Optimizing Aav-Mediated Neural Gene Therapy: A Next-Generation Neutralization Assay For Patient Stratification
Kovács Beatrix Helga
János Szentágothai Neurosciences Division
Dr. Bereczki Dániel
MTA TTK földszinti kis előadóterem
2026-09-07 10:00:00
Functional neurosciences
Dr. Sperlágh Beáta
Dr. Hillier Dániel
Barcsay-Veres Amarilla
Tobias Brügmann
Dr. Buzás Edit
Dr. Szalay Gergely
Dr. Alexa Anita
Adeno-associated virus (AAV) vectors are currently the leading platform for in vivo gene therapy. However, the widespread prevalence of pre-existing neutralizing antibodies (NAbs) against the AAV capsid in the human population poses a barrier to safety and efficacy. Consequently, sensitive and reliable screening of patients prior to treatment is essential. Despite this need, the currently used neutralization assays lack standardization and are often limited by two factors: the "serum matrix effect," where variable serum components artificially enhance viral transduction and mask low-level antibodies; and the lack of a consistent statistical framework, which often leads to subjective data interpretation.
To address these limitations, my doctoral thesis introduces coreTIA (core Transduction Inhibition Assay), a comprehensive framework that integrates an optimized experimental protocol with a rigorous statistical pipeline.
The experimental foundation of coreTIA is the Constant Serum Concentration (CSC) design, which maintains a constant serum environment across dilution points. This approach stabilizes assay baselines and unmasks low-level neutralizing activity. The optimization was validated across multiple AAV serotypes (AAV1, AAV5, AAV9) and species, including mice, cats, monkeys and humans.
Complementing the experimental workflow, I developed an open-source statistical framework for objective data analysis. Utilizing a Bayesian probabilistic model, this pipeline enables the precise estimation of 50% neutralizing dose (ND50) and quantifies measurement uncertainty, offering an alternative to subjective manual evaluation.
The sensitivity of the coreTIA platform was demonstrated by identifying pre-existing immunity in up to 21.7% of human samples that were classified as "seronegative" by conventional testing. Furthermore, in preclinical seroreversion studies, the assay extended the detection window of persisting antibodies by over a year. In conclusion, this harmonized platform provides a reliable tool for patient stratification, minimizing the risk of false-negative classification and immune-mediated treatment failure in gene therapy.
