Qualification Of An mRNA Expression Assay For GOI, Stability Insights, And Process Evolution Detection

By G. Iyer and S. Sleep

This study focuses on developing and qualifying a robust in vitro cell-based potency assay for an ophthalmic gene therapy vector, addressing the unique challenges in measuring rAAV product attributes such as quality, identity, purity, strength, and stability. Using multiplexed RT-qPCR, mRNA expression was analyzed in three phases: cell transduction, RNA isolation, and gene-specific detection. The assay was optimized and qualified for functional potency assessment, demonstrating high sensitivity, specificity, accuracy, precision, and linearity across RT-qPCR and total assay processes.

Additionally, trending analysis of mRNA expression patterns across stability, process comparability, and device compatibility studies provided insights into product performance and potential variations over time. This qualified assay supports product conformance in release testing, stability programs, and manufacturing changes, ensuring the consistency and functionality of gene therapy products throughout preclinical and clinical development phases.