CoreTIA: a modular, statistically robust transduction inhibition assay for AAV neutralization

Area of Science:

• * Molecular biology and virology
• * Gene therapy and drug development
• * Immunology and diagnostics

Background:

• * Pre-existing neutralizing antibodies (NAbs) against adeno-associated virus (AAV) pose a significant challenge to the efficacy of AAV-based gene therapies.
• * Current NAb detection assays lack standardization and do not adequately quantify uncertainty, hindering reliable cross-study comparisons and regulatory assessment.
• * This variability impedes the advancement of AAV gene therapy by complicating patient selection and treatment monitoring.

Purpose of the Study:

• * To introduce core Transduction Inhibition Assay (coreTIA), a standardized framework for quantifying AAV NAbs.
• * To provide a statistically robust analysis pipeline for precise NAb titer determination with quantified uncertainty.
• * To enhance reproducibility and consistency in NAb measurements across different studies and laboratories.

Main Methods:

• * Development of a modular experimental protocol for NAb detection using a transduction inhibition assay.
• * Implementation of a statistically rigorous analysis pipeline to calculate NAb titers and associated uncertainty.
• * Validation of the assay's performance across various adeno-associated virus (AAV) serotypes and refinement of key assay parameters.

Main Results:

• * coreTIA delivers precise and reproducible NAb titers with quantified uncertainty for each measurement.
• * The statistical framework allows for robust neutralization estimation even with incomplete dilution series, reducing the need for repeat testing and minimizing sample volume.
• * Consistent assay performance was demonstrated across different AAV serotypes, indicating broad applicability.

Conclusions:

• * coreTIA offers a standardized, statistically robust, and transparent method for measuring AAV NAbs, addressing a critical need in gene therapy.
• * The open-resource nature of coreTIA promotes assay harmonization and facilitates more reliable regulatory assessment of gene therapy candidates.
• * By overcoming a key barrier in NAb detection, coreTIA is expected to accelerate progress in adeno-associated virus gene therapy research and development.