Retinal Epithelial Neutralization Assay Optimizes AAV Serotype Selection for Ocular Gene Therapy

TL;DR

This study shows that using retinal cells improves the accuracy of predicting how antibodies block gene therapy in the eye, leading to better treatment choices.

Contribution

The study introduces a retinal epithelial-based assay that reveals antibody neutralization patterns specific to ocular gene therapy serotypes.

Findings

ARPE-19 cells showed 42–48% higher neutralizing antibody titers against AAV5/9 compared to 293T cells.

Female-derived sera showed significantly elevated neutralizing antibodies against specific serotypes in retinal cells.

Cross-neutralization patterns between AAV serotypes varied significantly between retinal and standard cell models.

Abstract

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.

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