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Retinal Epithelial Neutralization Assay Optimizes AAV Serotype Selection for Ocular Gene Therapy.
Yao Li1, Yujia Chen1, Nan Huo1
1Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China.
Viruses
|July 30, 2025
Summary
Pre-existing neutralizing antibodies (NAbs) hinder ocular gene therapy. Retinal cells, unlike standard models, reveal crucial NAb differences, improving AAV vector selection and patient outcomes.
Area of Science:
- Ophthalmology
- Gene Therapy
- Immunology
Background:
- Pre-existing neutralizing antibodies (NAbs) against adeno-associated virus (AAV) vectors are a major hurdle for ocular gene therapy.
- Standard NAb detection assays using non-ocular cells may not accurately reflect retinal transduction inhibition.
Purpose of the Study:
- To establish and compare NAb detection platforms using human retinal pigment epithelial (ARPE-19) cells versus standard 293T cells.
- To evaluate the impact of cell type on NAb titers and neutralization patterns for ocular AAV serotypes.
Main Methods:
- Developed parallel NAb detection platforms using ARPE-19 and 293T cells.
- Assessed clinical serum samples against AAV serotypes 2, 5, 8, and 9 using luciferase reporter-based transduction inhibition assays.
Main Results:
- ARPE-19 cells showed 42-48% higher NAb titers against AAV5/9 compared to 293T cells.
- Distinct serotype-specific neutralization hierarchies were observed between the two cell models.
- Female sera exhibited higher NAbs against certain serotypes in the ARPE-19 system, and cross-neutralization patterns differed significantly.
Conclusions:
- Physiologically relevant retinal cell models provide critical immunological data for ocular gene therapy.
- Retinal cell-based platforms are essential for optimizing AAV serotype selection, patient stratification, and predicting clinical outcomes.
