The detection of biologically relevant low-titer neutralizing antibodies against AAV require sensitive in vitro assays.

Patients with preexisting anti-adeno-associated virus 8 (AAV8) neutralizing antibodies (NAbs) are currently excluded from AAV8 gene therapy trials. Therefore, the assessment of biologically relevant AAV8-NAb titers is critical for product development in gene therapy. However, standardized assays have not been routinely used to determine anti-AAV8-NAb titers, contributing to a wide range of reported anti-AAV8 prevalences. Using a clinical in vitro NAb assay in a separate study, we found a higher than expected anti-AAV8-NAb prevalence in international cohorts of about 50%. This comparative study has a translational character confirming the biological relevance of anti-AAV8-antibody titers measured by this assay. We show the significance of low titer anti-AAV8 NAbs, and the relevance of the in vitro assay cut-off (1:5) compared to other assays. Importantly, we used internally standardized reagents and purified AAV8 constructs containing 90% full capsids to reduce the effect of empty capsids. We found that even very low anti-AAV8-NAb titers (<1:5) could efficiently hinder transduction in vivo, demonstrating the importance of sensitive NAb assays for clinical applications. The in vitro NAb assay was found to be more sensitive than an in vivo NAb assay, and thus more suitable for patient screening. Additionally, we showed that anti-AAV8-NAb titers below 1:5 were very rare, further supporting the in vitro assay. However, assays using a lower cut-off may still be useful to explain potential variances in transgene expression. These findings support the relevance of the higher than expected prevalence of anti-AAV8 NAbs, highlighting the need for strategies to circumvent preexisting anti-AAV8 NAbs.