An In Vitro RNA Editing-Based Reporter Assay for Transcriptional Activity of Therapeutic Gene in Gene Therapy Products.

The expression of therapeutic genes is critical for the efficacy of gene therapy products. However, existing methods such as immunological analysis at the protein level or reverse-transcription PCR at the RNA level are unable to accurately quantify the expression activity of the target gene. Herein, an in vitro RNA editing-based reporter assay was developed to detect specific mRNA. The designed sensor RNA could specifically identify the target mRNA, and the reporter gene was activated in a dose-dependent manner because of RNA editing mediated by endogenous adenosine deaminases acting on RNA. Of note, all sensors that targeted different regions, including the gene of interest, tag sequence, and 3' untranslated region, showed a dose-dependent response pattern. The sensor reporter assay, which was used for quantifying the transcriptional activity of recombinant adeno-associated virus-based gene therapy products, revealed excellent performance in terms of assay specificity, precision (inter-assay relative standard deviation < 15%), accuracy (90-115% recovery), and linearity (R > 0.99). The reporter assay could also be employed for other gene therapy vectors, including mRNA and recombinant lentivirus. Thus, a robust and reliable platform was developed for assessing the transcriptional activity of therapeutic genes, thereby offering a powerful tool for the quality control of gene therapy products.