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

Sem J Aronson,Robert S Bakker,Sascha Moenis,Remco Van Dijk,Giulia Bortolussi,Fanny Collaud,Xiaoxia Shi,Suzanne Duijst,Lysbeth Ten Bloemendaal,Giuseppe Ronzitti,Andrés F Muro,Federico Mingozzi,Ulrich Beuers,Piter J Bosma

doi:10.1016/j.omtm.2020.06.002

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 (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.

Highlights

Adeno-associated virus (AAV)-mediated gene therapy has shown great potential to treat inherited monogenetic disorders, as clinical trials performed in the last decade point out.[1,2,3,4,5,6,7,8] An increasing number of AAV-based gene therapy medicinal products will enter clinical development, because eventually these advanced therapies will become part of the therapeutic arsenal for rare genetic diseases
Here we present the validation of a quantitative AAV vector potency assay that detects both transgenic uridine diphosphoglucuronosyl transferase 1A1 (UGT1A1) expression and activity in a cell-based system
Detection of Intra-cellular UGT1A1 by Flow Cytometry To determine the transduction efficiency of AAV serotype 8 (AAV8)-hUGT1A1 vector batches, we developed a flow cytometry-based assay to quantify the percentage of UGT1A1-expressing cells

Summary

Introduction

Adeno-associated virus (AAV)-mediated gene therapy has shown great potential to treat inherited monogenetic disorders, as clinical trials performed in the last decade point out.[1,2,3,4,5,6,7,8] An increasing number of AAV-based gene therapy medicinal products will enter clinical development, because eventually these advanced therapies will become part of the therapeutic arsenal for rare genetic diseases. One of the challenging aspects during gene therapy development is the characterization and quantification of vector potency, being one of the crucial methods of product quality control and required for marketing authorization.[9] As defined by the European Medicines Agency (EMA),[10] vector potency is the measurement of the biological activity using a quantitative biological assay, which is linked to the relevant biological properties and the claimed mechanism of action. To avoid accumulation of this neurotoxic compound that can cause life-threatening bilirubin encephalopathy,[13] severely affected individuals depend on phototherapy up to 12 h/day, often followed by liver transplantation later in life.[14,15,16]

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