P01

Validation of a luminometric assay for the detection of human anti-AAV neutralization factors.

C Couzinié(1,2) N Jaulin(1) M Loirat(1) H Fonteneau(1,2) J Le Duff(1) G Gernoux(1) O Adjali(1)

1:TaRGeT - Translational Research in Gene Therapy, UMR 1089, Nantes Université, CHU de Nantes, INSERM, Nantes, 44200, France; 2:Capacités SAS, Nantes, 44200, France

In AAV-mediated gene therapies, pre-existing anti-AAV humoral immunity can hinder gene therapy efficacy and induce safety concerns. This is a major challenge, particularly for clinical trials, since over 50% of patients present anti-AAV neutralizing factors (NFs) depending on AAV serotypes and can be excluded from treatment eligibility.

Anti-AAV NFs are typically detected using a cell-based transduction inhibition assay. However, protocols remain not standardized in the AAV gene therapy community. Over the past 15 years, our team worked on the detection of both pre-existing and induced AAV neutralizing factors, and we developed a luminometric qualitative anti-AAV NF assay using standardized controls. Using a luciferase reporter gene system, our assay is adaptable to several AAV serotypes and species and enable the detection of anti-AAV neutralizing factors at titers as low as 1:5, aligned with the selection criteria commonly used in gene therapy studies.

In this context, in the present study we validated our method according to regulatory guidelines such as EMA-ICH M10 and FDA guidelines for bioanalytical method validation guidance for industry. Specifically, the method was evaluated for the detection of AAVrh10 neutralizing factors in human sera, with the assessment of applicable parameters within-in run and between-run accuracy and precision, dilution linearity, serum and vector stability and batch to batch variability.

In conclusion, this study provides a validated and reliable assay for research grade applications as well as regulatory studies such as toxicology studies and clinical trials.