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-hUGT1A1 vector, which is currently under clinical evaluation for the treatment of Crigler-Najjar syndrome. Potency of AAV8-hUGT1A1 was evaluated in vitro. 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 in vitro potency of various AAV8-hUGT1A1 batches was compared with their potency in vivo. After AAV8-hUGT1A1 transduction, quantification of UGT1A1-expressing cells shows a linear dose-response relation (R² = 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 (R² = 0.99) with adequate intra- and inter-day reproducibility in the low dose range (CV = 15.7% and 19.7%, respectively). Both in vitro potency assays reliably translate to in vivo efficacy of AAV8-hUGT1A1 vector lots. The described cell-based potency assay for AAV8-hUGT1A1 adequately determines transgenic UGT1A1 expression and activity, which is consistent with in vivo efficacy. This novel approach is suited for the determination of vector lot potency to support clinical-grade vector release.

临床级载体批次的效能评估对于支持腺相关病毒（AAV）载体的释放至关重要，并且是未来上市授权所必需的。我们已经开发并验证了一种基于细胞的定量效能测定方法，该方法可检测AAV8-h UGT1A1载体的转基因表达和活性，目前正在临床评估中用于治疗Crigler-Najjar综合征。在体外评估了AAV8-h UGT1A1的效力。转导人肝癌7（Huh7）细胞后，使用流式细胞仪对转基因阳性细胞进行定量，并通过胆红素结合测定法对转基因活性进行定量。比较了各种AAV8-hUGT1A1批次的体外效能与它们在体内的效能。在AAV8-h UGT1A1转导后，定量表达UGT1A1的细胞显示出线性的剂量反应关系（R ²  = 0.98），具有足够的测定内和日间重现性（变异系数[CV] = 11.0％和22.6％，分别）。因此，胆红素结合表现出线性的剂量-反应关系（R ²  = 0.99），在低剂量范围内（CV = 15.7％和19.7％），具有足够的日内和日间重现性。两种体外效价测定均可靠地转化为AAV8-h UGT1A1载体批次的体内功效。所述的AAV8-h UGT1A1的基于细胞的效能测定足够确定转基因UGT1A1的表达和活性，这与体内功效一致。这种新颖的方法适用于确定载体批次效价以支持临床级载体的释放。