taken by the European Food Safety Authority","volume":"133","author":"Cattaneo","year":"2023","journal-title":"Trends Food Sci. Technol."},{"key":"ref_77","first-page":"2212081s","article-title":"Incorporating New Approach Methodologies into Regulatory Nonclinical Pharmaceutical Safety Assessment","volume":"40","author":"Turner","year":"2023","journal-title":"ALTEX"},{"key":"ref_78","unstructured":"U.S. Environmental Protection Agency (2020). EPA New Approach Methods Work Plan: Reducing Use of Vertebrate Animals in Chemical Testing."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"e200908","DOI":"10.2903\/j.efsa.2022.e200908","article-title":"The Use of NAMs and Omics Data in Risk Assessment","volume":"20","author":"Miccoli","year":"2022","journal-title":"EFSA J."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"100197","DOI":"10.1016\/j.crtox.2024.100197","article-title":"IPSC-Derived and Patient-Derived Organoids: Applications and Challenges in Scalability and Reproducibility as Pre-Clinical Models","volume":"7","author":"Heinzelmann","year":"2024","journal-title":"Curr. Res. Toxicol."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/s41576-019-0100-z","article-title":"Induced Pluripotent Stem Cells in Disease Modelling and Drug Discovery","volume":"20","author":"Rowe","year":"2019","journal-title":"Nat. Rev. Genet."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1038\/nrn.2017.107","article-title":"The Use of Brain Organoids to Investigate Neural Development and Disease","volume":"18","author":"Kriegstein","year":"2017","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1038\/s41580-024-00804-1","article-title":"Modelling Human Brain Development and Disease with Organoids","volume":"26","author":"Birtele","year":"2025","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/534310a","article-title":"A Decade of: IPS Cells","volume":"534","author":"Scudellari","year":"2016","journal-title":"Nature"},{"key":"ref_85","doi-asserted-by":"crossref","unstructured":"Panferov, E., Dodina, M., Reshetnikov, V., Ryapolova, A., Ivanov, R., Karabelsky, A., and Minskaia, E. (2025). Induced Pluripotent (IPSC) and Mesenchymal (MSC) Stem Cells for In Vitro Disease Modeling and Regenerative Medicine. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26125617"},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1016\/j.stem.2015.09.001","article-title":"Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects","volume":"17","author":"Mertens","year":"2015","journal-title":"Cell Stem Cell"},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1038\/s41514-024-00161-5","article-title":"Strategies for Modeling Aging and Age-Related Diseases","volume":"10","author":"Jothi","year":"2024","journal-title":"npj Aging"},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"025120012","DOI":"10.36922\/OR025120012","article-title":"Organoids as a Platform for Personalized Antisense Oligonucleotide Screening: Advancing Precision Medicine","volume":"1","author":"Wan","year":"2025","journal-title":"Organoid Res."},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1038\/s41586-024-07310-6","article-title":"Antisense Oligonucleotide Therapeutic Approach for Timothy Syndrome","volume":"628","author":"Chen","year":"2024","journal-title":"Nature"},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1161\/CIRCULATIONAHA.123.068111","article-title":"Antisense Oligonucleotide Therapy for Calmodulinopathy","volume":"150","author":"Bortolin","year":"2024","journal-title":"Circulation"},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1038\/s41573-025-01182-9","article-title":"Application of New Approach Methodologies for Nonclinical Safety Assessment of Drug Candidates","volume":"24","author":"Beilmann","year":"2025","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_92","unstructured":"US Food and Drug Administration (2026). FDA Releases Draft Guidance on Alternatives to Animal Testing in Drug Development."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1038\/nrg3764","article-title":"Investigating Human Disease Using Stem Cell Models","volume":"15","author":"Sterneckert","year":"2014","journal-title":"Nat. Rev. Genet."},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"1247125","DOI":"10.1126\/science.1247125","article-title":"Organogenesis in a Dish: Modeling Development and Disease Using Organoid Technologies","volume":"345","author":"Lancaster","year":"2014","journal-title":"Science"},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1038\/s41586-020-1962-0","article-title":"Cell Stress in Cortical Organoids Impairs Molecular Subtype Specification","volume":"578","author":"Bhaduri","year":"2020","journal-title":"Nature"},{"key":"ref_96","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1038\/s41586-019-1289-x","article-title":"Individual Brain Organoids Reproducibly Form Cell Diversity of the Human Cerebral Cortex","volume":"570","author":"Velasco","year":"2019","journal-title":"Nature"},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.cell.2004.09.011","article-title":"CaV1.2 Calcium Channel Dysfunction Causes a Multisystem Disorder Including Arrhythmia and Autism","volume":"119","author":"Splawski","year":"2004","journal-title":"Cell"},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1016\/j.jacep.2017.08.007","article-title":"Clinical Outcomes and Modes of Death in Timothy Syndrome: A Multicenter International Study of a Rare Disorder","volume":"4","author":"Dufendach","year":"2018","journal-title":"JACC Clin. Electrophysiol."},{"key":"ref_99","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.pediatrneurol.2022.10.013","article-title":"A Cross-Sectional Study of the Neuropsychiatric Phenotype of CACNA1C-Related Disorder","volume":"138","author":"Levy","year":"2023","journal-title":"Pediatr. Neurol."},{"key":"ref_100","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1161\/CIRCULATIONAHA.112.001216","article-title":"Calmodulin Mutations Associated with Recurrent Cardiac Arrest in Infants","volume":"127","author":"Crotti","year":"2013","journal-title":"Circulation"},{"key":"ref_101","doi-asserted-by":"crossref","first-page":"2976","DOI":"10.1093\/eurheartj\/ehz463","article-title":"The International Calmodulinopathy Registry: Recording the Diverse Phenotypic Spectrum of Un-CALM Hearts","volume":"40","author":"Nyegaard","year":"2019","journal-title":"Eur. Heart J."},{"key":"ref_102","doi-asserted-by":"crossref","first-page":"2964","DOI":"10.1093\/eurheartj\/ehz311","article-title":"Calmodulin Mutations and Life-Threatening Cardiac Arrhythmias: Insights from the International Calmodulinopathy Registry","volume":"40","author":"Crotti","year":"2019","journal-title":"Eur. Heart J."},{"key":"ref_103","doi-asserted-by":"crossref","first-page":"13993","DOI":"10.1021\/acsnano.1c05099","article-title":"Delivery of Oligonucleotide Therapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular Vesicles","volume":"15","author":"Bost","year":"2021","journal-title":"ACS Nano"},{"key":"ref_104","doi-asserted-by":"crossref","first-page":"e13243","DOI":"10.15252\/emmm.202013243","article-title":"Delivery of Oligonucleotide-based Therapeutics: Challenges and Opportunities","volume":"13","author":"Hammond","year":"2021","journal-title":"EMBO Mol. Med."},{"key":"ref_105","doi-asserted-by":"crossref","first-page":"2529","DOI":"10.1093\/nar\/gkad067","article-title":"Chemistry, Structure and Function of Approved Oligonucleotide Therapeutics","volume":"51","author":"Egli","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_106","doi-asserted-by":"crossref","first-page":"1759","DOI":"10.1016\/j.ymthe.2020.06.015","article-title":"Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug","volume":"28","author":"Debacker","year":"2020","journal-title":"Mol. Ther."},{"key":"ref_107","doi-asserted-by":"crossref","first-page":"e2309472120","DOI":"10.1073\/pnas.2309472120","article-title":"Combinatorial Design of Ionizable Lipid Nanoparticles for Muscle-Selective MRNA Delivery with Minimized off-Target Effects","volume":"120","author":"Chen","year":"2023","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_108","doi-asserted-by":"crossref","first-page":"e13691","DOI":"10.1002\/jcsm.13691","article-title":"Targeting Drug Delivery System to Skeletal Muscles: A Comprehensive Review of Different Approaches","volume":"16","author":"Li","year":"2025","journal-title":"J. Cachexia. Sarcopenia Muscle"},{"key":"ref_109","doi-asserted-by":"crossref","first-page":"e2112546119","DOI":"10.1073\/pnas.2112546119","article-title":"Development of DG9 Peptide-Conjugated Single- and Multi-Exon Skipping Therapies for the Treatment of Duchenne Muscular Dystrophy","volume":"119","author":"Lim","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_110","doi-asserted-by":"crossref","first-page":"4477","DOI":"10.1038\/s41467-025-59494-8","article-title":"DG9 Boosts PMO Nuclear Uptake and Exon Skipping to Restore Dystrophic Muscle and Cardiac Function","volume":"16","author":"Shah","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_111","doi-asserted-by":"crossref","first-page":"960","DOI":"10.1126\/science.aaw7894","article-title":"Organoids-on-a-Chip","volume":"364","author":"Park","year":"2019","journal-title":"Science"},{"key":"ref_112","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1038\/s41551-022-00882-6","article-title":"A Multi-Organ Chip with Matured Tissue Niches Linked by Vascular Flow","volume":"6","author":"Teles","year":"2022","journal-title":"Nat. Biomed. Eng."},{"key":"ref_113","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1089\/nat.2022.0061","article-title":"Considerations in the Preclinical Assessment of the Safety of Antisense Oligonucleotides","volume":"33","author":"Goyenvalle","year":"2023","journal-title":"Nucleic Acid Ther."},{"key":"ref_114","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1124\/dmd.121.000418","article-title":"Adverse Drug Reactions and Toxicity of the Food and Drug Administration-Approved Antisense Oligonucleotide Drugs","volume":"50","author":"Alhamadani","year":"2022","journal-title":"Drug Metab. Dispos."},{"key":"ref_115","doi-asserted-by":"crossref","first-page":"762","DOI":"10.1038\/s41587-024-02244-w","article-title":"Template-Independent Enzymatic Synthesis of RNA Oligonucleotides","volume":"43","author":"Wiegand","year":"2025","journal-title":"Nat. Biotechnol."},{"key":"ref_116","unstructured":"World Health Organization (2025). Rare Diseases: A Global Health Priority for Equity and Inclusion, WHO. Executive Board 156th Session."},{"key":"ref_117","unstructured":"World Health Organization (2025). Rare Diseases: A Global Health Priority for Equity and Inclusion, WHO. Resolution WHA78.11, Seventy-Eighth World Health Assembly."},{"key":"ref_118","unstructured":"World Health Organization (2025). Universal Health Coverage: Rare Diseases: A Global Health Priority for Equity and Inclusion, WHO. Report by the Director-General, EB158\/13; Executive Board 158th Session."},{"key":"ref_119","unstructured":"Zubulake, Z. (2026, April 20). What FDA\u2019s NAM Guidance Means for Pharmaceutical Development. Available online: https:\/\/www.pharmtech.com\/view\/what-fda-s-nam-guidance-means-for-pharmaceutical-development."},{"key":"ref_120","doi-asserted-by":"crossref","unstructured":"OECD (2018). Guidance Document on Good In Vitro Method Practices (GIVIMP). OECD Ser. Test. Assess., 286, 172.","DOI":"10.1787\/eco_surveys-tun-2018-en"},{"key":"ref_121","unstructured":"US Food and Drug Administration (2025). Roadmap to Reducing Animal Testing in Preclinical Safety Studies."},{"key":"ref_122","unstructured":"US Food and Drug Administration (2026). Safety Assessment of Genome Editing in Human Gene Therapy Products Using Next-Generation Sequencing."},{"key":"ref_123","unstructured":"National Institutes of Health (2026, April 20). NIH Standardized Organoid Modeling (SOM) Center, Available online: https:\/\/www.nih.gov\/som."},{"key":"ref_124","unstructured":"National Institutes of Health (2026, April 20). NIH Establishes Nation\u2019s First Dedicated Organoid Development Center to Reduce Reliance on Animal Modeling, Available online: https:\/\/www.nih.gov\/news-events\/news-releases\/nih-establishes-nations-first-dedicated-organoid-development-center-reduce-reliance-animal-modeling."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/7\/780\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,5]],"date-time":"2026-07-05T04:18:11Z","timestamp":1783225091000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/7\/780"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,3]]},"references-count":124,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["genes17070780"],"URL":"https:\/\/doi.org\/10.3390\/genes17070780","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,3]]}}}