P02

A simple, rapid and robust bioluminescent assay for detecting anti-AAV neutralizing antibodies in serum samples

B Binkowski(1) M Seirup(1) I Prost(2) T Hoang(1) M Urh(1)

1:Promega Corporation; 2:Promega France

Adeno-associated viruses (AAVs) are widely used vectors to deliver transgenes for gene therapy. Individuals with pre-existing immunity to AAVs are less likely to benefit from gene therapy owing to the presence of neutralizing antibodies (NAbs) that reduce cellular uptake. Widespread, pre-existing immunity in human and animal subjects increases the need for sensitive assays to detect NAb activity in serum samples. Here we describe a cell-based transduction inhibition (TI) assay utilizing NanoLuc luciferase (Nluc), an enzyme engineered to be extremely bright and structurally stable. The superior brightness of Nluc (>700-fold brighter than firefly luciferase) enables the sensitive detection of NAb activity using low MOI values (100 – 10,000) and short incubation times (≤24 hours). The assay is tolerant to high levels of human serum, allowing dilutions as low as 1:3, and luminescence is measured following addition of an add-mix-read (homogeneous) detection reagent (Bio-Glo-AAV). We screened 1:3, 1:9, 1:27 & 1:81 dilutions of human serum samples (MOI = 10,000) to demonstrate the influence of sample dilution on hit rate. We also compared head-to-head with the analogous approach using firefly luciferase (Fluc) as the reporter, and we validated use of thaw-and-use HEK293 cells. Taken together, this rapid, highly sensitive and reliable assay enabled by Nluc AAV reporter technology precisely measures NAbs against AAVs in both human and animal serum.