Validation of a quantitative cell-based relative potency assay for LUXTURNA

Voretigene neparvovec-rzyl (Luxturna) is an AAV2 vector (AAV2-hRPE65v2) that expresses a cDNA encoding the human retinal pigment epithelium-specific 65 kDa protein (RPE65). It has been approved for the treatment of visual deficits associated with biallelic mutations in human RPE65 in the US, European Union (EU), and multiple other countries. To achieve regulatory approval, it was necessary to validate an assay demonstrating its biological activity or potency. The assay measures AAV2-h<i>RPE65</i>v2 transduction in HEK293 cells and the subsequent biological activity of the vector-encoded RPE65 protein in cell lysates. RPE65 converts all-<i>trans</i>-retinol to 11-<i>cis</i>-retinol, which is quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The assay was validated for seven characteristics, namely system and sample suitability, specificity, linearity, precision, relative accuracy, range, and robustness. The validated assay can be used to confirm the relative potency levels of different lots of Luxturna in the range of 50%-150% of a reference standard (defined as 100% potent). This represents the first report of validation studies supporting an <i>in vitro</i> cell-based relative potency assay for an AAV vector, which was used to evaluate lot-to-lot consistency, stability, and comparability following manufacturing changes and to successfully launch Luxturna, the first gene therapy approved in the US for a genetic disease.