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

Abstract

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 (R 2 = 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 2 = 0.99) with adequate intraand 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.

Inducible caspase 9-mediated suicide gene therapy using AAV6 vectors in a murine model of breast cancer

“…However, a number of factors, including vector saturation dose, where a dose of ∼5 × 10 10 vg/animal may be close to the saturation levels and which may have contributed to the similar regression pattern found in the WT and mutant vector–treated animals. 34 , 40 , 41 , 42 Although we noticed high AAV transduction in the mutant group, the same does not get reflected in terms of RTV, and we speculate that this discrepancy may stem from the limited availability of the dimerizer drug AP20187, which is integral to the ultimate cell kill effect. Future investigations focusing on dose optimization of AP20187 could help elucidate its impact on RTV.…”

CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy

“…Importantly, in vitro potency tests are less time-consuming, less labor-intensive, and cheaper than in vivo models. Successful potency assays were previously developed in liver [41] and retinal gene therapies [42].…”

Low efficacy of recombinant SV40 in Ugt1a1-/- mice with severe inherited hyperbilirubinemia

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