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

Area of Science:

• * Gene Therapy
• * Molecular Biology
• * Assay Development

Background:

• * Voretigene neparvovec-rzyl (Luxturna) is an AAV2 vector approved for treating inherited retinal diseases caused by RPE65 mutations.
• * Regulatory approval necessitates validated assays demonstrating biological activity and potency.
• * RPE65 protein is crucial for the visual cycle, converting all-trans-retinol to 11-cis-retinol.

Purpose of the Study:

• * To validate an in vitro cell-based assay for measuring the biological activity and relative potency of voretigene neparvovec-rzyl (AAV2-hRPE65v2).
• * To support regulatory approval and ensure lot-to-lot consistency of the gene therapy product.

Main Methods:

• * Transduction of HEK293 cells with AAV2-hRPE65v2 vector.
• * Quantification of vector-encoded RPE65 protein in cell lysates using liquid chromatography with tandem mass spectrometry (LC-MS/MS).
• * Validation of the assay across seven key characteristics: suitability, specificity, linearity, precision, accuracy, range, and robustness.

Main Results:

• * The validated assay accurately measures AAV2-hRPE65v2 transduction and subsequent RPE65 biological activity.
• * The assay demonstrated reliability for assessing relative potency within a 50%-150% range of a reference standard.
• * Successful validation supported lot-to-lot consistency, stability, and comparability assessments for Luxturna manufacturing.

Conclusions:

• * This study reports the first validation of an in vitro cell-based relative potency assay for an AAV vector.
• * The validated assay is critical for ensuring the quality and efficacy of Luxturna, the first gene therapy approved in the US for a genetic disease.
• * The assay facilitates manufacturing process improvements and regulatory compliance for gene therapies.