ArticleMolecules (Basel, Switzerland)2024
An In Vitro RNA Editing-Based Reporter Assay for Transcriptional Activity of Therapeutic Gene in Gene Therapy Products.
Article in Molecules (Basel, Switzerland), 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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
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