Li Yao, Chen Yujia, Huo Nan, Jia Zuyuan, Huang He, Zhao Zhenghao, Wu Shipo, Hou Lihua
Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China.
Viruses. 2025 Jul 15;17(7):988. doi: 10.3390/v17070988.
Adeno-associated virus (AAV) vectors face a critical translational challenge in ocular gene therapy due to pre-existing neutralizing antibodies (NAbs) whose seroprevalence limits patient eligibility. Standard NAb detection using non-ocular cell models (Human Embryonic Kidney 293T) may inadequately predict retinal transduction inhibition due to cell type-related variations in receptor usage and immunogenicity. This study established parallel NAb detection platforms utilizing human retinal pigment epithelial (ARPE-19) cells and standard 293T cells to systematically evaluate clinical serum samples against ophthalmologically relevant AAV serotypes (2, 5, 8, 9) via luciferase reporter-based transduction inhibition assays. Comparative analysis demonstrated ARPE-19 exhibited 42-48% higher NAb titers against AAV5/9 compared to 293T cells, with distinct serotype-biased neutralization hierarchies observed between cellular models. Furthermore, female-derived sera exhibited significantly elevated NAbs against particular serotypes in the ARPE-19 system. Critically, inter-serotype cross-neutralization correlation patterns differed substantially between cellular platforms. These findings demonstrate that physiologically relevant retinal cellular models provide essential immunological profiling data, revealing NAb characteristics obscured in standard assays. Consequently, employing retinal cell-based platforms is crucial for optimizing AAV serotype selection, patient stratification, and predicting clinical outcomes in ocular gene therapy.
由于预先存在的中和抗体（NAbs），腺相关病毒（AAV）载体在眼部基因治疗中面临着关键的转化挑战，这些中和抗体的血清流行率限制了患者的入选资格。使用非眼部细胞模型（人胚肾293T）进行的标准NAb检测可能无法充分预测视网膜转导抑制，因为受体使用和免疫原性存在细胞类型相关的差异。本研究建立了平行的NAb检测平台，利用人视网膜色素上皮（ARPE-19）细胞和标准的293T细胞，通过基于荧光素酶报告基因的转导抑制试验，系统地评估针对眼科相关AAV血清型（2、5、8、9）的临床血清样本。比较分析表明，与293T细胞相比，ARPE-19对AAV5/9的NAb滴度高42-48%，在细胞模型之间观察到明显的血清型偏向中和层次。此外，在ARPE-19系统中，女性来源的血清对特定血清型的NAb显著升高。至关重要的是，血清型间交叉中和相关模式在细胞平台之间存在很大差异。这些发现表明，生理相关的视网膜细胞模型提供了重要的免疫学分析数据，揭示了标准检测中掩盖的NAb特征。因此，采用基于视网膜细胞的平台对于优化AAV血清型选择、患者分层以及预测眼部基因治疗的临床结果至关重要。
