Beatrix Kovács, Viktória Szabó, Domonkos Horváth, Attila Balázs Dobos, Zoltán Zsolt Nagy, Wim Vanduffel, Zsuzsanna Szemlaky, Áron Szepesi, István Ulbert, Balázs Rózsa, Dániel Hillier
{"title":"用恒血清浓度法克服AAV中和试验中的基质效应。","authors":"Beatrix Kovács, Viktória Szabó, Domonkos Horváth, Attila Balázs Dobos, Zoltán Zsolt Nagy, Wim Vanduffel, Zsuzsanna Szemlaky, Áron Szepesi, István Ulbert, Balázs Rózsa, Dániel Hillier","doi":"10.1038/s41434-025-00567-0","DOIUrl":null,"url":null,"abstract":"<p><p>Sensitive quantification of adeno-associated virus (AAV) neutralizing antibodies (NAbs) is essential for gene therapy success. Conventional cell-based transduction inhibition assays often encounter matrix-induced artifacts resulting from variable serum content across dilutions, which artificially inflate transduction baselines and mask partial neutralization. To address this challenge, we developed the constant serum concentration (CSC) assay, which maintains constant serum levels across dilutions to stabilize assay baselines and enhance NAb detection sensitivity. Using human sera across multiple AAV serotypes, we demonstrated that CSC reclassified up to 21.7% of samples classified as non-neutralizing by a conventional variable serum concentration (VSC) assay format. This improved sensitivity was validated using monoclonal antibody and multi-species serum test benchmarks and enhanced the reliability of seronegative control pool selection. Importantly, CSC detected persistent seropositivity in preclinical seroreversion models up to one year longer than a conventional VSC assay. Since even low-level neutralizing antibodies can significantly impact gene therapy efficacy, these findings have direct implications for optimizing AAV redosing strategies and refining patient stratification. By addressing fundamental limitations in NAb quantification while maintaining procedural simplicity, the CSC assay provides crucial insights into antibody persistence with translational relevance across species and clinical settings.</p>","PeriodicalId":12699,"journal":{"name":"Gene Therapy","volume":" ","pages":""},"PeriodicalIF":4.5000,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Overcoming matrix effects in AAV neutralization assays with a constant serum concentration approach.\",\"authors\":\"Beatrix Kovács, Viktória Szabó, Domonkos Horváth, Attila Balázs Dobos, Zoltán Zsolt Nagy, Wim Vanduffel, Zsuzsanna Szemlaky, Áron Szepesi, István Ulbert, Balázs Rózsa, Dániel Hillier\",\"doi\":\"10.1038/s41434-025-00567-0\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Sensitive quantification of adeno-associated virus (AAV) neutralizing antibodies (NAbs) is essential for gene therapy success. Conventional cell-based transduction inhibition assays often encounter matrix-induced artifacts resulting from variable serum content across dilutions, which artificially inflate transduction baselines and mask partial neutralization. To address this challenge, we developed the constant serum concentration (CSC) assay, which maintains constant serum levels across dilutions to stabilize assay baselines and enhance NAb detection sensitivity. Using human sera across multiple AAV serotypes, we demonstrated that CSC reclassified up to 21.7% of samples classified as non-neutralizing by a conventional variable serum concentration (VSC) assay format. This improved sensitivity was validated using monoclonal antibody and multi-species serum test benchmarks and enhanced the reliability of seronegative control pool selection. Importantly, CSC detected persistent seropositivity in preclinical seroreversion models up to one year longer than a conventional VSC assay. Since even low-level neutralizing antibodies can significantly impact gene therapy efficacy, these findings have direct implications for optimizing AAV redosing strategies and refining patient stratification. By addressing fundamental limitations in NAb quantification while maintaining procedural simplicity, the CSC assay provides crucial insights into antibody persistence with translational relevance across species and clinical settings.</p>\",\"PeriodicalId\":12699,\"journal\":{\"name\":\"Gene Therapy\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":4.5000,\"publicationDate\":\"2025-09-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Gene Therapy\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1038/s41434-025-00567-0\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Gene Therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1038/s41434-025-00567-0","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Overcoming matrix effects in AAV neutralization assays with a constant serum concentration approach.
Sensitive quantification of adeno-associated virus (AAV) neutralizing antibodies (NAbs) is essential for gene therapy success. Conventional cell-based transduction inhibition assays often encounter matrix-induced artifacts resulting from variable serum content across dilutions, which artificially inflate transduction baselines and mask partial neutralization. To address this challenge, we developed the constant serum concentration (CSC) assay, which maintains constant serum levels across dilutions to stabilize assay baselines and enhance NAb detection sensitivity. Using human sera across multiple AAV serotypes, we demonstrated that CSC reclassified up to 21.7% of samples classified as non-neutralizing by a conventional variable serum concentration (VSC) assay format. This improved sensitivity was validated using monoclonal antibody and multi-species serum test benchmarks and enhanced the reliability of seronegative control pool selection. Importantly, CSC detected persistent seropositivity in preclinical seroreversion models up to one year longer than a conventional VSC assay. Since even low-level neutralizing antibodies can significantly impact gene therapy efficacy, these findings have direct implications for optimizing AAV redosing strategies and refining patient stratification. By addressing fundamental limitations in NAb quantification while maintaining procedural simplicity, the CSC assay provides crucial insights into antibody persistence with translational relevance across species and clinical settings.
期刊介绍:
Gene Therapy covers both the research and clinical applications of novel therapeutic techniques based on a genetic component. Over the last few decades, significant advances in technologies ranging from identifying novel genetic targets that cause disease through to clinical studies, which show therapeutic benefit, have elevated this multidisciplinary field to the forefront of modern medicine.