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Simple, rapid and robust bioluminescent cell-based assay for detecting neutralising antibodies against AAV in serum

E Kirby(1)

1:Promega Corporation

Adeno-associated viruses (AAV) are commonly used in gene therapy. But pre-existing immunity to AAVs can impede cellular uptake and trigger cytotoxic T-cell responses, compromising efficacy and safety. Therefore, a robust and reliable neutralising antibody (NAb) assay is necessary to determine eligibility for treatment as well as to identifying individuals for clinical trials.

Here we present a cell based NanoLuc® luciferase assay for detecting NAbs in serum. The assay improves on existing methods offering; higher sensitivity, lower MOI required (100–3000), shorter 24h assay time and detection using a standard luminometer.

We tested 60 human serum samples for NAbs against a panel of AAV serotypes 1—10 and engineered variant AAV-DJ. A large percentage of the samples had pre-existing NAbs to at least one serotype and of these, the majority displayed NAbs against multiple AAV serotypes. Fewer samples were seropositive for AAV-DJ than for any of the parental serotypes highlighting the benefit of capsid engineering. The assays broad dynamic range allows the serum samples to be categorised into 4 groups: negative, low, medium and high NAbtitres. Samples from each group were assayed for neutralising titres with ND50 values agreeing with their assigned groups.

To demonstrate robustness, we conducted a study with 40 mice divided into 4 groups of AAV9 at varying doses. This showed a strong correlation of NAblevels to the AAV viral loads.

Our NanoLuc® AAV reporter assay is fast, highly sensitive and reliable for NAb detection against AAVs in serum. Further validation is needed before clinical use.

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