A Quantitative In Vitro Potency Assay for Adeno-Associated Virus Vectors Encoding for the UGT1A1 Transgene.

Potency assessment of clinical-grade vector lots is crucial to support adeno-associated virus (AAV) vector release and is required for future marketing authorization. We have developed and validated a cell-based, quantitative potency assay that detects both transgenic expression and activity of an AAV8-h vector, which is currently under clinical evaluation for the treatment of Crigler-Najjar syndrome. Potency of AAV8-h was evaluated . After transduction of human hepatoma 7 (Huh7) cells, transgene-positive cells were quantified using flow cytometry and transgenic activity by a bilirubin conjugation assay. The potency of various AAV8-hUGT1A1 batches was compared with their potency . After AAV8-h transduction, quantification of UGT1A1-expressing cells shows a linear dose-response relation (R = 0.98) with adequate intra-assay and inter-day reproducibility (coefficient of variation [CV] = 11.0% and 22.6%, respectively). In accordance, bilirubin conjugation shows a linear dose-response relation (R = 0.99) with adequate intra- and inter-day reproducibility in the low dose range (CV = 15.7% and 19.7%, respectively). Both potency assays reliably translate to efficacy of AAV8-h vector lots. The described cell-based potency assay for AAV8-h adequately determines transgenic UGT1A1 expression and activity, which is consistent with efficacy. This novel approach is suited for the determination of vector lot potency to support clinical-grade vector release.