用恒血清浓度法克服AAV中和试验中的基质效应。

IF 4.5 3区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Beatrix Kovács, Viktória Szabó, Domonkos Horváth, Attila Balázs Dobos, Zoltán Zsolt Nagy, Wim Vanduffel, Zsuzsanna Szemlaky, Áron Szepesi, István Ulbert, Balázs Rózsa, Dániel Hillier
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引用次数: 0

摘要

腺相关病毒(AAV)中和抗体(nab)的敏感定量对基因治疗的成功至关重要。传统的基于细胞的转导抑制试验经常遇到基质诱导的伪像,这些伪像是由不同稀释度的血清含量引起的,人为地夸大了转导基线并掩盖了部分中和作用。为了应对这一挑战,我们开发了恒定血清浓度(CSC)测定法,该方法在不同稀释度下保持恒定的血清浓度,以稳定测定基线并提高NAb检测灵敏度。使用多种AAV血清型的人血清,我们证明CSC通过传统的可变血清浓度(VSC)检测格式重新分类了高达21.7%的非中和样本。使用单克隆抗体和多种血清测试基准验证了这种提高的敏感性,并提高了血清阴性对照池选择的可靠性。重要的是,CSC在临床前血清逆转模型中检测到持续血清阳性的时间比传统的VSC检测长一年。由于即使是低水平的中和抗体也能显著影响基因治疗效果,因此这些发现对优化AAV再给药策略和改进患者分层具有直接意义。通过解决NAb定量的基本限制,同时保持程序的简单性,CSC分析提供了跨物种和临床环境翻译相关性的抗体持久性的重要见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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.

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来源期刊
Gene Therapy
Gene Therapy 医学-生化与分子生物学
CiteScore
9.70
自引率
2.00%
发文量
67
审稿时长
4-8 weeks
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.
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